Interaction of receptor type of protein tyrosine phosphatase sigma (RPTPσ) with a glycosaminoglycan library

Interaction of receptor type of protein tyrosine phosphatase sigma (RPTPσ) with a... Abstract Receptor type of protein tyrosine phosphatase sigma (RPTPσ) functions as a glycosaminoglycan (GAG) receptor of neuronal cells in both the central and peripheral nervous systems. Both chondroitin sulphate (CS) and heparan sulphate (HS) are important constituents of GAG ligands for RPTPσ, although they have opposite effects on neuronal cells. CS inhibits neurite outgrowth and neural regeneration through RPTPσ, whereas HS enhances them. We prepared recombinant RPTPσ N-terminal fragment containing the GAG binding site and various types of biotin-conjugated GAG (CS and HS) with chemical modification and chemo-enzymatic synthesis. Then interaction of the RPTPσ N-terminal fragment was analysed using GAG-biotin immobilized on streptavidin sensor chips by surface plasmon resonance. Interaction of RPTPσ with the CS library was highly correlated to the degree of disulphated disaccharide E unit, which had two sulphate groups at C-4 and C-6 positions of the N-acetylgalactosamine residue (CSE). The optimum molecular mass of CSE was suggested to be approximately 10 kDa. Heparin showed higher affinity to RPTPσ than the CS library. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology, but also provide medical application to regulate neural regeneration. chondroitin, glycosaminoglycans, heparin, RPTPσ, SPR In the extracellular matrix (ECM) in the central and peripheral nervous systems, chondroitin sulphate (CS) and heparan sulphate (HS) play important roles in the maintenance of the neuronal network and synapse formation; however, they exhibit opposite effects. A series of evidence supports that CS inhibits axonal growth and nerve regeneration through receptor type of protein tyrosine phosphatase sigma (RPTPσ) (1, 2), whereas HS promotes these processes (3, 4). For instance, CS accumulation in the glial scar after neuronal injury limited axonal growth and regeneration of the neuronal network. Injection of chondroitinase ABC to the lesion promoted axonal regeneration and functional recovery after spinal cord injury (5, 6). CS N-acetylgalactosaminyltransferase-1 knockout mice, which show decreased CS synthesis and increased HS levels, experience better restoration of the neuronal network after spinal cord injury than wild-type mice (7). Glycosaminoglycan (GAG) is a linear acidic polysaccharide composed of repeating disaccharide units and is modified with sulphate groups at various positions on the sugar residues (Fig. 1A) (8). Whereas hyaluronan (HA), composed of a repeating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) disaccharide unit, lacks sulphate modification, CS, composed of a GlcA and N-acetylgalactosamine (GalNAc) disaccharide unit, is modified with sulphate groups at various positions of the sugar residues. The major disaccharide structures of CS are as follows: a non-sulphated unit (0S, GlcA-GalNAc), a monosulphated unit at the C-4 position of the GalNAc residue (4S, GlcA-GalNAc4S), a monosulphated unit at the C-6 position of GalNAc (6S, GlcA-GalNAc6S), a disulphated unit at the C-4 and C-6 positions of GalNAc (diSE, GlcA-GalNAc4S6S), a disulphated unit at the C-2 position of GlcA and the C-4 position of GalNAc (diSB, GlcA2S-GalNAc4S), a disulphated unit at the C-2 position of GlcA and the C-6 position of GalNAc (diSD, GlcA2S-GalNAc6S), and a trisulfated disaccharide unit (triS, GlcA2S-GalNAc4S6S). Some GlcA residues are epimerized to iduronic acid (IdoA); the chain containing IdoA residues is designated as dermatan sulphate (DS). Fig. 1 View largeDownload slide Structures of GAGs and RPTPσ. (A) Structures of disaccharide units of HA, CS/DS and HS/HP. R1, R2 and R3 in the CS/DS table indicate O-substituted groups at the C-6, C-4 positions of the GalNAc residue, and C-2 position of the GlcA/IdoA residue, respectively. R1, R2 and R3 in the HS/HP table indicate O- or N-substituted groups at the C-6 position of the GlcN residue, C-2 position of the GlcA/IdoA residue and C-2 position of GlcN residue, respectively. (B) Domain organization of human RPTPσ isoform 4. Signals indicate the following: N-, amino terminus; S, signal peptide; Ig, immunoglobulin-like domains (1–3); FN3, fibronectin type-III domains (1–5); TM, transmembrane domain; phase, phosphatase domains (D1-D2) and -C, carboxy terminus. (C) SDS-PAGE with CBB (a and b) and western blotting with anti-RPTPσ antibody (c and d) for crude HaloTag-binding protein (70 kDa, a and c) and TEV-treated purified RPTPσ N-terminal fragment (36 kDa, b and d). Fig. 1 View largeDownload slide Structures of GAGs and RPTPσ. (A) Structures of disaccharide units of HA, CS/DS and HS/HP. R1, R2 and R3 in the CS/DS table indicate O-substituted groups at the C-6, C-4 positions of the GalNAc residue, and C-2 position of the GlcA/IdoA residue, respectively. R1, R2 and R3 in the HS/HP table indicate O- or N-substituted groups at the C-6 position of the GlcN residue, C-2 position of the GlcA/IdoA residue and C-2 position of GlcN residue, respectively. (B) Domain organization of human RPTPσ isoform 4. Signals indicate the following: N-, amino terminus; S, signal peptide; Ig, immunoglobulin-like domains (1–3); FN3, fibronectin type-III domains (1–5); TM, transmembrane domain; phase, phosphatase domains (D1-D2) and -C, carboxy terminus. (C) SDS-PAGE with CBB (a and b) and western blotting with anti-RPTPσ antibody (c and d) for crude HaloTag-binding protein (70 kDa, a and c) and TEV-treated purified RPTPσ N-terminal fragment (36 kDa, b and d). HS and heparin (HP) are composed of GlcA/IdoA and N-acetylated or N-sulfonated glucosamine (GlcNAc/GlcNS) disaccharide units with O-sulfation at various positions of the sugar residues. The major disaccharide structures of HS/HP are as follows: a non-sulphated unit (0S, GlcA/IdoA-GlcNAc), a mono N-sulfonated unit (NS, GlcA/IdoA-GlcNS), a monosulphated unit at the C-2 position of the GlcA/IdoA residue (US, GlcA2S/IdoA2S-GlcNAc), a monosulphated unit at the C-6 position of the GlcNAc residue (6S, GlcA/IdoA-GlcNAc6S), a disulphated unit at the C-6 position of the GlcNS residue (N6diS, GlcA/IdoA-GlcNS6S), a disulphated unit at the C-2 position of the GlcA/IdoA and GlcNS residues (NUdiS, GlcA2S/IdoA2S-GlcNS), a disulphated unit at the C-2 position of GlcA/IdoA and the C-6 position of GlcNAc (U6diS, GlcA2S/IdoA2S-GlcNAc6S), and a trisulphated disaccharide unit (triS, GlcA2S/IdoA2S-GlcNS6S) (Fig. 1A). CS and HS functions are mediated through their receptors, which are expressed in neurons, the major receptor being RPTPσ (9). RPTPσ comprises an intracellular region containing two tandem catalytic domains and an extracellular region containing three immunoglobulin (Ig)-like domains followed by five fibronectin type III (FN3) domains (Fig. 1B). The GAG-binding site is located in a N-terminal Ig1 domain, which contains a cluster of basic amino acid residues (3). The region containing three Ig-like domains interacts with HP to form oligomers, and with CS to form a monomer (4). This distinctive receptor clustering caused by GAGs is thought to regulate axon growth and neuron network regeneration. However, it is still unclear how diverse GAG species specifically affect the clustering and downstream signal transduction. Previously, we constructed CS/DS and HS/HP library with defined structure using chemical modification and polysaccharide synthesis enzymes (10, 11). Here, we confirmed the effect of CS and HP on neurite outgrowth using GAG phospholipid conjugates. We also investigated the interaction of the GAG-binding fragment of RPTPσ with different GAG species in our GAG library. Analysis using surface plasmon resonance (SPR) revealed that their interaction was highly correlated with the degree of diSE unit in CS and the sulfation degree (SD) of HS/HP. Highly sulphated HP possessed four-fold higher affinity to RPTPσ fragment than the diSE-rich CS (CSE). Materials and Methods Materials Chondroitin (CH) prepared by desulfation of CS, native CSE from squid cartilage, DS from pig skin, partially depolymerized HA from chick comb, HS from porcine aorta, HP from pig intestine, N-acetylheparosan (HPS) from Escherichia coli strain K5 (12), HP desulfated at the C-2 positions of the GlcA/IdoA residues (2DS-HP), HP desulfated at the C-6 positions of the GlcNAc/GlcNS residues (6DS-HP), HP completely desulfated at most positions of sugar residues and N-resulfonated at C-2 positions of GlcN residues (CDSNS-HP), HP N-desulfated and N-acetylated at the C-2 positions of GlcNS residues (NDSNAc-HP), chondroitinase ABC, and heparitinase I, II and III were obtained from Seikagaku (Tokyo, Japan). Chondroitin hexasaccharide (CH6) prepared by partial digestion of CH (13), recombinant chondroitin polymerase from E. coli strain K4 (K4CP) (14), K4CP mutants (15), recombinant CS sulfotransferases: chondroitin 4-sulfotransferase-1 (C4ST-1) (16), chondroitin 6-sulfotransferase-1 (C6ST-1) (17), GalNAc4S 6-sulfotransferase (GalNac4S-6ST) (18), and uronosyl 2-sulfotransferase (UA2ST) (19), HS 2-sulfotramsferase (HS2ST) (20), HS 6-sulfotramsferase-1 (HS6ST-1) (21), and phosphatidylethanolamine (PE)-conjugated GAG derivatives (22, 23) were prepared as described previously: CH-PE (average molecular mass (Mr) 10k, major disaccharide composition 0S 100%), CSA-PE (Mr 15k, 4S 74%, 6S 22%), CSC-PE (Mr 20k, 4S 22%, 6S 63%, diSd 10%), CSE-PE (Mr 50k, 4S 20%, 6S 8%, diSe 66%), HS-PE (Mr 20k, 0S 46%, 6S 15%, NS 21%, N6diS 8%, NUdiS 6%, triS 3%) and HP-PE (Mr 10k, N6diS 15%, NUdiS 7%, triS 78%). Adenosine 3′-phosphate 5′-phosphosulfate (PAPS), uridine 5′-diphospho-α-d-N-acetylgalactosamine (UDP-GalNAc) and uridine 5′-diphospho-α-d-glucuronate (UDP-GlcA) were from Yamasa (Choshi, Japan). Streptavidin-conjugated sensor chips (SA chips) for the Biacore biosensor were purchased from GE Healthcare (Pittsburgh, PA). Preparation of GAG species and their biotin conjugates The chemo-enzymatically synthesized CS library was constructed as described previously (10). Briefly, CH poly- and oligosaccharides were synthesized from CH6 with K4CP or K4CP mutants. CH octasaccharide (CH8) and dodecasaccharide (CH12) were synthesized with two K4CP mutants and two donor substrates (UDP-GalNAc or UDP-GlcA) by an alternative elongation reaction (15). The average molecular masses (Mr) of prepared CH polymers were 3, 7, 10, 20, 100, and 150 kDa. CS species with different sulfation patterns were synthesized using the CH polymers and oligomers as acceptor substrates, PAPS as a donor substrate and various recombinant chondroitin sulfotransferases (C4ST-1, C6ST-1, GalNac4S-6ST and UA2ST) as catalytic enzymes. For example, the CH species with diverse chain lengths were sulphated at C-4 positions of the GalNAc residues with C4ST-1 to obtain CSA species. CSC was synthesized with C6ST-1. CSAC was synthesized by simultaneous reaction of C4ST-1 and C6ST-1. CSAD was synthesized with UA2ST from CSAC. CSE species with diverse chain lengths were synthesized with GalNAc4S6ST from CSA species having various chain lengths. CSDE was synthesized with GalNAc4S6ST from CSAD. CST was synthesized from CSE using UA2ST. Desulfated and sulphated HP derivatives were prepared as described previously (24–26). Briefly, NDSNAc-HP was prepared by N-desulfation with dimethyl sulfoxide-methanol at 20°C followed by N-acetylation with acetic acid anhydride. 2DS-HP was obtained by freeze drying under slightly alkaline conditions. 6DS-HP was obtained by N-methyl-N-(trimethylsilyl) trifluoroacetamide treatment of HP. CDSNS-HP was prepared by solvolytic desulfation and trimethyl sulphur trioxide complex treatment of HP. 2-Sulfated HPS (2S-HPS) and 6-sulfated HPS (6S-HPS) were prepared from HPS with HS2ST and HS6ST-1 recombinant enzymes, respectively. The GAG oligo- and polysaccharides were conjugated with hexamethylenediamine (HMDA) at the reducing ends by the reductive amination method before or after chain elongation and sulfation reaction (10). Then, they were modified with sulfo-NHS-activated biotin reagent (sulfo-NHS-LC-biotin, Pierce, Rockford, IL) at the amine group of the HMDA residue. Mr, disaccharide composition and SD (number of sulphate groups per disaccharide unit) of the GAG-biotin conjugates are summarized in Table I. Table I. Biotin-conjugated GAG species and their disaccharide compositions GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – Note: –, not detected. Table I. Biotin-conjugated GAG species and their disaccharide compositions GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – Note: –, not detected. Neurite outgrowth assay Animal experiments were approved by Animal Care and Use Committee for Aichi Medical University and followed the ethical standard formulated in the Helsinki Declaration. Rat cerebellar granule neurons (CGNs) were collected as reported previously (27) from cerebella of Sprague-Dawley rats, purchased from Japan SLC (Hamamatsu, Japan), at postnatal days 7–9. CGNs (1.5 × 105 cells) were seeded onto cover glass (18 × 18 mm, Matsunami, Tokyo, Japan) coated with 25 μg/ml poly-l-lysine (Sigma, St. Louis, MO) and 5 μg/ml laminin (Becton Dickinson, Bedford, MA) in 35 mm Petri dishes and cultured at 37°C in Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco), 2 mM glutamine, 20 mM KCl, 50 U/ml penicillin and 50 μg/ml streptomycin. Five hours later after cell seeding, CH-PE, CSA-PE, CSC-PE, CSE-PE, HS-PE or HP-PE was added to the culture at a final concentration of 10 μg/ml, followed by further incubation at 37°C for 19 h. The neurons were fixed with 4% paraformaldehyde in PBS and washed with PBS, followed by blocking with 1% bovine serum albumin in PBS for 30 min. Subsequently, the fixed cells were treated with anti-neuron-specific β-tubulin antibody (Covance, Princeton, NJ) at room temperature for 1 h. After rinsing with PBS, neurons were incubated with a fluorescence-labelled secondary antibody (Alexa Fluor 488-conjugated anti-mouse IgG goat serum, Invitrogen) at room temperature for 45 min to visualize neurite outgrowth. The stained cells on the cover glass were mounted with Fluoromount Aqueous medium (Sigma) and inspected using an Axio Vision fluorescent microscope (Carl Zeiss). The relative amount of neurite outgrowth was quantified based on the area of β-tubulin-positive cells per total cells in a field of vision. Expression and purification of RPTPσ N-terminal fragment A plasmid pFN21A-B4243 containing the full sequence of human RPTPσ isoform 4 (accession no. AB209333 and NP_570925) was obtained from Kazusa DNA Research Institute (Kisarazu, Japan). For expression of the N-terminal Ig 1-3 fragment (amino acid 30-321) of RPTPσ, the DNA encoding the N-terminal fragment was amplified by PCR using the pFN21A-B4243 plasmid as a template; forward primer, 5'-GCTAGCGATCGCCATGGAAGAGCCCCCCAGGTTTATCAAAGA-3' and reverse primer, 5'- TCGAGAGCTCGAGATTTCACCGTGATCTGAGCAACCGCCTC-3'. The Sgf1 and Eco1CR1 cleavage sites are underlined. The PCR product was subcloned into a pFC14K HaloTag CMV Flexi expression vector. The inserted DNA sequence was confirmed with a DNA sequencer (3130 Genetic Analyzer, Applied Biosystems, Brancurg, NJ). N-terminal RPTPσ fragment-inserted expression plasmid (pFC14K-RPTPσ(30-321)-HaloTag) was transfected into HEK293T cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The transfected HEK293T cells were incubated in the culture medium at 37°C for 2–4 days. The cells were homogenized with a glass homogenizer and the extract was collected by centrifugation. The recombinant protein containing HaloTag at the C-terminus was adsorbed with HaloLink Resin (Promega). The RPTPσ N-terminal fragment was purified by treatment with TEV protease (Promega) to release the fragment from the HaloTag fusion protein that covalently bound to the HaloLink resin. (Fig. 1C). SPR analysis Interactions of the recombinant RPTPσ N-terminal fragment with the various GAG species were analysed using an SPR biosensor (BIAcore T200; GE Healthcare) (28). Briefly, the GAG-biotin conjugates (10 µg/ml) were immobilized in flow cells 2–4 of the SA chip. Biotin (100 pmol/ml) was used as a control in flow cell 1 of the chip. GAG-biotin and biotin solution (70 μl) were injected into SA chips at a flow rate of 5 μl/min in 50 mM phosphate buffer (pH 7.2) containing 0.05% Tween 20. Responses of immobilized samples were recorded after washing (60 s, three times) with a solution of 1 M NaCl containing 50 mM NaOH. Binding assays were performed at 25°C at a constant flow rate of 30 µl/min during both the association and dissociation phases using 10 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 3 mM EDTA and 0.005% Tween 20 (HBS-EP). RPTPσ N-terminal fragment analyte solutions (25, 50, 100, 200, 400, and 800 nM) in HBS-EP were injected into the GAG ligand-immobilized flow cells for 60 s (association), followed by injection of HBS-EP buffer for 180 s (dissociation). Regeneration of the sensor chip surface was accomplished by an injection of 20 µl of HBS-EP containing 2 M NaCl. The sensorgrams were recorded, and the kinetic parameters calculated from association and dissociation curves were determined using BIA Evaluation 4.1 software (GE Healthcare). Compositional analysis of GAG derivatives The GAG samples (10 pmol–1 nmol) were digested with chondroitinase ABC (10 mU) for HA and CS/DS or with heparitinase I, -II and -III mixed enzymes (10 mU each) for HS/HP at 37°C for 1 h. The digested products containing unsaturated disaccharides were analysed using a fluorometric post-column HPLC system as reported previously (29). SDS-PAGE and western blotting The protein samples were applied to 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with the primary antibody followed by treatment with a peroxidase-conjugated secondary antibody. The reacted proteins were detected with chemiluminescence reagent and LAS-400 detector (GE Healthcare). Statistical analysis Statistical analyses were performed with an unpaired Student’s t-test. The level of significance is indicated by asterisks: *, P < 0.05 and **, P < 0.01. The error bars show standard deviations. Results and Discussion Effect of GAG-PE on neurite outgrowth of CGNs CS and HS were reported to regulate axonal growth and neuronal regeneration through RPTPσ, although with opposite effects (3, 4). To confirm this, we investigated the effect of CS and HS/HP on neurite outgrowth of rat CGNs using CSE-PE and HP-PE. Rat CGNs express RPTPσ, similar to most of mammalian neurons (9). Treatment with CSE-PE inhibited neurite outgrowth, whereas HP-PE enhanced it, when they were added 5 h after cell seeding (Fig. 2). When CS-PE was added before cell seeding, it substantially inhibited cell adhesion. Interestingly, free polysaccharides (CSE and HP) showed no significant effect on neurite outgrowth up to 50 μg/ml (data not shown). Other PE-modified GAG derivatives had no statistically significant effects, although CSA-PE and CSC-PE showed a tendency to inhibit neurite outgrowth and HS-PE showed a tendency to enhance it (Fig. 2D). Fig. 2 View largeDownload slide Neurite outgrowth stained with anti-neuron-specific β-tubulin. (A) Control rat CGNs. (B) CSE-PE-treated CGNs. (C) HP-PE-treated CGNs. Scale bars, 100 μm. (D) Relative neurite outgrowth (%) of rat neurons treated with buffer (control), CH-PE, CSA-PE, CSC-PE, CSE-PE, HS-PE and HP-PE; the values obtained with control cells are taken as 100%. Bars represent the standard deviations determined in five independent experiments. *, P < 0.05; **, P < 0.005. (E) Molecular structure of CS-PE (dipalmitoylphosphatidylethanolamine-conjugated chondroitin sulphate). Fig. 2 View largeDownload slide Neurite outgrowth stained with anti-neuron-specific β-tubulin. (A) Control rat CGNs. (B) CSE-PE-treated CGNs. (C) HP-PE-treated CGNs. Scale bars, 100 μm. (D) Relative neurite outgrowth (%) of rat neurons treated with buffer (control), CH-PE, CSA-PE, CSC-PE, CSE-PE, HS-PE and HP-PE; the values obtained with control cells are taken as 100%. Bars represent the standard deviations determined in five independent experiments. *, P < 0.05; **, P < 0.005. (E) Molecular structure of CS-PE (dipalmitoylphosphatidylethanolamine-conjugated chondroitin sulphate). GAG must be immobilized to a solid material, such as culture dish, when its cellular activities are to be analysed. Because free GAG is usually unable to bind to solid materials, it does not exhibit cellular activity. Conjugated proteins help GAGs to exert their functions. In fact, some studies evaluated GAG function by using proteoglycans (native GAGs and core-protein complexes) (1, 4) or artificial GAG-protein complexes (27) rather than free GAGs. Previously, we generated PE-conjugated GAG (22) that could bind to culture dishes via hydrophobic interactions. We then investigated its effects, and found that CS-PE promoted axonal elongation at low concentrations (0.1–1.0 μg/ml) and inhibited neurite extension and adhesion at concentrations higher than 1.0 μg/ml (30). We also found that CSE-PE-coated polystyrene beads repulsed the growth cones of chick retinal neurons (31, 32). Previous studies (33, 34) indicated that CSE had high affinity to RPTPσ and inhibition of neurite outgrowth using CS polymers and anti-CS antibodies. In this study, using CSE-PE and HP-PE, we found that CSE and HP have opposite effects on neurite outgrowth of rat CGNs. Immobilization of biotin-conjugated GAG library to SA-coated sensor chips The synthesized biotin-conjugated various GAG species with various chain sizes and disaccharide compositions (Table I) were immobilized onto SA-coated sensor chips for SPR analysis. The GAG species were divided into three groups: HA and various CS/DS species, various CSE species and HS/HP derivatives. The first group was composed of HA generated by partial digestion with testicular hyaluronidase, CH synthesized with K4CP, DS isolated from pig skin and seven synthetic CS species (CSA, CSC, CSAC, CSAD, CSE, CSDE and CST) that were generated from CH (10 kDa) by various sulfations with corresponding sulfotransferases. The second group consisted of CSE species having various chain sizes (8-mer, 12-mer, 7 kDa, 10 kDa, 20 kDa, 50 kDa, 100 kDa and 150 kDa) and various degrees of diSE (9–88%), which were synthesized with K4CP, C4ST-1 and GalNAc4S-6ST, except CSE (50 kDa) isolated from squid cartilage. The third group consisted of HP, NDSNAc-HP, 2DS-HP, 6DS-HP, CDSNS-HP, HS from porcine aorta, HPS from E. coli strain K5, 2S-HPS and 6S-HPS. The GAG species were conjugated with HMDA and then biotin groups at the reducing end of the sugar chains. The solution of GAG-biotin conjugates (10 μg/ml) was immobilized to SA sensor chips for 840 s. Responses of immobilized GAG-biotins were detected in the range of 300–600 RU and those of biotin as a control were not detected significantly. All biotinylated samples were successfully immobilized. SPR analysis of the interaction between RPTPσ N-terminal fragment and immobilized GAGs We analysed the interaction of the purified RPTPσ N-terminal fragment with GAG-immobilized SA chips using a Biacore T200 SPR analyser. Various concentrations (25–800 nM) of the RPTPσ N-terminal fragment were injected to the GAG-immobilized sensor chips. The association and dissociation curves of the sensorgrams were analysed and kinetic parameters were determined using BIA Evaluation 4.1 software (GE Healthcare). The response curves of the sensorgrams fitted better to a two-state reaction model than to a 1:1 binding model (Fig. 3). Therefore, we adopted the kinetic parameters calculated from the two-state reaction model (Table II). Table II. Kinetic parameters of RPTPσ affinity to GAG-immobilized chips GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — a“–” means affinity was too low; therefore, the kinetic parameters were not indicated. bMaxRU indicates maximum response (RU) with injection of 800 nM RPTPσ fragment injection, with ± standard deviation (SD, n = 3) shown. c“–” indicates that MaxRU was nearly zero or had a negative value. Table II. Kinetic parameters of RPTPσ affinity to GAG-immobilized chips GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — a“–” means affinity was too low; therefore, the kinetic parameters were not indicated. bMaxRU indicates maximum response (RU) with injection of 800 nM RPTPσ fragment injection, with ± standard deviation (SD, n = 3) shown. c“–” indicates that MaxRU was nearly zero or had a negative value. Fig. 3 View largeDownload slide Comparison of SPR evaluation models. Sensorgrams of the several concentrations (25, 50, 100, 200, 400 and 800 nM) of the RPTPσ fragment with CSE- (A and B) and HP- (C and D) immobilized chips are shown, with fitting by 1:1 binding model (A and C) and two-state reaction model (B and D) on SPR. The coloured solid lines represent curves for actual measurements and black broken lines represent calculated curves. Fig. 3 View largeDownload slide Comparison of SPR evaluation models. Sensorgrams of the several concentrations (25, 50, 100, 200, 400 and 800 nM) of the RPTPσ fragment with CSE- (A and B) and HP- (C and D) immobilized chips are shown, with fitting by 1:1 binding model (A and C) and two-state reaction model (B and D) on SPR. The coloured solid lines represent curves for actual measurements and black broken lines represent calculated curves. Two-state reaction model describes a 1:1 binding of analyte (A, RPTPσ) to immobilized ligand (B, GAG) forming AB, followed by a conformational change that stabilizes the complex (AB*) as shown by the formula below (Software Handbook of Biacore T200, GE Healthcare). A+Bkd1ka1ABkd2ka2AB* In this equation, ka1 [M−1 s−1] is association rate constant for analyte binding, kd1 [s−1] is the dissociation constant for analyte from the complex, ka2 [s−1] is the forward rate constant for the conformational change and kd2 [s−1] is the reverse rate constant for the conformational change. The overall equilibrium dissociation constant Kd [M] is given by the following equation: Kd=kd1/ka1×kd2/(kd2+ka2) The first half of this equation indicates the 1:1 binding and the second half indicates the conformational change. For various GAG chips, kinetic parameters of RPTPσ interactions were calculated with the two-state reaction model (Table II). Most values of the second half of the equation were in the order of 10−1–10−2, indicating that the second half did not contribute to the interaction significantly. In contrast, the values of the first half greatly changed in the order of 10−4–10−7, suggesting that the first half contributed to fluctuations of the interaction between RPTPσ and GAG species. However, it should be noted that the actual conformational change could not be studied in this study and should be investigated using other techniques such as NMR, IR and CD spectroscopies. Sensorgrams for the highest concentration (800 nM) of the RPTPσ fragment on the various GAG-immobilized chips are shown in Fig. 4. Maximum response (MaxRU) defined as the highest response value at the highest concentration (800 nM) of injected RPTPσ fragment in GAG-immobilized chip was determined from the response curve (Table II). The values of MaxRU and Kd are plotted against several properties of GAG samples in Fig. 5. Fig. 4 View largeDownload slide Sensorgrams of RPTPσ with various GAG chips on SPR. The sensorgrams for a high concentration (800 nM) of the RPTPσ N-terminal fragment with the sensor chips of group 1 CS species (A), CSE long chains of group 2 (B), CSE short chains of group 2 (C) and group 3 HP derivatives (D) are shown. (A) Red, black, blue, green orange and grey lines show CSE, CST, CSDE, CSAD, CSA and DS, respectively. (B) Red, brown, orange, grey, blue, black, green and yellow lines show CSE10k a, CSE10k b, CSE10k c, CSE10k d, CSE20k, CSE50k, CSE100k and CSE150k, respectively. (C) Red, blue, orange, green and grey lines show CSE7k a, CSE7k b, CSE7k c, CSE12 and CSE8, respectively. (D) Red, blue, orange, black, grey and green lines show HP, NDSNAc-HP, 2DS-HP, 6DS-HP, CSDNS-HP and HS, respectively. Fig. 4 View largeDownload slide Sensorgrams of RPTPσ with various GAG chips on SPR. The sensorgrams for a high concentration (800 nM) of the RPTPσ N-terminal fragment with the sensor chips of group 1 CS species (A), CSE long chains of group 2 (B), CSE short chains of group 2 (C) and group 3 HP derivatives (D) are shown. (A) Red, black, blue, green orange and grey lines show CSE, CST, CSDE, CSAD, CSA and DS, respectively. (B) Red, brown, orange, grey, blue, black, green and yellow lines show CSE10k a, CSE10k b, CSE10k c, CSE10k d, CSE20k, CSE50k, CSE100k and CSE150k, respectively. (C) Red, blue, orange, green and grey lines show CSE7k a, CSE7k b, CSE7k c, CSE12 and CSE8, respectively. (D) Red, blue, orange, black, grey and green lines show HP, NDSNAc-HP, 2DS-HP, 6DS-HP, CSDNS-HP and HS, respectively. Fig. 5 View largeDownload slide Kinetic analyses of RPTPσ N-terminal fragment with various GAG chips on SPR. (A) Plot of MaxRU versus SD values of GAG group 1. Grey circle, HA and CH; grey square, CSC, grey triangle, CSAC; white triangle, DS; black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (B) Plot of Kd versus SD values of GAG group 1. Black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (C) Plot of MaxRU versus diSE% of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; white circle, CSE polymers (CSE20k, 50k, 100k and 150k); and white triangle, CSE oligomers (CSE8, CSE12). (D) Plot of Kd versus diSE% values of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; and white circle, CSE polymers (CSE20k, 50k, 100k and 150k). (E). Plot of MaxRU versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (F) Plot of Kd versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (G) Plot of MaxRU versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP; white square, CDSNS-HP; grey triangle, HS, grey circle, 2S-HPS; grey triangle, 6S-HPS and grey square, HPS. (H) Plot of Kd versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP and white square, CDSNS-HP. Fig. 5 View largeDownload slide Kinetic analyses of RPTPσ N-terminal fragment with various GAG chips on SPR. (A) Plot of MaxRU versus SD values of GAG group 1. Grey circle, HA and CH; grey square, CSC, grey triangle, CSAC; white triangle, DS; black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (B) Plot of Kd versus SD values of GAG group 1. Black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (C) Plot of MaxRU versus diSE% of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; white circle, CSE polymers (CSE20k, 50k, 100k and 150k); and white triangle, CSE oligomers (CSE8, CSE12). (D) Plot of Kd versus diSE% values of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; and white circle, CSE polymers (CSE20k, 50k, 100k and 150k). (E). Plot of MaxRU versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (F) Plot of Kd versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (G) Plot of MaxRU versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP; white square, CDSNS-HP; grey triangle, HS, grey circle, 2S-HPS; grey triangle, 6S-HPS and grey square, HPS. (H) Plot of Kd versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP and white square, CDSNS-HP. Sensorgrams of group 1 composed of HA and CS/DS (Fig. 4A and Supplementary Fig. S1) showed diverse curves for GAG species. The values of MaxRU of group 1 (HA and CS/DS) generally correlated with the SD (Fig. 5A). Kd values of group 1 GAG species were negatively correlated with SD (Fig. 5B). Highly sulphated CS species, CST (SD = 2.07) and CSE (SD = 1.85), which had a content of 87–89% multi-sulphated disaccharide units, showed similarly high affinity (MaxRU ≈ 900 and Kd ≈ 5 × 10−8 M). GlcA 2-O-sulfation in CST appeared to contribute less to the affinity of RPTPσ. Highly sulphated CS, CSDE (SD = 1.47) and CSAD (SD = 1.41), showed relatively high responses. CSDE containing diSE unit had a higher response than CSAD, although they possessed similar SD. The group of CS having one sulphate per disaccharide unit, CSA (SD = 0.98), CSC (SD = 0.98), CSAC (SD = 0.99) and DS (SD = 1.05), and non-sulphated saccharides, HA and CH, hardly showed SPR responses. Their kinetic parameters could not be calculated because the quality of the response curves was too low for kinetic evaluation. These results suggest that diSE unit might be critical for interaction between RPTPσ and CS. We then analysed the interaction of the RPTPσ fragment with group 2 sensor chips, which had various CSE species immobilized on them (Fig. 4B, C, and Supplementary Fig. S2). In general, the MaxRU values of the CSE species correlated with the ratio of diSE (diSE%, Fig. 5C) and their Kd values negatively correlated with diSE% (Fig. 5D). CSE octasaccharide (CSE8) showed a low MaxRU value, although it contained considerable diSE units (48%). Similarly sulphated dodecasaccharide (CSE12, diSE: 47%) showed more potent binding capacity (MaxRU = 290) than CSE8. Three species of CSE7k with Mr 7 kDa, 28-mer average sugar chains and different diSE contents (43%, 30%, and 12%, respectively); four species of CSE10k with Mr 10 kDa, 46-mer and different diSE contents (87%, 73%, 61% and 9%, respectively); and four species of CSE polysaccharide with Mr 20, 50, 100 and 150 kDa and exhibited interactions as strong as their diSE contents. Plots of the MaxRU and Kd values against Mr of the group 2 CSE species showed complicated patterns. When the CSE species were divided into four groups, i.e. low (12–15%), relatively low (20–30%), medium (40–50%) and high (> 60%) diSE ratios, the correlation was obvious between affinity and molecular size (Fig. 5E and F). The CSE species having high (> 60%) and medium (40–50%) diSE ratios exhibited the highest affinity when Mr was 10 kDa, but the affinity was lower for either shorter or longer sugar chains. CSE species with low (12–15%) and relatively low (20–30%) diSE ratios showed low affinity and indistinct correlation between kinetic parameters and Mr of CSE species. Next, we analysed the interaction of RPTPσ with HS/HP derivatives (group 3). Sensorgrams of the group 3 ligands are shown in Fig. 4D and Supplementary Fig. S3. MaxRU values of this group correlated with SD (Fig. 5G). Their Kd values were negatively correlated with SD (Fig. 5H). HP (SD = 2.6), exhibiting the highest sulfation levels in this study, possessed the highest affinity for RPTPσ fragment. It showed a 4-fold higher MaxRU value and over 2-fold lower Kd value than CSE10k, which possessed the highest activity among CS species (Table II). Similarly, a highly sulphated subgroup of HP derivatives NDSNAc-HP (SD = 1.90), 2DS-HP (SD = 1.60) and 6DS-HP (SD = 1.86) exhibited high affinity to RPTPσ. Most of the disaccharide content of HP is triS, whereas that of NDSAc-HP, 2DS-HP and 6DS-HP is U6diS, N6diS and NUdiS, respectively. This indicates that affinity of HP with RPTPσ might not be reduced when one sulphate group at any position of N-sulfation of GlcN, 2-sulfation of GlcA/IdoA and 6-sulfation of GlcN is deleted. In contrast, low-sulphated HS/HP derivatives (SD < 1.0), CDSNS-HP, HS, 2S-HPS, 6S-HPS and HPS, had low affinity to RPTPσ. Conclusion In this study, we investigated the relationship between GAG structure and binding activity of neuronal receptor RPTPσ using our artificial GAG library. CSE chains having high diSE units and Mr 10 kDa and HP derivatives having high content of diS units were critical for RPTPσ affinity. HP derivatives had 4-fold higher affinity than CSE species, but it is not clear why CS and HP exhibit opposite activity. Large quantity of CSPG is produced in injured central nervous systems, but HSPG is not. Because of the difference of CS and HS production, CS is sought to be dominant effector on RPTPσ. These GAG activities may be associated with cluster formation and phosphatase activity of the receptor, different intracellular signalling after GAG-receptor interaction, and/or another phenomenon, which should be identified in future. Using PE-conjugated GAGs, We confirmed that CSE inhibits and HP enhances neurite outgrowth of rat CGNs. We demonstrated that a biotin-conjugated GAG library composed of chemoenzymatically synthesized CS species and chemically modified HP derivatives contained useful ligands for characterization of GAG-binding materials, such as RPTPσ. The SPR kinetic interaction study of RPTPσ using our GAG library revealed high-affinity structures of CS and HP. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology but also provide medical applications to regulate neural regeneration. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank Prof. Kenji Kadomatsu and Dr Kazuma Sakamoto (Nagoya University) for teaching us the isolation method for rat CGNs and Yoshimi Shintoku for expert technical assistance. Funding This work was supported in part by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Number 17K07352 to NS. Conflict of Interest None declared. References 1 Shen Y. , Tenney A.P. , Busch S.A. , Horn K.P. , Cuascut F.X. , Liu K. , He Z. , Silver J. , Flanagan J.G. ( 2009 ) PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration . Science 326 , 592 – 596 Google Scholar CrossRef Search ADS PubMed 2 Gardner R.T. , Habecker B.A. ( 2013 ) Infarct-derived chondroitin sulfate proteoglycans prevent sympathetic reinnervation after cardiac ischemia-reperfusion injury . J. Neurosci . 33 , 7175 – 7183 Google Scholar CrossRef Search ADS PubMed 3 Coles C.H. , Mitakidis N. , Zhang P. , Elegheert J. , Lu W. , Stoker A.W. , Nakagawa T. , Craig A.M. , Jones E.Y. , Aricescu A.R. ( 2014 ) Structural basis for extracellular cis and trans RPTPsigma signal competition in synaptogenesis . Nat. Commun . 5 , 5209 Google Scholar CrossRef Search ADS PubMed 4 Coles C.H. , Shen Y. , Tenney A.P. , Siebold C. , Sutton G.C. , Lu W. , Gallagher J.T. , Jones E.Y. , Flanagan J.G. , Aricescu A.R. ( 2011 ) Proteoglycan-specific molecular switch for RPTPsigma clustering and neuronal extension . Science 332 , 484 – 488 Google Scholar CrossRef Search ADS PubMed 5 Bradbury E.J. , Moon L.D. , Popat R.J. , King V.R. , Bennett G.S. , Patel P.N. , Fawcett J.W. , McMahon S.B. ( 2002 ) Chondroitinase ABC promotes functional recovery after spinal cord injury . Nature 416 , 636 – 640 Google Scholar CrossRef Search ADS PubMed 6 Bartus K. , James N.D. , Didangelos A. , Bosch K.D. , Verhaagen J. , Yanez-Munoz R.J. , Rogers J.H. , Schneider B.L. , Muir E.M. , Bradbury E.J. ( 2014 ) Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury . J. Neurosci . 34 , 4822 – 4836 Google Scholar CrossRef Search ADS PubMed 7 Takeuchi K. , Yoshioka N. , Higa Onaga S. , Watanabe Y. , Miyata S. , Wada Y. , Kudo C. , Okada M. , Ohko K. , Oda K. , Sato T. , Yokoyama M. , Matsushita N. , Nakamura M. , Okano H. , Sakimura K. , Kawano H. , Kitagawa H. , Igarashi M. ( 2013 ) Chondroitin sulphate N-acetylgalactosaminyl-transferase-1 inhibits recovery from neural injury . Nat. Commun . 4 , 2740 Google Scholar CrossRef Search ADS PubMed 8 Esko J.D. , Kimata K. , Lindahl U. ( 2009 ) Proteoglycans and Sulfated Glycosaminoglycans in Essentials of Glycobiology , 2nd ed ( Varki A. , Cummings R.D. , Esko J.D. , Freeze H.H. , Stanley P. , Bertozzi C.R. , Hart G.W. , Etzler M.E. , eds.) pp. 229 – 248 . Cold Spring Harbor , New York 9 Wang H. , Yan H. , Canoll P.D. , Silvennoinen O. , Schlessinger J. , Musacchio J.M. ( 1995 ) Expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma) in the nervous system of the developing and adult rat . J. Neurosci. Res. 41 , 297 – 310 Google Scholar CrossRef Search ADS PubMed 10 Sugiura N. , Shioiri T. , Chiba M. , Sato T. , Narimatsu H. , Kimata K. , Watanabe H. ( 2012 ) Construction of a chondroitin sulfate library with defined structures and analysis of molecular interactions . J. Biol. Chem. 287 , 43390 – 43400 Google Scholar CrossRef Search ADS PubMed 11 Ashikari-Hada S. , Habuchi H. , Kariya Y. , Itoh N. , Reddi A.H. , Kimata K. ( 2004 ) Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library . J. Biol. Chem . 279 , 12346 – 12354 Google Scholar CrossRef Search ADS PubMed 12 Vann W.F. , Schmidt M.A. , Jann B. , Jann K. ( 1981 ) The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli O10: K5: H4 . Eur. J. Biochem. 116 , 359 – 364 Google Scholar CrossRef Search ADS PubMed 13 Sugiura N. , Shimokata S. , Watanabe H. , Kimata K. ( 2007 ) MS analysis of chondroitin polymerization: effects of Mn2+ ions on the stability of UDP-sugars and chondroitin synthesis . Anal. Biochem . 365 , 62 – 73 Google Scholar CrossRef Search ADS PubMed 14 Ninomiya T. , Sugiura N. , Tawada A. , Sugimoto K. , Watanabe H. , Kimata K. ( 2002 ) Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4 . J. Biol. Chem. 277 , 21567 – 21575 Google Scholar CrossRef Search ADS PubMed 15 Sugiura N. , Shimokata S. , Minamisawa T. , Hirabayashi J. , Kimata K. , Watanabe H. ( 2008 ) Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants . Glycoconj. J. . 25 , 521 – 530 Google Scholar CrossRef Search ADS PubMed 16 Okuda T. , Mita S. , Yamauchi S. , Matsubara T. , Yagi F. , Yamamori D. , Fukuta M. , Kuroiwa A. , Matsuda Y. , Habuchi O. ( 2000 ) Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase . J. Biochem. (Tokyo) 128 , 763 – 770 Google Scholar CrossRef Search ADS 17 Fukuta M. , Kobayashi Y. , Uchimura K. , Kimata K. , Habuchi O. ( 1998 ) Molecular cloning and expression of human chondroitin 6-sulfotransferase . Biochim. Biophys. Acta 1399 , 57 – 61 Google Scholar CrossRef Search ADS PubMed 18 Ohtake S. , Ito Y. , Fukuta M. , Habuchi O. ( 2001 ) Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene . J. Biol. Chem. 276 , 43894 – 43900 Google Scholar CrossRef Search ADS PubMed 19 Kobayashi M. , Sugumaran G. , Liu J. , Shworak N.W. , Silbert J.E. , Rosenberg R.D. ( 1999 ) Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan/chondroitin sulfate . J. Biol. Chem. 274 , 10474 – 10480 Google Scholar CrossRef Search ADS PubMed 20 Kobayashi M. , Habuchi H. , Yoneda M. , Habuchi O. , Kimata K. ( 1997 ) Molecular cloning and expression of Chinese hamster overy cell heparan sulfate 2-sulfotransferase . J. Biol. Chem. 272 , 13980 – 13985 Google Scholar CrossRef Search ADS PubMed 21 Habuchi H. , Tanaka M. , Habuchi O. , Yoshida K. , Suzuki H. , Ban K. , Kimata K. ( 2000 ) The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine . J. Biol. Chem. 275 , 2859 – 2868 Google Scholar CrossRef Search ADS PubMed 22 Sugiura N. , Sakurai K. , Hori Y. , Karasawa K. , Suzuki S. , Kimata K. ( 1993 ) Preparation of lipid-derivatized glycosaminoglycans to probe a regulatory function of the carbohydrate moieties of proteoglycans in cell-matrix interaction . J. Biol. Chem . 268 , 15779 – 15787 Google Scholar PubMed 23 Sugiura N. , Kimata K. ( 1994 ) Syntheses and functions of neoproteoglycans: lipid-derivatized chondroitin sulfate with antiadhesion activity . Methods Enzymol . 247 , 362 – 373 Google Scholar CrossRef Search ADS PubMed 24 Nagasawa K. , Inoue Y. , Kamata T. ( 1977 ) Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol . Carbohydr. Res. 58 , 47 – 55 Google Scholar CrossRef Search ADS PubMed 25 Kariya Y. , Kyogashima M. , Suzuki K. , Isomura T. , Sakamoto T. , Horie K. , Ishihara M. , Takano R. , Kamei K. , Hara S. ( 2000 ) Preparation of completely 6-O-desulfated heparin and its ability to enhance activity of basic fibroblast growth factor . J. Biol. Chem. 275 , 25949 – 25958 Google Scholar CrossRef Search ADS PubMed 26 Uchiyama H. , Nagasawa K. ( 1991 ) Changes in the structure and biological property of N—O sulfate- transferred, N-resulfated heparin . J. Biol. Chem. 266 , 6756 – 6760 Google Scholar PubMed 27 Imagama S. , Sakamoto K. , Tauchi R. , Shinjo R. , Ohgomori T. , Ito Z. , Zhang H. , Nishida Y. , Asami N. , Takeshita S. , Sugiura N. , Watanabe H. , Yamashita T. , Ishiguro N. , Matsuyama Y. , Kadomatsu K. ( 2011 ) Keratan sulfate restricts neural plasticity after spinal cord injury . J. Neurosci . 31 , 17091 – 17102 Google Scholar CrossRef Search ADS PubMed 28 Sugiura N. , Clausen T.M. , Shioiri T. , Gustavsson T. , Watanabe H. , Salanti A. ( 2016 ) Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library . Glycoconj. J. 33 , 985 – 994 Google Scholar CrossRef Search ADS PubMed 29 Toyoda H. , Kinoshita-Toyoda A. , Selleck S.B. ( 2000 ) Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo . J. Biol. Chem. 275 , 2269 – 2275 Google Scholar CrossRef Search ADS PubMed 30 Oohira A. , Kushima Y. , Tokita Y. , Sugiura N. , Sakurai K. , Suzuki S. , Kimata K. ( 2000 ) Effects of lipid-derivatized glycosaminoglycans (GAGs), a novel probe for functional analyses of GAGs, on cell-to-substratum adhesion and neurite elongation in primary cultures of fetal rat hippocampal neurons . Arch. Biochem. Biophys . 378 , 78 – 83 Google Scholar CrossRef Search ADS PubMed 31 Shimbo M. , Ando S. , Sugiura N. , Kimata K. , Ichijo H. ( 2013 ) Moderate repulsive effects of E-unit-containing chondroitin sulfate (CSE) on behavior of retinal growth cones . Brain Res . 1491 , 34 – 43 Google Scholar CrossRef Search ADS PubMed 32 Ichijo H. , Sugiura N. , Kimata K. ( 2013 ) Application of chondroitin sulfate derivatives for understanding axonal guidance in the nervous system during development . Polymers 5 , 254 – 268 Google Scholar CrossRef Search ADS 33 Brown J.M. , Xia J. , Zhuang B. , Cho K.S. , Rogers C.J. , Gama C.I. , Rawat M. , Tully S.E. , Uetani N. , Mason D.E. , Tremblay M.L. , Peters E.C. , Habuchi O. , Chen D.F. , Hsieh-Wilson L.C. ( 2012 ) A sulfated carbohydrate epitope inhibits axon regeneration after injury. Proc. Natl. Acad. Sci. U.S.A . 109 , 4768 – 4773 Google Scholar CrossRef Search ADS 34 Dickendesher T.L. , Baldwin K.T. , Mironova Y.A. , Koriyama Y. , Raiker S.J. , Askew K.L. , Wood A. , Geoffroy C.G. , Zheng B. , Liepmann C.D. , Katagiri Y. , Benowitz L.I. , Geller H.M. , Giger R.J. ( 2012 ) NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans . Nat. Neurosci. 15 , 703 – 712 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations CGN cerebellar granule neuron CH chondroitin CS chondroitin sulphate DS dermatan sulphate GAG glycosaminoglycan GalNAc N-acetylgalactosamine GlcA glucuronic acid GlcNAc N-acetylglucosamine HMDA hexamethylenediamine HP heparin HPS N-acetylheparosan HS heparan sulphate IdoA iduronic acid MaxRU maximum response Mr average molecular mass RPTPσ receptor type of protein tyrosine phosphatase sigma SD sulphation degree SPR surface plasmon resonance © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Interaction of receptor type of protein tyrosine phosphatase sigma (RPTPσ) with a glycosaminoglycan library

Loading next page...
 
/lp/ou_press/interaction-of-receptor-type-of-protein-tyrosine-phosphatase-sigma-BRuLrWC1GR
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
ISSN
0021-924X
eISSN
1756-2651
D.O.I.
10.1093/jb/mvy027
Publisher site
See Article on Publisher Site

Abstract

Abstract Receptor type of protein tyrosine phosphatase sigma (RPTPσ) functions as a glycosaminoglycan (GAG) receptor of neuronal cells in both the central and peripheral nervous systems. Both chondroitin sulphate (CS) and heparan sulphate (HS) are important constituents of GAG ligands for RPTPσ, although they have opposite effects on neuronal cells. CS inhibits neurite outgrowth and neural regeneration through RPTPσ, whereas HS enhances them. We prepared recombinant RPTPσ N-terminal fragment containing the GAG binding site and various types of biotin-conjugated GAG (CS and HS) with chemical modification and chemo-enzymatic synthesis. Then interaction of the RPTPσ N-terminal fragment was analysed using GAG-biotin immobilized on streptavidin sensor chips by surface plasmon resonance. Interaction of RPTPσ with the CS library was highly correlated to the degree of disulphated disaccharide E unit, which had two sulphate groups at C-4 and C-6 positions of the N-acetylgalactosamine residue (CSE). The optimum molecular mass of CSE was suggested to be approximately 10 kDa. Heparin showed higher affinity to RPTPσ than the CS library. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology, but also provide medical application to regulate neural regeneration. chondroitin, glycosaminoglycans, heparin, RPTPσ, SPR In the extracellular matrix (ECM) in the central and peripheral nervous systems, chondroitin sulphate (CS) and heparan sulphate (HS) play important roles in the maintenance of the neuronal network and synapse formation; however, they exhibit opposite effects. A series of evidence supports that CS inhibits axonal growth and nerve regeneration through receptor type of protein tyrosine phosphatase sigma (RPTPσ) (1, 2), whereas HS promotes these processes (3, 4). For instance, CS accumulation in the glial scar after neuronal injury limited axonal growth and regeneration of the neuronal network. Injection of chondroitinase ABC to the lesion promoted axonal regeneration and functional recovery after spinal cord injury (5, 6). CS N-acetylgalactosaminyltransferase-1 knockout mice, which show decreased CS synthesis and increased HS levels, experience better restoration of the neuronal network after spinal cord injury than wild-type mice (7). Glycosaminoglycan (GAG) is a linear acidic polysaccharide composed of repeating disaccharide units and is modified with sulphate groups at various positions on the sugar residues (Fig. 1A) (8). Whereas hyaluronan (HA), composed of a repeating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) disaccharide unit, lacks sulphate modification, CS, composed of a GlcA and N-acetylgalactosamine (GalNAc) disaccharide unit, is modified with sulphate groups at various positions of the sugar residues. The major disaccharide structures of CS are as follows: a non-sulphated unit (0S, GlcA-GalNAc), a monosulphated unit at the C-4 position of the GalNAc residue (4S, GlcA-GalNAc4S), a monosulphated unit at the C-6 position of GalNAc (6S, GlcA-GalNAc6S), a disulphated unit at the C-4 and C-6 positions of GalNAc (diSE, GlcA-GalNAc4S6S), a disulphated unit at the C-2 position of GlcA and the C-4 position of GalNAc (diSB, GlcA2S-GalNAc4S), a disulphated unit at the C-2 position of GlcA and the C-6 position of GalNAc (diSD, GlcA2S-GalNAc6S), and a trisulfated disaccharide unit (triS, GlcA2S-GalNAc4S6S). Some GlcA residues are epimerized to iduronic acid (IdoA); the chain containing IdoA residues is designated as dermatan sulphate (DS). Fig. 1 View largeDownload slide Structures of GAGs and RPTPσ. (A) Structures of disaccharide units of HA, CS/DS and HS/HP. R1, R2 and R3 in the CS/DS table indicate O-substituted groups at the C-6, C-4 positions of the GalNAc residue, and C-2 position of the GlcA/IdoA residue, respectively. R1, R2 and R3 in the HS/HP table indicate O- or N-substituted groups at the C-6 position of the GlcN residue, C-2 position of the GlcA/IdoA residue and C-2 position of GlcN residue, respectively. (B) Domain organization of human RPTPσ isoform 4. Signals indicate the following: N-, amino terminus; S, signal peptide; Ig, immunoglobulin-like domains (1–3); FN3, fibronectin type-III domains (1–5); TM, transmembrane domain; phase, phosphatase domains (D1-D2) and -C, carboxy terminus. (C) SDS-PAGE with CBB (a and b) and western blotting with anti-RPTPσ antibody (c and d) for crude HaloTag-binding protein (70 kDa, a and c) and TEV-treated purified RPTPσ N-terminal fragment (36 kDa, b and d). Fig. 1 View largeDownload slide Structures of GAGs and RPTPσ. (A) Structures of disaccharide units of HA, CS/DS and HS/HP. R1, R2 and R3 in the CS/DS table indicate O-substituted groups at the C-6, C-4 positions of the GalNAc residue, and C-2 position of the GlcA/IdoA residue, respectively. R1, R2 and R3 in the HS/HP table indicate O- or N-substituted groups at the C-6 position of the GlcN residue, C-2 position of the GlcA/IdoA residue and C-2 position of GlcN residue, respectively. (B) Domain organization of human RPTPσ isoform 4. Signals indicate the following: N-, amino terminus; S, signal peptide; Ig, immunoglobulin-like domains (1–3); FN3, fibronectin type-III domains (1–5); TM, transmembrane domain; phase, phosphatase domains (D1-D2) and -C, carboxy terminus. (C) SDS-PAGE with CBB (a and b) and western blotting with anti-RPTPσ antibody (c and d) for crude HaloTag-binding protein (70 kDa, a and c) and TEV-treated purified RPTPσ N-terminal fragment (36 kDa, b and d). HS and heparin (HP) are composed of GlcA/IdoA and N-acetylated or N-sulfonated glucosamine (GlcNAc/GlcNS) disaccharide units with O-sulfation at various positions of the sugar residues. The major disaccharide structures of HS/HP are as follows: a non-sulphated unit (0S, GlcA/IdoA-GlcNAc), a mono N-sulfonated unit (NS, GlcA/IdoA-GlcNS), a monosulphated unit at the C-2 position of the GlcA/IdoA residue (US, GlcA2S/IdoA2S-GlcNAc), a monosulphated unit at the C-6 position of the GlcNAc residue (6S, GlcA/IdoA-GlcNAc6S), a disulphated unit at the C-6 position of the GlcNS residue (N6diS, GlcA/IdoA-GlcNS6S), a disulphated unit at the C-2 position of the GlcA/IdoA and GlcNS residues (NUdiS, GlcA2S/IdoA2S-GlcNS), a disulphated unit at the C-2 position of GlcA/IdoA and the C-6 position of GlcNAc (U6diS, GlcA2S/IdoA2S-GlcNAc6S), and a trisulphated disaccharide unit (triS, GlcA2S/IdoA2S-GlcNS6S) (Fig. 1A). CS and HS functions are mediated through their receptors, which are expressed in neurons, the major receptor being RPTPσ (9). RPTPσ comprises an intracellular region containing two tandem catalytic domains and an extracellular region containing three immunoglobulin (Ig)-like domains followed by five fibronectin type III (FN3) domains (Fig. 1B). The GAG-binding site is located in a N-terminal Ig1 domain, which contains a cluster of basic amino acid residues (3). The region containing three Ig-like domains interacts with HP to form oligomers, and with CS to form a monomer (4). This distinctive receptor clustering caused by GAGs is thought to regulate axon growth and neuron network regeneration. However, it is still unclear how diverse GAG species specifically affect the clustering and downstream signal transduction. Previously, we constructed CS/DS and HS/HP library with defined structure using chemical modification and polysaccharide synthesis enzymes (10, 11). Here, we confirmed the effect of CS and HP on neurite outgrowth using GAG phospholipid conjugates. We also investigated the interaction of the GAG-binding fragment of RPTPσ with different GAG species in our GAG library. Analysis using surface plasmon resonance (SPR) revealed that their interaction was highly correlated with the degree of diSE unit in CS and the sulfation degree (SD) of HS/HP. Highly sulphated HP possessed four-fold higher affinity to RPTPσ fragment than the diSE-rich CS (CSE). Materials and Methods Materials Chondroitin (CH) prepared by desulfation of CS, native CSE from squid cartilage, DS from pig skin, partially depolymerized HA from chick comb, HS from porcine aorta, HP from pig intestine, N-acetylheparosan (HPS) from Escherichia coli strain K5 (12), HP desulfated at the C-2 positions of the GlcA/IdoA residues (2DS-HP), HP desulfated at the C-6 positions of the GlcNAc/GlcNS residues (6DS-HP), HP completely desulfated at most positions of sugar residues and N-resulfonated at C-2 positions of GlcN residues (CDSNS-HP), HP N-desulfated and N-acetylated at the C-2 positions of GlcNS residues (NDSNAc-HP), chondroitinase ABC, and heparitinase I, II and III were obtained from Seikagaku (Tokyo, Japan). Chondroitin hexasaccharide (CH6) prepared by partial digestion of CH (13), recombinant chondroitin polymerase from E. coli strain K4 (K4CP) (14), K4CP mutants (15), recombinant CS sulfotransferases: chondroitin 4-sulfotransferase-1 (C4ST-1) (16), chondroitin 6-sulfotransferase-1 (C6ST-1) (17), GalNAc4S 6-sulfotransferase (GalNac4S-6ST) (18), and uronosyl 2-sulfotransferase (UA2ST) (19), HS 2-sulfotramsferase (HS2ST) (20), HS 6-sulfotramsferase-1 (HS6ST-1) (21), and phosphatidylethanolamine (PE)-conjugated GAG derivatives (22, 23) were prepared as described previously: CH-PE (average molecular mass (Mr) 10k, major disaccharide composition 0S 100%), CSA-PE (Mr 15k, 4S 74%, 6S 22%), CSC-PE (Mr 20k, 4S 22%, 6S 63%, diSd 10%), CSE-PE (Mr 50k, 4S 20%, 6S 8%, diSe 66%), HS-PE (Mr 20k, 0S 46%, 6S 15%, NS 21%, N6diS 8%, NUdiS 6%, triS 3%) and HP-PE (Mr 10k, N6diS 15%, NUdiS 7%, triS 78%). Adenosine 3′-phosphate 5′-phosphosulfate (PAPS), uridine 5′-diphospho-α-d-N-acetylgalactosamine (UDP-GalNAc) and uridine 5′-diphospho-α-d-glucuronate (UDP-GlcA) were from Yamasa (Choshi, Japan). Streptavidin-conjugated sensor chips (SA chips) for the Biacore biosensor were purchased from GE Healthcare (Pittsburgh, PA). Preparation of GAG species and their biotin conjugates The chemo-enzymatically synthesized CS library was constructed as described previously (10). Briefly, CH poly- and oligosaccharides were synthesized from CH6 with K4CP or K4CP mutants. CH octasaccharide (CH8) and dodecasaccharide (CH12) were synthesized with two K4CP mutants and two donor substrates (UDP-GalNAc or UDP-GlcA) by an alternative elongation reaction (15). The average molecular masses (Mr) of prepared CH polymers were 3, 7, 10, 20, 100, and 150 kDa. CS species with different sulfation patterns were synthesized using the CH polymers and oligomers as acceptor substrates, PAPS as a donor substrate and various recombinant chondroitin sulfotransferases (C4ST-1, C6ST-1, GalNac4S-6ST and UA2ST) as catalytic enzymes. For example, the CH species with diverse chain lengths were sulphated at C-4 positions of the GalNAc residues with C4ST-1 to obtain CSA species. CSC was synthesized with C6ST-1. CSAC was synthesized by simultaneous reaction of C4ST-1 and C6ST-1. CSAD was synthesized with UA2ST from CSAC. CSE species with diverse chain lengths were synthesized with GalNAc4S6ST from CSA species having various chain lengths. CSDE was synthesized with GalNAc4S6ST from CSAD. CST was synthesized from CSE using UA2ST. Desulfated and sulphated HP derivatives were prepared as described previously (24–26). Briefly, NDSNAc-HP was prepared by N-desulfation with dimethyl sulfoxide-methanol at 20°C followed by N-acetylation with acetic acid anhydride. 2DS-HP was obtained by freeze drying under slightly alkaline conditions. 6DS-HP was obtained by N-methyl-N-(trimethylsilyl) trifluoroacetamide treatment of HP. CDSNS-HP was prepared by solvolytic desulfation and trimethyl sulphur trioxide complex treatment of HP. 2-Sulfated HPS (2S-HPS) and 6-sulfated HPS (6S-HPS) were prepared from HPS with HS2ST and HS6ST-1 recombinant enzymes, respectively. The GAG oligo- and polysaccharides were conjugated with hexamethylenediamine (HMDA) at the reducing ends by the reductive amination method before or after chain elongation and sulfation reaction (10). Then, they were modified with sulfo-NHS-activated biotin reagent (sulfo-NHS-LC-biotin, Pierce, Rockford, IL) at the amine group of the HMDA residue. Mr, disaccharide composition and SD (number of sulphate groups per disaccharide unit) of the GAG-biotin conjugates are summarized in Table I. Table I. Biotin-conjugated GAG species and their disaccharide compositions GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – Note: –, not detected. Table I. Biotin-conjugated GAG species and their disaccharide compositions GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – GAG Group 1 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS HA 27k 0.00 100 – – – – – – CH 10k 0.00 100 – – – – – – DS 20k 1.05 0.6 93.6 – – 5.8 – – CSA 10k 0.98 1.9 98.1 – – – – – CSC 10k 0.98 1.6 – 98.4 – – – – CSAC 10k 0.99 1.0 44.7 54.3 – – – – CSAD 10k 1.41 1.3 44.5 12.2 – – 41.9 – CSE 10k 1.85 2.6 10.0 – 87.3 – – – CSDE 10k 1.47 2.0 48.3 0.9 7.7 – 41.1 – CST 10k 2.07 1.1 9.5 – 71.1 – – 18.3 GAG Group 2 Mr (kDa) SD Disaccharide compositions (%) 0S 4S 6S diSe diSb diSd triS CSE8 2.5k 1.43 4.6 47.7 – 47.7 – – – CSE12 3k 1.34 13.0 39.6 – 47.4 – – – CSE7k a 7k 1.38 21.0 36.4 – 42.7 – – – CSE7k b 7k 0.87 43.4 26.8 – 29.9 – – – CSE7k c 7k 0.85 27.0 61.1 – 12.0 – – – CSE10k a 10k 1.85 2.6 10.0 – 87.3 – – – CSE10k b 10k 1.59 13.7 13.7 – 72.6 – – – CSE10k c 10k 1.40 20.5 18.6 – 60.9 – – – CSE10k d 10k 0.89 20.4 70.4 – 9.2 – – – CSE20k 20k 1.18 4.7 72.3 – 23.0 – – – CSE50k 50k 1.61 2.6 26.3 7.3 63.8 – – – CSE100k 100k 1.37 4.2 54.7 – 41.1 – – – CSE150k 150k 1.13 2.5 81.7 – 15.0 – – – GAG Group 3 Mr (kDa) SD Disaccharide compositions (%) 0S NS US 6S N6diS NUdiS U6diS triS HP 10k 2.63 8.3 2.8 – 1.8 3.5 – – 83.7 NDSNAc-HP 10k 1.90 4.8 – – 7.5 – – 80.4 7.3 2DS-HP 10k 1.60 5.2 26.3 – 3.7 64.8 – – – 6DS-HP 10k 1.86 6.8 – – – – 93.2 – – CDSNS-HP 10k 0.95 10.2 78.6 – – 3.9 4.4 – – HS 20k 0.63 53.9 25.1 – 9.0 5.4 2.0 – 4.7 2S-HPS 20k 0.06 94.2 – 5.8 – – – – – 6S-HPS 20k 0.02 98.4 – – 1.6 – – – – HPS 20k 0.00 100 – – – – – – – Note: –, not detected. Neurite outgrowth assay Animal experiments were approved by Animal Care and Use Committee for Aichi Medical University and followed the ethical standard formulated in the Helsinki Declaration. Rat cerebellar granule neurons (CGNs) were collected as reported previously (27) from cerebella of Sprague-Dawley rats, purchased from Japan SLC (Hamamatsu, Japan), at postnatal days 7–9. CGNs (1.5 × 105 cells) were seeded onto cover glass (18 × 18 mm, Matsunami, Tokyo, Japan) coated with 25 μg/ml poly-l-lysine (Sigma, St. Louis, MO) and 5 μg/ml laminin (Becton Dickinson, Bedford, MA) in 35 mm Petri dishes and cultured at 37°C in Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco), 2 mM glutamine, 20 mM KCl, 50 U/ml penicillin and 50 μg/ml streptomycin. Five hours later after cell seeding, CH-PE, CSA-PE, CSC-PE, CSE-PE, HS-PE or HP-PE was added to the culture at a final concentration of 10 μg/ml, followed by further incubation at 37°C for 19 h. The neurons were fixed with 4% paraformaldehyde in PBS and washed with PBS, followed by blocking with 1% bovine serum albumin in PBS for 30 min. Subsequently, the fixed cells were treated with anti-neuron-specific β-tubulin antibody (Covance, Princeton, NJ) at room temperature for 1 h. After rinsing with PBS, neurons were incubated with a fluorescence-labelled secondary antibody (Alexa Fluor 488-conjugated anti-mouse IgG goat serum, Invitrogen) at room temperature for 45 min to visualize neurite outgrowth. The stained cells on the cover glass were mounted with Fluoromount Aqueous medium (Sigma) and inspected using an Axio Vision fluorescent microscope (Carl Zeiss). The relative amount of neurite outgrowth was quantified based on the area of β-tubulin-positive cells per total cells in a field of vision. Expression and purification of RPTPσ N-terminal fragment A plasmid pFN21A-B4243 containing the full sequence of human RPTPσ isoform 4 (accession no. AB209333 and NP_570925) was obtained from Kazusa DNA Research Institute (Kisarazu, Japan). For expression of the N-terminal Ig 1-3 fragment (amino acid 30-321) of RPTPσ, the DNA encoding the N-terminal fragment was amplified by PCR using the pFN21A-B4243 plasmid as a template; forward primer, 5'-GCTAGCGATCGCCATGGAAGAGCCCCCCAGGTTTATCAAAGA-3' and reverse primer, 5'- TCGAGAGCTCGAGATTTCACCGTGATCTGAGCAACCGCCTC-3'. The Sgf1 and Eco1CR1 cleavage sites are underlined. The PCR product was subcloned into a pFC14K HaloTag CMV Flexi expression vector. The inserted DNA sequence was confirmed with a DNA sequencer (3130 Genetic Analyzer, Applied Biosystems, Brancurg, NJ). N-terminal RPTPσ fragment-inserted expression plasmid (pFC14K-RPTPσ(30-321)-HaloTag) was transfected into HEK293T cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The transfected HEK293T cells were incubated in the culture medium at 37°C for 2–4 days. The cells were homogenized with a glass homogenizer and the extract was collected by centrifugation. The recombinant protein containing HaloTag at the C-terminus was adsorbed with HaloLink Resin (Promega). The RPTPσ N-terminal fragment was purified by treatment with TEV protease (Promega) to release the fragment from the HaloTag fusion protein that covalently bound to the HaloLink resin. (Fig. 1C). SPR analysis Interactions of the recombinant RPTPσ N-terminal fragment with the various GAG species were analysed using an SPR biosensor (BIAcore T200; GE Healthcare) (28). Briefly, the GAG-biotin conjugates (10 µg/ml) were immobilized in flow cells 2–4 of the SA chip. Biotin (100 pmol/ml) was used as a control in flow cell 1 of the chip. GAG-biotin and biotin solution (70 μl) were injected into SA chips at a flow rate of 5 μl/min in 50 mM phosphate buffer (pH 7.2) containing 0.05% Tween 20. Responses of immobilized samples were recorded after washing (60 s, three times) with a solution of 1 M NaCl containing 50 mM NaOH. Binding assays were performed at 25°C at a constant flow rate of 30 µl/min during both the association and dissociation phases using 10 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 3 mM EDTA and 0.005% Tween 20 (HBS-EP). RPTPσ N-terminal fragment analyte solutions (25, 50, 100, 200, 400, and 800 nM) in HBS-EP were injected into the GAG ligand-immobilized flow cells for 60 s (association), followed by injection of HBS-EP buffer for 180 s (dissociation). Regeneration of the sensor chip surface was accomplished by an injection of 20 µl of HBS-EP containing 2 M NaCl. The sensorgrams were recorded, and the kinetic parameters calculated from association and dissociation curves were determined using BIA Evaluation 4.1 software (GE Healthcare). Compositional analysis of GAG derivatives The GAG samples (10 pmol–1 nmol) were digested with chondroitinase ABC (10 mU) for HA and CS/DS or with heparitinase I, -II and -III mixed enzymes (10 mU each) for HS/HP at 37°C for 1 h. The digested products containing unsaturated disaccharides were analysed using a fluorometric post-column HPLC system as reported previously (29). SDS-PAGE and western blotting The protein samples were applied to 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with the primary antibody followed by treatment with a peroxidase-conjugated secondary antibody. The reacted proteins were detected with chemiluminescence reagent and LAS-400 detector (GE Healthcare). Statistical analysis Statistical analyses were performed with an unpaired Student’s t-test. The level of significance is indicated by asterisks: *, P < 0.05 and **, P < 0.01. The error bars show standard deviations. Results and Discussion Effect of GAG-PE on neurite outgrowth of CGNs CS and HS were reported to regulate axonal growth and neuronal regeneration through RPTPσ, although with opposite effects (3, 4). To confirm this, we investigated the effect of CS and HS/HP on neurite outgrowth of rat CGNs using CSE-PE and HP-PE. Rat CGNs express RPTPσ, similar to most of mammalian neurons (9). Treatment with CSE-PE inhibited neurite outgrowth, whereas HP-PE enhanced it, when they were added 5 h after cell seeding (Fig. 2). When CS-PE was added before cell seeding, it substantially inhibited cell adhesion. Interestingly, free polysaccharides (CSE and HP) showed no significant effect on neurite outgrowth up to 50 μg/ml (data not shown). Other PE-modified GAG derivatives had no statistically significant effects, although CSA-PE and CSC-PE showed a tendency to inhibit neurite outgrowth and HS-PE showed a tendency to enhance it (Fig. 2D). Fig. 2 View largeDownload slide Neurite outgrowth stained with anti-neuron-specific β-tubulin. (A) Control rat CGNs. (B) CSE-PE-treated CGNs. (C) HP-PE-treated CGNs. Scale bars, 100 μm. (D) Relative neurite outgrowth (%) of rat neurons treated with buffer (control), CH-PE, CSA-PE, CSC-PE, CSE-PE, HS-PE and HP-PE; the values obtained with control cells are taken as 100%. Bars represent the standard deviations determined in five independent experiments. *, P < 0.05; **, P < 0.005. (E) Molecular structure of CS-PE (dipalmitoylphosphatidylethanolamine-conjugated chondroitin sulphate). Fig. 2 View largeDownload slide Neurite outgrowth stained with anti-neuron-specific β-tubulin. (A) Control rat CGNs. (B) CSE-PE-treated CGNs. (C) HP-PE-treated CGNs. Scale bars, 100 μm. (D) Relative neurite outgrowth (%) of rat neurons treated with buffer (control), CH-PE, CSA-PE, CSC-PE, CSE-PE, HS-PE and HP-PE; the values obtained with control cells are taken as 100%. Bars represent the standard deviations determined in five independent experiments. *, P < 0.05; **, P < 0.005. (E) Molecular structure of CS-PE (dipalmitoylphosphatidylethanolamine-conjugated chondroitin sulphate). GAG must be immobilized to a solid material, such as culture dish, when its cellular activities are to be analysed. Because free GAG is usually unable to bind to solid materials, it does not exhibit cellular activity. Conjugated proteins help GAGs to exert their functions. In fact, some studies evaluated GAG function by using proteoglycans (native GAGs and core-protein complexes) (1, 4) or artificial GAG-protein complexes (27) rather than free GAGs. Previously, we generated PE-conjugated GAG (22) that could bind to culture dishes via hydrophobic interactions. We then investigated its effects, and found that CS-PE promoted axonal elongation at low concentrations (0.1–1.0 μg/ml) and inhibited neurite extension and adhesion at concentrations higher than 1.0 μg/ml (30). We also found that CSE-PE-coated polystyrene beads repulsed the growth cones of chick retinal neurons (31, 32). Previous studies (33, 34) indicated that CSE had high affinity to RPTPσ and inhibition of neurite outgrowth using CS polymers and anti-CS antibodies. In this study, using CSE-PE and HP-PE, we found that CSE and HP have opposite effects on neurite outgrowth of rat CGNs. Immobilization of biotin-conjugated GAG library to SA-coated sensor chips The synthesized biotin-conjugated various GAG species with various chain sizes and disaccharide compositions (Table I) were immobilized onto SA-coated sensor chips for SPR analysis. The GAG species were divided into three groups: HA and various CS/DS species, various CSE species and HS/HP derivatives. The first group was composed of HA generated by partial digestion with testicular hyaluronidase, CH synthesized with K4CP, DS isolated from pig skin and seven synthetic CS species (CSA, CSC, CSAC, CSAD, CSE, CSDE and CST) that were generated from CH (10 kDa) by various sulfations with corresponding sulfotransferases. The second group consisted of CSE species having various chain sizes (8-mer, 12-mer, 7 kDa, 10 kDa, 20 kDa, 50 kDa, 100 kDa and 150 kDa) and various degrees of diSE (9–88%), which were synthesized with K4CP, C4ST-1 and GalNAc4S-6ST, except CSE (50 kDa) isolated from squid cartilage. The third group consisted of HP, NDSNAc-HP, 2DS-HP, 6DS-HP, CDSNS-HP, HS from porcine aorta, HPS from E. coli strain K5, 2S-HPS and 6S-HPS. The GAG species were conjugated with HMDA and then biotin groups at the reducing end of the sugar chains. The solution of GAG-biotin conjugates (10 μg/ml) was immobilized to SA sensor chips for 840 s. Responses of immobilized GAG-biotins were detected in the range of 300–600 RU and those of biotin as a control were not detected significantly. All biotinylated samples were successfully immobilized. SPR analysis of the interaction between RPTPσ N-terminal fragment and immobilized GAGs We analysed the interaction of the purified RPTPσ N-terminal fragment with GAG-immobilized SA chips using a Biacore T200 SPR analyser. Various concentrations (25–800 nM) of the RPTPσ N-terminal fragment were injected to the GAG-immobilized sensor chips. The association and dissociation curves of the sensorgrams were analysed and kinetic parameters were determined using BIA Evaluation 4.1 software (GE Healthcare). The response curves of the sensorgrams fitted better to a two-state reaction model than to a 1:1 binding model (Fig. 3). Therefore, we adopted the kinetic parameters calculated from the two-state reaction model (Table II). Table II. Kinetic parameters of RPTPσ affinity to GAG-immobilized chips GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — a“–” means affinity was too low; therefore, the kinetic parameters were not indicated. bMaxRU indicates maximum response (RU) with injection of 800 nM RPTPσ fragment injection, with ± standard deviation (SD, n = 3) shown. c“–” indicates that MaxRU was nearly zero or had a negative value. Table II. Kinetic parameters of RPTPσ affinity to GAG-immobilized chips GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — GAG kd1/ka1 kd1/(kd2+ka2) Kd (M) MaxRU ± SDb Group 1     HA –a – – —c     CH – – – —     DS – – – 82.0 ± 7.8     CSA 6.82 × 10−5 4.66 × 10−1 3.18 × 10−5 191.2 ± 12.4     CSC – – – —     CSAC – – – 66.9 ± 11.2     CSAD 1.89 × 10−4 5.28 × 10−2 9.96 × 10−6 457.8 ± 77.5     CSE 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSDE 4.46 × 10−6 3.67 × 10−2 1.63 × 10−7 597.8 ± 52.0     CST 5.16 × 10−7 8.90 × 10−2 4.59 × 10−8 908.9 ± 20.5 Group 2     CSE8 – – – 49.8 ± 1.8     CSE12 5.01 × 10−6 4.48 × 10−1 2.24 × 10−6 289.5 ± 33.0     CSE7k a 7.53 × 10−6 7.22 × 10−2 5.44 × 10−7 792.6 ± 107.2     CSE7k b 1.88 × 10−5 9.87 × 10−1 1.85 × 10−5 100.4 ± 8.2     CSE7k c 4.03 × 10−5 9.67 × 10−1 3.89 × 10−5 99.2 ± 6.6     CSE10k a 2.79 × 10−6 2.14 × 10−2 5.98 × 10−8 891.3 ± 69.8     CSE10k b 2.77 × 10−6 6.78 × 10−2 1.88 × 10−7 963.8 ± 39.4     CSE10k c 9.16 × 10−6 1.74 × 10−2 1.59 × 10−7 551.9 ± 39.4     CSE10k d 6.36 × 10−6 9.68 × 10−1 6.15 × 10−6 32.6 ± 7.4     CSE20k 3.96 × 10−4 2.06 × 10−1 3.65 × 10−5 223.2 ± 25.5     CSE50k 7.38 × 10−5 6.81 × 10−2 5.03 × 10−6 554.4 ± 60.0     CSE100k 1.89 × 10−4 6.54 × 10−2 1.67 × 10−5 321.9 ± 17.9     CSE150k 2.84 × 10−5 2.39 × 10−1 7.07 × 10−6 90.1 ± 9.7 Group 3     HP 8.27 × 10−7 3.18 × 10−2 2.63 × 10−8 3832.3 ± 321.6     NDSNAc-HP 1.44 × 10−6 9.72 × 10−2 1.40 × 10−7 2059.7 ± 145.4     2DS-HP 9.84 × 10−7 3.39 × 10−2 3.34 × 10−8 2906.5 ± 105.0     6DS-HP 4.93 × 10−7 4.16 × 10−2 2.05 × 10−8 2097.6 ± 192.5     CDSNS-HP 5.69 × 10−6 6.45 × 10−2 3.67 × 10−7 364.1 ± 36.0     HS – – – 132.5 ± 10.3     2S-HPS – – – —     6S-HPS – – – —     HPS – – – — a“–” means affinity was too low; therefore, the kinetic parameters were not indicated. bMaxRU indicates maximum response (RU) with injection of 800 nM RPTPσ fragment injection, with ± standard deviation (SD, n = 3) shown. c“–” indicates that MaxRU was nearly zero or had a negative value. Fig. 3 View largeDownload slide Comparison of SPR evaluation models. Sensorgrams of the several concentrations (25, 50, 100, 200, 400 and 800 nM) of the RPTPσ fragment with CSE- (A and B) and HP- (C and D) immobilized chips are shown, with fitting by 1:1 binding model (A and C) and two-state reaction model (B and D) on SPR. The coloured solid lines represent curves for actual measurements and black broken lines represent calculated curves. Fig. 3 View largeDownload slide Comparison of SPR evaluation models. Sensorgrams of the several concentrations (25, 50, 100, 200, 400 and 800 nM) of the RPTPσ fragment with CSE- (A and B) and HP- (C and D) immobilized chips are shown, with fitting by 1:1 binding model (A and C) and two-state reaction model (B and D) on SPR. The coloured solid lines represent curves for actual measurements and black broken lines represent calculated curves. Two-state reaction model describes a 1:1 binding of analyte (A, RPTPσ) to immobilized ligand (B, GAG) forming AB, followed by a conformational change that stabilizes the complex (AB*) as shown by the formula below (Software Handbook of Biacore T200, GE Healthcare). A+Bkd1ka1ABkd2ka2AB* In this equation, ka1 [M−1 s−1] is association rate constant for analyte binding, kd1 [s−1] is the dissociation constant for analyte from the complex, ka2 [s−1] is the forward rate constant for the conformational change and kd2 [s−1] is the reverse rate constant for the conformational change. The overall equilibrium dissociation constant Kd [M] is given by the following equation: Kd=kd1/ka1×kd2/(kd2+ka2) The first half of this equation indicates the 1:1 binding and the second half indicates the conformational change. For various GAG chips, kinetic parameters of RPTPσ interactions were calculated with the two-state reaction model (Table II). Most values of the second half of the equation were in the order of 10−1–10−2, indicating that the second half did not contribute to the interaction significantly. In contrast, the values of the first half greatly changed in the order of 10−4–10−7, suggesting that the first half contributed to fluctuations of the interaction between RPTPσ and GAG species. However, it should be noted that the actual conformational change could not be studied in this study and should be investigated using other techniques such as NMR, IR and CD spectroscopies. Sensorgrams for the highest concentration (800 nM) of the RPTPσ fragment on the various GAG-immobilized chips are shown in Fig. 4. Maximum response (MaxRU) defined as the highest response value at the highest concentration (800 nM) of injected RPTPσ fragment in GAG-immobilized chip was determined from the response curve (Table II). The values of MaxRU and Kd are plotted against several properties of GAG samples in Fig. 5. Fig. 4 View largeDownload slide Sensorgrams of RPTPσ with various GAG chips on SPR. The sensorgrams for a high concentration (800 nM) of the RPTPσ N-terminal fragment with the sensor chips of group 1 CS species (A), CSE long chains of group 2 (B), CSE short chains of group 2 (C) and group 3 HP derivatives (D) are shown. (A) Red, black, blue, green orange and grey lines show CSE, CST, CSDE, CSAD, CSA and DS, respectively. (B) Red, brown, orange, grey, blue, black, green and yellow lines show CSE10k a, CSE10k b, CSE10k c, CSE10k d, CSE20k, CSE50k, CSE100k and CSE150k, respectively. (C) Red, blue, orange, green and grey lines show CSE7k a, CSE7k b, CSE7k c, CSE12 and CSE8, respectively. (D) Red, blue, orange, black, grey and green lines show HP, NDSNAc-HP, 2DS-HP, 6DS-HP, CSDNS-HP and HS, respectively. Fig. 4 View largeDownload slide Sensorgrams of RPTPσ with various GAG chips on SPR. The sensorgrams for a high concentration (800 nM) of the RPTPσ N-terminal fragment with the sensor chips of group 1 CS species (A), CSE long chains of group 2 (B), CSE short chains of group 2 (C) and group 3 HP derivatives (D) are shown. (A) Red, black, blue, green orange and grey lines show CSE, CST, CSDE, CSAD, CSA and DS, respectively. (B) Red, brown, orange, grey, blue, black, green and yellow lines show CSE10k a, CSE10k b, CSE10k c, CSE10k d, CSE20k, CSE50k, CSE100k and CSE150k, respectively. (C) Red, blue, orange, green and grey lines show CSE7k a, CSE7k b, CSE7k c, CSE12 and CSE8, respectively. (D) Red, blue, orange, black, grey and green lines show HP, NDSNAc-HP, 2DS-HP, 6DS-HP, CSDNS-HP and HS, respectively. Fig. 5 View largeDownload slide Kinetic analyses of RPTPσ N-terminal fragment with various GAG chips on SPR. (A) Plot of MaxRU versus SD values of GAG group 1. Grey circle, HA and CH; grey square, CSC, grey triangle, CSAC; white triangle, DS; black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (B) Plot of Kd versus SD values of GAG group 1. Black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (C) Plot of MaxRU versus diSE% of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; white circle, CSE polymers (CSE20k, 50k, 100k and 150k); and white triangle, CSE oligomers (CSE8, CSE12). (D) Plot of Kd versus diSE% values of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; and white circle, CSE polymers (CSE20k, 50k, 100k and 150k). (E). Plot of MaxRU versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (F) Plot of Kd versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (G) Plot of MaxRU versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP; white square, CDSNS-HP; grey triangle, HS, grey circle, 2S-HPS; grey triangle, 6S-HPS and grey square, HPS. (H) Plot of Kd versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP and white square, CDSNS-HP. Fig. 5 View largeDownload slide Kinetic analyses of RPTPσ N-terminal fragment with various GAG chips on SPR. (A) Plot of MaxRU versus SD values of GAG group 1. Grey circle, HA and CH; grey square, CSC, grey triangle, CSAC; white triangle, DS; black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (B) Plot of Kd versus SD values of GAG group 1. Black triangle, CSA; white circle, CSAD; white square, CSDE; black circle, CSE; and black square, CST. (C) Plot of MaxRU versus diSE% of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; white circle, CSE polymers (CSE20k, 50k, 100k and 150k); and white triangle, CSE oligomers (CSE8, CSE12). (D) Plot of Kd versus diSE% values of GAG group 2. Black circle, CSE7k a–c; black square, CSE10k a–d; and white circle, CSE polymers (CSE20k, 50k, 100k and 150k). (E). Plot of MaxRU versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (F) Plot of Kd versus Mr of GAG group 2. White circle, CSE with low diSE composition (12–15%); white triangle, CSE with relatively low diSE composition (20–30%); black circle, CSE with medium diSE composition (40–50%); and black square, CSE with high diSE composition (>60%). (G) Plot of MaxRU versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP; white square, CDSNS-HP; grey triangle, HS, grey circle, 2S-HPS; grey triangle, 6S-HPS and grey square, HPS. (H) Plot of Kd versus SD values of GAG group 3. Black square, HP; black circle, 2DS-HP; white circle, 6DS-HP; black triangle, NDSNAc-HP and white square, CDSNS-HP. Sensorgrams of group 1 composed of HA and CS/DS (Fig. 4A and Supplementary Fig. S1) showed diverse curves for GAG species. The values of MaxRU of group 1 (HA and CS/DS) generally correlated with the SD (Fig. 5A). Kd values of group 1 GAG species were negatively correlated with SD (Fig. 5B). Highly sulphated CS species, CST (SD = 2.07) and CSE (SD = 1.85), which had a content of 87–89% multi-sulphated disaccharide units, showed similarly high affinity (MaxRU ≈ 900 and Kd ≈ 5 × 10−8 M). GlcA 2-O-sulfation in CST appeared to contribute less to the affinity of RPTPσ. Highly sulphated CS, CSDE (SD = 1.47) and CSAD (SD = 1.41), showed relatively high responses. CSDE containing diSE unit had a higher response than CSAD, although they possessed similar SD. The group of CS having one sulphate per disaccharide unit, CSA (SD = 0.98), CSC (SD = 0.98), CSAC (SD = 0.99) and DS (SD = 1.05), and non-sulphated saccharides, HA and CH, hardly showed SPR responses. Their kinetic parameters could not be calculated because the quality of the response curves was too low for kinetic evaluation. These results suggest that diSE unit might be critical for interaction between RPTPσ and CS. We then analysed the interaction of the RPTPσ fragment with group 2 sensor chips, which had various CSE species immobilized on them (Fig. 4B, C, and Supplementary Fig. S2). In general, the MaxRU values of the CSE species correlated with the ratio of diSE (diSE%, Fig. 5C) and their Kd values negatively correlated with diSE% (Fig. 5D). CSE octasaccharide (CSE8) showed a low MaxRU value, although it contained considerable diSE units (48%). Similarly sulphated dodecasaccharide (CSE12, diSE: 47%) showed more potent binding capacity (MaxRU = 290) than CSE8. Three species of CSE7k with Mr 7 kDa, 28-mer average sugar chains and different diSE contents (43%, 30%, and 12%, respectively); four species of CSE10k with Mr 10 kDa, 46-mer and different diSE contents (87%, 73%, 61% and 9%, respectively); and four species of CSE polysaccharide with Mr 20, 50, 100 and 150 kDa and exhibited interactions as strong as their diSE contents. Plots of the MaxRU and Kd values against Mr of the group 2 CSE species showed complicated patterns. When the CSE species were divided into four groups, i.e. low (12–15%), relatively low (20–30%), medium (40–50%) and high (> 60%) diSE ratios, the correlation was obvious between affinity and molecular size (Fig. 5E and F). The CSE species having high (> 60%) and medium (40–50%) diSE ratios exhibited the highest affinity when Mr was 10 kDa, but the affinity was lower for either shorter or longer sugar chains. CSE species with low (12–15%) and relatively low (20–30%) diSE ratios showed low affinity and indistinct correlation between kinetic parameters and Mr of CSE species. Next, we analysed the interaction of RPTPσ with HS/HP derivatives (group 3). Sensorgrams of the group 3 ligands are shown in Fig. 4D and Supplementary Fig. S3. MaxRU values of this group correlated with SD (Fig. 5G). Their Kd values were negatively correlated with SD (Fig. 5H). HP (SD = 2.6), exhibiting the highest sulfation levels in this study, possessed the highest affinity for RPTPσ fragment. It showed a 4-fold higher MaxRU value and over 2-fold lower Kd value than CSE10k, which possessed the highest activity among CS species (Table II). Similarly, a highly sulphated subgroup of HP derivatives NDSNAc-HP (SD = 1.90), 2DS-HP (SD = 1.60) and 6DS-HP (SD = 1.86) exhibited high affinity to RPTPσ. Most of the disaccharide content of HP is triS, whereas that of NDSAc-HP, 2DS-HP and 6DS-HP is U6diS, N6diS and NUdiS, respectively. This indicates that affinity of HP with RPTPσ might not be reduced when one sulphate group at any position of N-sulfation of GlcN, 2-sulfation of GlcA/IdoA and 6-sulfation of GlcN is deleted. In contrast, low-sulphated HS/HP derivatives (SD < 1.0), CDSNS-HP, HS, 2S-HPS, 6S-HPS and HPS, had low affinity to RPTPσ. Conclusion In this study, we investigated the relationship between GAG structure and binding activity of neuronal receptor RPTPσ using our artificial GAG library. CSE chains having high diSE units and Mr 10 kDa and HP derivatives having high content of diS units were critical for RPTPσ affinity. HP derivatives had 4-fold higher affinity than CSE species, but it is not clear why CS and HP exhibit opposite activity. Large quantity of CSPG is produced in injured central nervous systems, but HSPG is not. Because of the difference of CS and HS production, CS is sought to be dominant effector on RPTPσ. These GAG activities may be associated with cluster formation and phosphatase activity of the receptor, different intracellular signalling after GAG-receptor interaction, and/or another phenomenon, which should be identified in future. Using PE-conjugated GAGs, We confirmed that CSE inhibits and HP enhances neurite outgrowth of rat CGNs. We demonstrated that a biotin-conjugated GAG library composed of chemoenzymatically synthesized CS species and chemically modified HP derivatives contained useful ligands for characterization of GAG-binding materials, such as RPTPσ. The SPR kinetic interaction study of RPTPσ using our GAG library revealed high-affinity structures of CS and HP. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology but also provide medical applications to regulate neural regeneration. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank Prof. Kenji Kadomatsu and Dr Kazuma Sakamoto (Nagoya University) for teaching us the isolation method for rat CGNs and Yoshimi Shintoku for expert technical assistance. Funding This work was supported in part by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Number 17K07352 to NS. Conflict of Interest None declared. References 1 Shen Y. , Tenney A.P. , Busch S.A. , Horn K.P. , Cuascut F.X. , Liu K. , He Z. , Silver J. , Flanagan J.G. ( 2009 ) PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration . Science 326 , 592 – 596 Google Scholar CrossRef Search ADS PubMed 2 Gardner R.T. , Habecker B.A. ( 2013 ) Infarct-derived chondroitin sulfate proteoglycans prevent sympathetic reinnervation after cardiac ischemia-reperfusion injury . J. Neurosci . 33 , 7175 – 7183 Google Scholar CrossRef Search ADS PubMed 3 Coles C.H. , Mitakidis N. , Zhang P. , Elegheert J. , Lu W. , Stoker A.W. , Nakagawa T. , Craig A.M. , Jones E.Y. , Aricescu A.R. ( 2014 ) Structural basis for extracellular cis and trans RPTPsigma signal competition in synaptogenesis . Nat. Commun . 5 , 5209 Google Scholar CrossRef Search ADS PubMed 4 Coles C.H. , Shen Y. , Tenney A.P. , Siebold C. , Sutton G.C. , Lu W. , Gallagher J.T. , Jones E.Y. , Flanagan J.G. , Aricescu A.R. ( 2011 ) Proteoglycan-specific molecular switch for RPTPsigma clustering and neuronal extension . Science 332 , 484 – 488 Google Scholar CrossRef Search ADS PubMed 5 Bradbury E.J. , Moon L.D. , Popat R.J. , King V.R. , Bennett G.S. , Patel P.N. , Fawcett J.W. , McMahon S.B. ( 2002 ) Chondroitinase ABC promotes functional recovery after spinal cord injury . Nature 416 , 636 – 640 Google Scholar CrossRef Search ADS PubMed 6 Bartus K. , James N.D. , Didangelos A. , Bosch K.D. , Verhaagen J. , Yanez-Munoz R.J. , Rogers J.H. , Schneider B.L. , Muir E.M. , Bradbury E.J. ( 2014 ) Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury . J. Neurosci . 34 , 4822 – 4836 Google Scholar CrossRef Search ADS PubMed 7 Takeuchi K. , Yoshioka N. , Higa Onaga S. , Watanabe Y. , Miyata S. , Wada Y. , Kudo C. , Okada M. , Ohko K. , Oda K. , Sato T. , Yokoyama M. , Matsushita N. , Nakamura M. , Okano H. , Sakimura K. , Kawano H. , Kitagawa H. , Igarashi M. ( 2013 ) Chondroitin sulphate N-acetylgalactosaminyl-transferase-1 inhibits recovery from neural injury . Nat. Commun . 4 , 2740 Google Scholar CrossRef Search ADS PubMed 8 Esko J.D. , Kimata K. , Lindahl U. ( 2009 ) Proteoglycans and Sulfated Glycosaminoglycans in Essentials of Glycobiology , 2nd ed ( Varki A. , Cummings R.D. , Esko J.D. , Freeze H.H. , Stanley P. , Bertozzi C.R. , Hart G.W. , Etzler M.E. , eds.) pp. 229 – 248 . Cold Spring Harbor , New York 9 Wang H. , Yan H. , Canoll P.D. , Silvennoinen O. , Schlessinger J. , Musacchio J.M. ( 1995 ) Expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma) in the nervous system of the developing and adult rat . J. Neurosci. Res. 41 , 297 – 310 Google Scholar CrossRef Search ADS PubMed 10 Sugiura N. , Shioiri T. , Chiba M. , Sato T. , Narimatsu H. , Kimata K. , Watanabe H. ( 2012 ) Construction of a chondroitin sulfate library with defined structures and analysis of molecular interactions . J. Biol. Chem. 287 , 43390 – 43400 Google Scholar CrossRef Search ADS PubMed 11 Ashikari-Hada S. , Habuchi H. , Kariya Y. , Itoh N. , Reddi A.H. , Kimata K. ( 2004 ) Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library . J. Biol. Chem . 279 , 12346 – 12354 Google Scholar CrossRef Search ADS PubMed 12 Vann W.F. , Schmidt M.A. , Jann B. , Jann K. ( 1981 ) The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli O10: K5: H4 . Eur. J. Biochem. 116 , 359 – 364 Google Scholar CrossRef Search ADS PubMed 13 Sugiura N. , Shimokata S. , Watanabe H. , Kimata K. ( 2007 ) MS analysis of chondroitin polymerization: effects of Mn2+ ions on the stability of UDP-sugars and chondroitin synthesis . Anal. Biochem . 365 , 62 – 73 Google Scholar CrossRef Search ADS PubMed 14 Ninomiya T. , Sugiura N. , Tawada A. , Sugimoto K. , Watanabe H. , Kimata K. ( 2002 ) Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4 . J. Biol. Chem. 277 , 21567 – 21575 Google Scholar CrossRef Search ADS PubMed 15 Sugiura N. , Shimokata S. , Minamisawa T. , Hirabayashi J. , Kimata K. , Watanabe H. ( 2008 ) Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants . Glycoconj. J. . 25 , 521 – 530 Google Scholar CrossRef Search ADS PubMed 16 Okuda T. , Mita S. , Yamauchi S. , Matsubara T. , Yagi F. , Yamamori D. , Fukuta M. , Kuroiwa A. , Matsuda Y. , Habuchi O. ( 2000 ) Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase . J. Biochem. (Tokyo) 128 , 763 – 770 Google Scholar CrossRef Search ADS 17 Fukuta M. , Kobayashi Y. , Uchimura K. , Kimata K. , Habuchi O. ( 1998 ) Molecular cloning and expression of human chondroitin 6-sulfotransferase . Biochim. Biophys. Acta 1399 , 57 – 61 Google Scholar CrossRef Search ADS PubMed 18 Ohtake S. , Ito Y. , Fukuta M. , Habuchi O. ( 2001 ) Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene . J. Biol. Chem. 276 , 43894 – 43900 Google Scholar CrossRef Search ADS PubMed 19 Kobayashi M. , Sugumaran G. , Liu J. , Shworak N.W. , Silbert J.E. , Rosenberg R.D. ( 1999 ) Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan/chondroitin sulfate . J. Biol. Chem. 274 , 10474 – 10480 Google Scholar CrossRef Search ADS PubMed 20 Kobayashi M. , Habuchi H. , Yoneda M. , Habuchi O. , Kimata K. ( 1997 ) Molecular cloning and expression of Chinese hamster overy cell heparan sulfate 2-sulfotransferase . J. Biol. Chem. 272 , 13980 – 13985 Google Scholar CrossRef Search ADS PubMed 21 Habuchi H. , Tanaka M. , Habuchi O. , Yoshida K. , Suzuki H. , Ban K. , Kimata K. ( 2000 ) The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine . J. Biol. Chem. 275 , 2859 – 2868 Google Scholar CrossRef Search ADS PubMed 22 Sugiura N. , Sakurai K. , Hori Y. , Karasawa K. , Suzuki S. , Kimata K. ( 1993 ) Preparation of lipid-derivatized glycosaminoglycans to probe a regulatory function of the carbohydrate moieties of proteoglycans in cell-matrix interaction . J. Biol. Chem . 268 , 15779 – 15787 Google Scholar PubMed 23 Sugiura N. , Kimata K. ( 1994 ) Syntheses and functions of neoproteoglycans: lipid-derivatized chondroitin sulfate with antiadhesion activity . Methods Enzymol . 247 , 362 – 373 Google Scholar CrossRef Search ADS PubMed 24 Nagasawa K. , Inoue Y. , Kamata T. ( 1977 ) Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol . Carbohydr. Res. 58 , 47 – 55 Google Scholar CrossRef Search ADS PubMed 25 Kariya Y. , Kyogashima M. , Suzuki K. , Isomura T. , Sakamoto T. , Horie K. , Ishihara M. , Takano R. , Kamei K. , Hara S. ( 2000 ) Preparation of completely 6-O-desulfated heparin and its ability to enhance activity of basic fibroblast growth factor . J. Biol. Chem. 275 , 25949 – 25958 Google Scholar CrossRef Search ADS PubMed 26 Uchiyama H. , Nagasawa K. ( 1991 ) Changes in the structure and biological property of N—O sulfate- transferred, N-resulfated heparin . J. Biol. Chem. 266 , 6756 – 6760 Google Scholar PubMed 27 Imagama S. , Sakamoto K. , Tauchi R. , Shinjo R. , Ohgomori T. , Ito Z. , Zhang H. , Nishida Y. , Asami N. , Takeshita S. , Sugiura N. , Watanabe H. , Yamashita T. , Ishiguro N. , Matsuyama Y. , Kadomatsu K. ( 2011 ) Keratan sulfate restricts neural plasticity after spinal cord injury . J. Neurosci . 31 , 17091 – 17102 Google Scholar CrossRef Search ADS PubMed 28 Sugiura N. , Clausen T.M. , Shioiri T. , Gustavsson T. , Watanabe H. , Salanti A. ( 2016 ) Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library . Glycoconj. J. 33 , 985 – 994 Google Scholar CrossRef Search ADS PubMed 29 Toyoda H. , Kinoshita-Toyoda A. , Selleck S.B. ( 2000 ) Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo . J. Biol. Chem. 275 , 2269 – 2275 Google Scholar CrossRef Search ADS PubMed 30 Oohira A. , Kushima Y. , Tokita Y. , Sugiura N. , Sakurai K. , Suzuki S. , Kimata K. ( 2000 ) Effects of lipid-derivatized glycosaminoglycans (GAGs), a novel probe for functional analyses of GAGs, on cell-to-substratum adhesion and neurite elongation in primary cultures of fetal rat hippocampal neurons . Arch. Biochem. Biophys . 378 , 78 – 83 Google Scholar CrossRef Search ADS PubMed 31 Shimbo M. , Ando S. , Sugiura N. , Kimata K. , Ichijo H. ( 2013 ) Moderate repulsive effects of E-unit-containing chondroitin sulfate (CSE) on behavior of retinal growth cones . Brain Res . 1491 , 34 – 43 Google Scholar CrossRef Search ADS PubMed 32 Ichijo H. , Sugiura N. , Kimata K. ( 2013 ) Application of chondroitin sulfate derivatives for understanding axonal guidance in the nervous system during development . Polymers 5 , 254 – 268 Google Scholar CrossRef Search ADS 33 Brown J.M. , Xia J. , Zhuang B. , Cho K.S. , Rogers C.J. , Gama C.I. , Rawat M. , Tully S.E. , Uetani N. , Mason D.E. , Tremblay M.L. , Peters E.C. , Habuchi O. , Chen D.F. , Hsieh-Wilson L.C. ( 2012 ) A sulfated carbohydrate epitope inhibits axon regeneration after injury. Proc. Natl. Acad. Sci. U.S.A . 109 , 4768 – 4773 Google Scholar CrossRef Search ADS 34 Dickendesher T.L. , Baldwin K.T. , Mironova Y.A. , Koriyama Y. , Raiker S.J. , Askew K.L. , Wood A. , Geoffroy C.G. , Zheng B. , Liepmann C.D. , Katagiri Y. , Benowitz L.I. , Geller H.M. , Giger R.J. ( 2012 ) NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans . Nat. Neurosci. 15 , 703 – 712 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations CGN cerebellar granule neuron CH chondroitin CS chondroitin sulphate DS dermatan sulphate GAG glycosaminoglycan GalNAc N-acetylgalactosamine GlcA glucuronic acid GlcNAc N-acetylglucosamine HMDA hexamethylenediamine HP heparin HPS N-acetylheparosan HS heparan sulphate IdoA iduronic acid MaxRU maximum response Mr average molecular mass RPTPσ receptor type of protein tyrosine phosphatase sigma SD sulphation degree SPR surface plasmon resonance © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of BiochemistryOxford University Press

Published: Feb 6, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off