Structural analysis of neutral glycosphingolipids from the silkworm Bombyx mori and the difference in ceramide composition between larvae and pupae

Structural analysis of neutral glycosphingolipids from the silkworm Bombyx mori and the... Abstract Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcβ1Cer, Manβ4Glcβ1Cer, GlcNAcβ3Manβ4Glcβ1Cer, Galβ3Manβ4Glcβ1Cer, GalNAcα4Galβ3Manβ4Glcβ1Cer, GlcNAcβ3Galβ3Manβ4Glcβ1Cer, Galα4Galβ3Manβ4Glcβ1Cer and (GalNAcα4)1–4 GalNAcα4Galβ3Manβ4Glcβ1Cer. Lin‐ear elongation of α4-GalNAc was observed at the non-reducing end of Galβ3Manβ4Glcβ1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcβ3Manβ4Glcβ1Cer, a characteristic GSL–glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis. development, fatty acid, glycolipid, mass spectrometry, sphingolipids Glycosphingolipids (GSLs) contribute to various biological processes and functions. They are biosynthesized by several glycosyltransferases in a systematic sequential process. The analysis of the correlation between the glycosyltransferase gene and GSL expression patterns is important to study the functions of GSLs. Chemical structural analysis and expression patterns of GSL are major determinants of the phenotype of an organism. Such analyses are aided by detailed knowledge of the genome, proteome and phenome of organisms (1) such as Drosophila melanogaster (2–4) and Caenorhabditis elegans (5–8). In Drosophila, the expression and biosynthesis of GSLs have been studied in well-known developmental pathways and the nervous system, revealing essential functions of GSLs (9). The silkworm Bombyx mori, which has been domesticated for silk production since ancient times, is a well-established lepidopteran animal model for which substantial research on its developmental biology, genetics, molecular biology and immunology has been conducted. The silkworm B. mori (order Lepidoptera, class Insecta, phylum Arthropoda) exhibits complete (holometabolous) metamorphosis in the stages of the ovum, larva (fifth instar), pupa (in cocoon) and imago. Various studies on innate immunity in invertebrates have utilized muscle contraction in silkworm larvae to demonstrate the activation of insect cytokines by humoral and cellular components (10). Several databases on silkworms provide genome-wide sequences, cDNAs and expressed sequence tags (11–14). Therefore, silkworms are a valuable model for comparing DNA sequences within the same genus (e.g. versus flies) or with other genera such as roundworms. In addition, an established transgenic silkworm strain has been developed for the in vivo evaluation of human protein–protein interactions (15). Furthermore, the body size of silkworms is an advantage in GSL research. Extracted GSLs cannot be amplified; therefore, experiments require many animal models to obtain sufficient amounts of GSLs for analyses. A fifth instar silkworm larva is approximately 5 cm in body length, 1 cm in diameter and over 4 g in weight. Therefore, silkworms are advantageous for conducting comparative studies of GSL expression, structure and function across different stages in Arthropod development. In this study, we described the common GSLs extracted from the larvae and pupae of silkworms and compared them with GSLs of the cell line High-Five™ established from ovarian cells of the cabbage lopper Trichoplusia ni (order Lepidoptera, class Insecta, phylum Arthropoda) (16), the fly Lucilia caesar (17), the fly Calliphora vicina (18) and the moth Manduca sexta (19), which have characteristic arthro-series sugar chains (At3Cer: GlcNAcβ3Manβ4Glcβ1Cer). The comparison of larval and pupal GSLs revealed developmental differences in ceramide compositions. Materials and Methods Bulk extraction and isolation of neutral GSLs from dried pupae for structural characterization Dried pupae (2.0 kg) without cocoons were purchased from a silk string manufacturing corporation (Usui-seishi, Annaka, Gunma, Japan). Whole lipids were extracted from a sample using a mixture of chloroform: methanol (2:1 and 1:1, v/v) and chloroform:methanol:water (1:1:0.2, v/v/v). The extracts were combined and dried, and the dried lipids were subjected to mild alkaline hydrolysis to prepare the sphingolipid fraction as previously reported (20). Alkaline-stable products were added to a diethylaminoethyl (DEAE)-Sephadex A-25 column (CH3COO− form; GE Healthcare UK Led., Buckinghamshire, UK) to remove acidic compounds. Neutral and polar compounds were eluted with a mixture of chloroform:methanol:water (30:60:8, v/v/v). The mixture was then subjected to QAE-Sephadex A-25 chromatography (OH− form; GE Healthcare Co.) for the removal of contaminant polar compounds. Neutral compounds were eluted with five column volumes of chloroform:methanol:water (30:60:8, v/v/v). The eluates were completely dried and acetylated by pyridine:acetic anhydride (3:2, v/v) for further purification of neutral GSLs by magnesium silicate column chromatography (Florisil, 60–100 mesh; Nakalai Tesque, Kyoto) (21). The columns were successively eluted with six column volumes of n-hexane: dichloroethane (1:4, v/v), 1 vol of pure dichloroethane, 3 vol of dichloroethane: acetone (1:1, v/v), 3 vol of dichloroethane:methanol (3:1, v/v), 3 vol of dichloroethane:methanol:water (2:8:1, v/v/v), and 3 vol of chloroform:methanol:water (6:4:1, v/v/v). The fractions of acetylated GSLs eluted by dichloroethane:acetone (1:1) and dichloroethane:methanol (3:1) were combined, evaporated to dryness and deacetylated with 0.5 M KOH in methanol at 37°C for 6 h. The reaction mixture was dialyzed against tap water overnight and evaporated to dryness (yield of 173 mg). The neutral GSL fraction (120 mg) was added to a column of porous silica gel (1.8 × 90 cm, Iatrobeads 6RS-8060; Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) pre-equilibrated with chloroform:methanol:water (80:20:1, v/v/v). This column was treated with two consecutive linear gradient systems of chloroform:methanol:water (80:20:1, v/v/v 665 ml–50:50:5, 755 ml and 50:50:5, 680 ml–20:80:10, 755 ml). The effluent was collected in 3-ml fractions, and an aliquot from each tube was assayed by thin-layer chromatography (TLC) with a solvent system of chloroform:methanol:water (60:40:10, v/v/v) and orcinol:H2SO4 reagent for detecting sugars. On the basis of the TLC appearance, neutral GSLs expressed in substantial quantities were fractionated into 16 fractions. Several fractions (CTeSs, CPSs, CHpSs and COSs) required further purification using a high-performance liquid chromatography (HPLC) system (Shimadzu LC-9A) with an Iatrobeads column (1.0 × 30 cm) and solvent systems of 1-propanol:water:28% ammonium hydroxide (75:8.5:5 v/v/v for CTSs, 75:12.5:5 v/v/v for CTeSs and CPSs, 75:18.5:5 v/v/v for CHpS and 75:17.5:5 v/v/v for COS) at a flow rate of 1 ml/min. Extraction and fractionation of neutral GSLs for the comparison of GSL expression patterns Third instar larvae were purchased from a silkworm breeding company (Kougensha Co. Ltd, Matsumoto, Nagano, Japan) and bred to fifth instar larvae and pupae. Starting materials used for were 429 larvae (total: 1825 g) and 200 pupae (total: 239 g). Whole lipids were extracted from lyophilized samples (431.9 g for larvae and 57.3 g for pupae). Neutral GSLs were isolated using a similar method utilized for the bulk extraction and isolation of neutral GSLs from dried pupae for structural characterization, except if starting from lyophilized material. The neutral GSL fractions (yield: 192 mg for larvae and 30 mg for pupae) were analysed by TLC as described below. Solvents for TLC analysis TLC was performed using silica gel 60 (Merck KGaA) and visualized by spraying the chromatogram with the orcinol–H2SO4 reagent followed by heating at 110°C. The following solvent systems were utilized for development: chloroform:methanol:water (60:40:10, v/v/v) and 1-propanol:water:28% ammonium hydroxide (75:25:5, v/v/v). Compositional sugar analysis by gas chromatography (GC) Compositional sugar analysis of each purified GSL was conducted following derivatization to trimethylsilyl (TMSi) methyl glycosides using GC (Shimadzu GC-18A). The GC system included a capillary column that was chemically bonded (Shimadzu HiCap-CBP 5, 0.22 mm × 25 m) with temperature programmed at 2°C/min from 140 to 230 °C. The ceramide compositions of GSLs were determined by analysing fatty acid methyl ester and TMSi-sphingoids (22) on the same GC capillary column programmed at 2 °C/min from 170 to 240 °C for fatty acids and at 2 °C/min from 200 to 240 °C for sphingoids (23). Sugar linkage analysis Samples (∼200 μg) of purified GSLs were permethylated with NaOH and CH3I in dimethyl sulfoxide (24). Partially methylated GSLs were hydrolyzed with a 0.3 ml mixture of HCl:water:acetic acid (8.0:0.5:1.5, v/v/v) at 80 °C for 18 h. Hydrolysates were dried under an N2 stream and were reduced with 1% NaBH4 in 0.01 M NaOH overnight. Reaction products were dried and then acetylated with a mixture of acetic anhydride:pyridine (1:1, v/v) at 100 °C for 10 min (25). Partially methylated alditol acetates (PMAAs) obtained were analysed by GC and GC-mass spectrometry (MS) using the same Shimadzu HiCap-CBP 5 capillary column as described above. Electron ionization mass spectra were acquired using a Shimadzu GCMS-QP 5050 gas chromatograph-mass spectrometer under the following conditions: oven temperature, 80 °C (2 min) to 180 °C (20 °C/min) to 240 °C (4 °C/min); interface temperature, 250 °C; injection port temperature, 240 °C; helium gas pressure, 100 kPa; ionizing voltage, 70 eV; and ionizing current, 60 μA. Peaks on gas chromatograms were identified by comparing them with retention times and physicochemical data from NIST/EPA/NIH Mass Spectral Library NIST 11 (Shimadzu, Tokyo, Japan). Proton nuclear magnetic resonance spectroscopy (1 H-NMR) 1 H NMR spectra of purified GSLs were obtained using a JEOL JNM-ECS-400 400 MHz 1 H NMR spectrometer at an operating temperature of 60 °C. GSL samples (1 mg) were dissolved in 0.5 ml of dimethyl sulfoxide-d6 containing 2% D2O. Chemical shifts were referenced to solvent signals (δH 2.49 ppm) in d6-demethylsulfoxide. Matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS) MALDI-TOF MS analysis of purified GSLs was performed using a Shimadzu AXIMA Confidence MALDI-TOF mass spectrometer that was operating in the positive-ion reflectron mode for full MS analysis and post-source decay (PSD) mode for MS/MS analysis. Data were acquired in both modes using a UV nitrogen laser (337 nm) and in the automatic measurement mode at an acceleration voltage of 20 kV. Positive-ion mass spectra were acquired with 100 profiles/run at ∼80 power for the reflectron mode and with 100 shots/run at ∼90 power in a curved field reflectron in the PSD mode. Precursor ions were selected using an ion gate width of ∼3 m/z units in the PSD mode. Purified GSL (4–10 μg) dissolved in chloroform:methanol (2:1, v/v) solution was spotted on a sample plate and air dried. Totally, 0.5 μl of matrix solution was spotted on sample wells followed by air drying. The matrix solution used was α-cyano-4-hydroxycinnamic acid (Shimadzu GLC, Kyoto, Japan) at a saturated concentration [5 mg in 1 mL of ethanol:water (1:1, v/v)]. External mass calibration was provided by the [M + H]+ ions of bradykinin fragment 1–7 (757.40 Da; Sigma Chemical Co.) and ACTH fragment 18–39 (2465.20 Da; Sigma Chemical Co). Results Three starting materials were used: dried pupae, which were purchased from a silk string manufacturing corporation, and larvae and pupae, which were bred in our laboratory. For the structural characterization of GSLs, besides the yield of GSL, dried pupae were used as they can be easily obtained in bulk amounts for lipid extraction. (Bred) larvae and (bred) pupae were used for the comparison of GSL expression patterns. Purification and fractionation of neutral GSLs from dried pupae Neutral GSLs from dried pupae were separated into 16 fractions by silica gel chromatography using a gradient solvent system of chloroform:methanol:water (Fig. 1A). Several fractions of neutral GSLs were further separated by TLC into two or three GSL species using a propanol:water:ammonium hydroxide solvent system (Fig. 1B). After purification and separation (Supplementary Fig. S1), fraction (Fr.) 3 (Fr. 3, lane 3 in Fig. 1) yielded ceramide trisaccharide (CTS) 1 (CTS1, Fr. 3-1) and CTS2 (Fr. 3-5); Fr. 6 (lane 6) yielded ceramide tetrasaccharide 1 (CTeS1, Fr. 6-3), CTeS2 (Fr. 6-5) and CTeS3 (Fr. 6-7); Fr. 7 (lane 7) yielded ceramide pentasaccharide (CPS, Fr. 7-4); Fr. 11 (lane 11) yielded ceramide heptasaccharide (CHpS, Fr. 11-2-3); and Fr. 13 (lane 13) yielded ceramide octasaccharide (COS, Fr. 13-4). Ceramide mono- (CMS), di- (CDS) and hexasaccharide (CHS) were obtained by first-step silica gel chromatography. In addition, fractions containing minor components were observed by TLC; however, the yields were insufficient for chemical structural analysis. Fig. 1 View largeDownload slide Thin-layer chromatograms of neutral GSL fractions from the silkworm B. mori. Lane T, neutral GSL fraction obtained by Florisil column chromatography through acetylation and deacetylation; lanes 1–16, neutral GSL fractions separated by Iatrobeads column chromatography using a gradient elution system with chloroform:methanol:water. The plate in A was developed with chloroform:methanol:water (60:40:10, v/v/v) and that in B by propanol:water:28% ammonium hydroxide (75:25:5, v/v/v). The spots were visualized by the orcinol–H2SO4 reagent. Fig. 1 View largeDownload slide Thin-layer chromatograms of neutral GSL fractions from the silkworm B. mori. Lane T, neutral GSL fraction obtained by Florisil column chromatography through acetylation and deacetylation; lanes 1–16, neutral GSL fractions separated by Iatrobeads column chromatography using a gradient elution system with chloroform:methanol:water. The plate in A was developed with chloroform:methanol:water (60:40:10, v/v/v) and that in B by propanol:water:28% ammonium hydroxide (75:25:5, v/v/v). The spots were visualized by the orcinol–H2SO4 reagent. Characterization of the ceramide tetrasaccharides CTeS1, CTeS2 and CTeS3 from dried pupae GSLs containing HexNAc (CTeS1 and CTeS2) were eluted faster than constituent Hex (CTeS3) by Iatrobeads column chromatography using the propanol:water:ammonium hydroxide solvent system. The GSL containing GlcNAc (CTeS1) eluted faster than the GSL containing GalNAc (CTeS2). Purified CTeS2 (2.1 mg) was the major CTeS component, whereas CTeS1 (1.0 mg) and CTeS3 (0.9 mg) were minor components of the 120 mg dried neutral GSL mixture in pupae; however, crude side-fractions also contained these GSLs. Sugar analyses indicated that all three CTeSs contained Man, Gal and Glc as common components, whereas CTeS1 contained only GlcNAc and CTeS2 contained only GalNAc. In methylation analysis (Table I and Supplementary Fig. S2), common PMAA derivatives corresponding to 3-linked Man (1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannnitol, 3-Man) and 4-linked Glc (1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgluctitol, 4-Glc) were detected in all three CTeSs as indicated by a 2-fold higher single peak using a low polar capillary column (Supplementary Fig. S2). PMAAs from each monosaccharide at the non-reducing end and the penultimate Gal differed among CTeSs, with terminal-GlcNAc (1,5-di-O-acetyl-3,4,6-tri-O-methyl-N-acetylglucosaminitol, t-GlcNAc) and 3-linked Gal (1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol, 3-Gal) in CTeS1, terminal-GalNAc (1,5-di-O-acetyl-3,4,6-tri-O-methyl-N-acetylgalactosaminitol, t-GalNAc) and 4-linked Gal (1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol, 4-Gal) in CTeS2, and terminal-Gal (1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol, t-Gal) and 4-Gal in CTeS3. Table I. PMAA analysis of neutral glycosphingolipids (GSLs) from the silkworm B. mori CMS  t-Glc                    CDS    t-Man    4-Glc              CTS1            3-Man + 4-Glc    t-GlcNAc      CTS2      t-Gal      3-Man + 4-Glc          CTeS1            3-Man + 4-Glc  3-Gal  t-GlcNAc      CTeS2          4-Gal  3-Man + 4-Glc      t-GalNAc    CTeS3      t-Gal    4-Gal  3-Man + 4-Glc          CPS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHpS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  COS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CMS  t-Glc                    CDS    t-Man    4-Glc              CTS1            3-Man + 4-Glc    t-GlcNAc      CTS2      t-Gal      3-Man + 4-Glc          CTeS1            3-Man + 4-Glc  3-Gal  t-GlcNAc      CTeS2          4-Gal  3-Man + 4-Glc      t-GalNAc    CTeS3      t-Gal    4-Gal  3-Man + 4-Glc          CPS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHpS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  COS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  Results summarized as detectable peaks according to the order of retention using a HiCap-CBP 5 capillary column for GC (Supplementary Fig. S2). The abbreviations indicate the following: t-Glc; terminal Glc, t-Man; terminal Man, 4-Glc; 4-linked Glc, 3-Man; 3-linked Man, t-GlcNAc; terminal GlcNAc, t-Gal; terminal Gal, 4-Gal; 4-linked Gal, and 4-GalNAc; 4-linked GalNAc. PMAA of 3-Man and 4-Glc could not be separated by this column. The 1 H NMR spectra of the three CTeSs (Fig. 2) exhibited three typical major H-1 resonances with chemical shifts and J1,2 coupling constants for β-Glc (4.19 ppm; J1,2 = 7.3–7.8 Hz), β-Gal (4.31 or 4.32 ppm; J1,2 = 6.9–7.8 Hz) and β-Man (4.53 or 4.55 ppm; J1,2 = ∼1 Hz), whereas the terminal sugar residue exhibited distinct shifts and J1,2 coupling constants as follows (Table II): β-GlcNAc (4.64 ppm; J1,2 = 7.8 Hz) for CTeS1, α-GalNAc (4.84 ppm; J1,2 = 3.7 Hz) for CTeS2, and α-Gal (4.82 ppm; J1,2 = 3.7 Hz) for CTeS3. Table II. Chemical shifts and J1,2 coupling constants of protons in neutral GSLs at 60 °C CMS            Glc1-  Chemical shifts (ppm)            4.14  Coupling constants (Hz)            7.8  Chemical shifts (ppm)            4.13  Coupling constants (Hz)            7.8  CDS          Man1-  4Glc1-  Chemical shifts (ppm)          4.50  4.18  Coupling constants (Hz)          a  8.2  Chemical shifts (ppm)            4.15  Coupling constants (Hz)            7.8  CTS1        GlcNAc1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.54  4.52  4.16  Coupling constants (Hz)        7.3  a  7.8  CTS2        Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.26  4.54  4.19  Coupling constants (Hz)        7.3  a  7.8  CTeS1      GlcNAc1-  3Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.64  4.31  4.54  4.19  Coupling constants (Hz)      7.8  7.8  a  7.8  CTeS2      GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.84  4.32  4.55  4.19  Coupling constants (Hz)      3.7  6.9  a  7.8  CTeS3      Gal1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.82  4.31  4.53  4.19  Coupling constants (Hz)      3.7  7.3  a  7.3  CPS    GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)    4.87  4.90  4.32  4.55  4.19  Coupling constants (Hz)    3.7  3.7  7.3  a  7.8  CHS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.89  4.93  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  3.7  6.9  a  7.8  CHpS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.7  3.7  b  6.4  a  7.8  COS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  b  6.9  a  8.2  CMS            Glc1-  Chemical shifts (ppm)            4.14  Coupling constants (Hz)            7.8  Chemical shifts (ppm)            4.13  Coupling constants (Hz)            7.8  CDS          Man1-  4Glc1-  Chemical shifts (ppm)          4.50  4.18  Coupling constants (Hz)          a  8.2  Chemical shifts (ppm)            4.15  Coupling constants (Hz)            7.8  CTS1        GlcNAc1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.54  4.52  4.16  Coupling constants (Hz)        7.3  a  7.8  CTS2        Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.26  4.54  4.19  Coupling constants (Hz)        7.3  a  7.8  CTeS1      GlcNAc1-  3Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.64  4.31  4.54  4.19  Coupling constants (Hz)      7.8  7.8  a  7.8  CTeS2      GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.84  4.32  4.55  4.19  Coupling constants (Hz)      3.7  6.9  a  7.8  CTeS3      Gal1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.82  4.31  4.53  4.19  Coupling constants (Hz)      3.7  7.3  a  7.3  CPS    GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)    4.87  4.90  4.32  4.55  4.19  Coupling constants (Hz)    3.7  3.7  7.3  a  7.8  CHS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.89  4.93  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  3.7  6.9  a  7.8  CHpS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.7  3.7  b  6.4  a  7.8  COS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  b  6.9  a  8.2  aSinglet. bBroad singlet. Fig. 2 View largeDownload slide Anomeric proton regions of 1 H NMR spectra for neutral GSLs. Spectra obtained in DMSO-d6 containing 2% D2O at an operating temperature of 60 °C. Chemical shifts and J1,2 coupling constants are summarized in Table II. Sph-4 and -5 indicate vinyl H-4 and H-5 multiplets, respectively, from the (E)-4-sphinganine base. Fig. 2 View largeDownload slide Anomeric proton regions of 1 H NMR spectra for neutral GSLs. Spectra obtained in DMSO-d6 containing 2% D2O at an operating temperature of 60 °C. Chemical shifts and J1,2 coupling constants are summarized in Table II. Sph-4 and -5 indicate vinyl H-4 and H-5 multiplets, respectively, from the (E)-4-sphinganine base. Gas chromatography and GC-MS revealed that the ceramide constituents (Table III) of the three CTeSs were composed of tetradecasphingenine (14:1) as the major sphingoid and 2-hydroxy C20:0 and C22:0 acids (h20:0 and h22:0) as the major fatty acids. Furthermore, from the 1 H NMR spectra, characteristic coupled vinyl H-4 and H-5 multiplets at ∼5.38 and 5.58 ppm, respectively, indicated that these ceramides contained an (E)-4-sphingenine base. Table III. Ceramide composition of neutral GSLs Fatty acid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS  C14:0  –  3.1  tr.  3.3  1.1  tr.  –  1.8  –  tr.  0.7  C15:0  –  –  tr.  1.7  –  –  –  tr.  –  –  –  C16:0  tr.  3.5  3.6  4.5  2.9  2.5  tr.  5.3  tr.  1.3  3.1  C17:0  –  –  2.1  3.0  –  –  –  tr.  –  –  –  C18:0  1.9  6.8  6.2  8.0  2.4  1.8  1.6  1.6  2.5  2.0  4.0  C19:0  tr.  tr.  tr.  1.4  –  –  –  tr.  1.1  tr.  1.8  C20:0  12.1  22.3  33.9  4.0  2.8  1.3  1.2  1.9  7.3  7.9  11.6  C21:0  tr.  tr.  1.0  tr.  –  –  –  tr.  1.4  tr.  1.6  C22:0  15.0  26.0  46.3  4.6  4.0  tr.  1.0  3.1  13.6  12.2  10.7  C23:0  1.0  tr.  1.3  tr.  –  –  –  tr.  1.4  tr.  1.8  C24:0  3.6  1.6  3.3  tr.  –  –  7.4  –  –  1.2  1.9  h16:0  tr.  2.6  –  –  –  –  –  –  –  –  –  h18:0  2.1  3.0  –  –  –  1.6  1.4  –  –  1.1  1.3  h19:0  1.0  tr.  –  –  –  1.4  1.4  –  –  1.3  2.9  h20:0  24.3  12.2  1.0  16.9  17.3  26.2  28.5  20.9  19.7  23.3  20.6  h21:0  2.6  tr.  –  3.2  3.6  4.4  4.6  3.9  –  3.7  4.0  h22:0  30.5  17.3  1.3  38.0  48.2  49.5  48.0  47.7  41.8  37.8  28.0  h23:0  1.7  tr.  –  6.3  9.3  6.7  –  7.8  6.5  4.9  3.6  h24:0  4.2  1.6  –  5.1  8.4  4.6  4.9  6.0  4.7  3.3  2.4  Total  100  100  100  100  100  100  100  100  100  100  100    Sphingoid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS    d14:1  59.0  76.4  72.9  61.9  60.2  75.1  81.2  68.1  64.5  66.5  66.7  d14:0  16.4  6.8  5.6  9.3  14.5  11.2  10.1  12.1  11.7  13.9  3.7  d15:1  –  –  2.7  3.1  –  –  –  –  3.6  –  –  d15:0  –  –  –  2.0  –  –  –  –  –  –  –  d16:1  12.4  11.2  11.1  9.5  11.2  7.8  5.5  10.5  11.1  9.8  7.4  d16:0  12.2  5.6  7.7  14.2  14.1  5.9  3.2  9.3  9.1  9.8  22.2  Total  100  100  100  100  100  100  100  100  100  100  100  Fatty acid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS  C14:0  –  3.1  tr.  3.3  1.1  tr.  –  1.8  –  tr.  0.7  C15:0  –  –  tr.  1.7  –  –  –  tr.  –  –  –  C16:0  tr.  3.5  3.6  4.5  2.9  2.5  tr.  5.3  tr.  1.3  3.1  C17:0  –  –  2.1  3.0  –  –  –  tr.  –  –  –  C18:0  1.9  6.8  6.2  8.0  2.4  1.8  1.6  1.6  2.5  2.0  4.0  C19:0  tr.  tr.  tr.  1.4  –  –  –  tr.  1.1  tr.  1.8  C20:0  12.1  22.3  33.9  4.0  2.8  1.3  1.2  1.9  7.3  7.9  11.6  C21:0  tr.  tr.  1.0  tr.  –  –  –  tr.  1.4  tr.  1.6  C22:0  15.0  26.0  46.3  4.6  4.0  tr.  1.0  3.1  13.6  12.2  10.7  C23:0  1.0  tr.  1.3  tr.  –  –  –  tr.  1.4  tr.  1.8  C24:0  3.6  1.6  3.3  tr.  –  –  7.4  –  –  1.2  1.9  h16:0  tr.  2.6  –  –  –  –  –  –  –  –  –  h18:0  2.1  3.0  –  –  –  1.6  1.4  –  –  1.1  1.3  h19:0  1.0  tr.  –  –  –  1.4  1.4  –  –  1.3  2.9  h20:0  24.3  12.2  1.0  16.9  17.3  26.2  28.5  20.9  19.7  23.3  20.6  h21:0  2.6  tr.  –  3.2  3.6  4.4  4.6  3.9  –  3.7  4.0  h22:0  30.5  17.3  1.3  38.0  48.2  49.5  48.0  47.7  41.8  37.8  28.0  h23:0  1.7  tr.  –  6.3  9.3  6.7  –  7.8  6.5  4.9  3.6  h24:0  4.2  1.6  –  5.1  8.4  4.6  4.9  6.0  4.7  3.3  2.4  Total  100  100  100  100  100  100  100  100  100  100  100    Sphingoid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS    d14:1  59.0  76.4  72.9  61.9  60.2  75.1  81.2  68.1  64.5  66.5  66.7  d14:0  16.4  6.8  5.6  9.3  14.5  11.2  10.1  12.1  11.7  13.9  3.7  d15:1  –  –  2.7  3.1  –  –  –  –  3.6  –  –  d15:0  –  –  –  2.0  –  –  –  –  –  –  –  d16:1  12.4  11.2  11.1  9.5  11.2  7.8  5.5  10.5  11.1  9.8  7.4  d16:0  12.2  5.6  7.7  14.2  14.1  5.9  3.2  9.3  9.1  9.8  22.2  Total  100  100  100  100  100  100  100  100  100  100  100  Results were calculated from the peak area of GC as detected by FID. h, 2-hydroxy fatty acid; d, dihydroxysphingoid; tr., trace; -, not detected. The putative structures of the three CTeSs were confirmed by the positive-ion reflectron mode of MALDI-TOF MS analysis, as shown in Fig. 3 and the detected peaks listed in Table IV. Two major peaks differing by 28 U were observed in all three CTeS spectra, with shifts reflecting the presence of distinct ceramide species consisting of d14:1 sphingoid and h20:0 and h22:0 fatty acids. The mass spectra of CTeS1 and CTeS2 exhibited sodium adduct ion species, [M + Na]+, at m/z 1265.2 and 1293.2, coinciding with HexNAc1Hex3Cer containing mainly h20:0-d14:1 and h22:0-d14:1 ceramides. The [M + Na]+ ions of CTeS3 at m/z 1224.2 and 1252.2 coincided with the mass values of Hex4Cer containing mainly h20:0-d14:1 and h22:0-d14:1 ceramides. The [M + Na]+ ions of CTeS1 at m/z 1309.2 and of CTeS3 at m/z 1268.2 coincided with a d14:0-h23:0 ceramide (peak f in Fig. 3). Table IV. Summary of MALDI-TOF MS analysis of major positive ions from neutral GSLs Ceramide    CMS  CDS      Theoretical  Observed  Theoretical  Observed      C20:0-d14:1    722.56  722.8  884.61  884.9      C21:0-d14:1    736.57  736.8  898.62        C22:0-d14:1  C20:0-d16:1  750.59  750.8  912.64  912.9      C22:0-d14:0  C20:0-d16:0  752.60  752.8  914.65        C23:0-d14:1  C21:0-d16:1  764.60  764.8  926.65        h20:0-d14:1    738.55  738.8  900.60  900.9      h21:0-d14:0    754.58  754.7  916.63        h22:0-d14:1  h20:0-d16:1  766.58  766.8  928.63  928.9      h23:0-d14:0  h21:0-d16:0  782.61  782.8  944.67              CTS1  CTS2        Ceramide  Theoretical  Observed  Theoretical  Observed        C20:0-d14:1    1087.69  a:1087.9  1046.66        C22:0-d14:1  C20:0-d16:1  1115.72  b:1115.9  1074.69        h20:0-d14:1    1103.68    1062.66  1062.9      h22:0-d14:1  h20:0-d16:1  1131.71    1090.69  1090.9            CTeS1  CTeS2  CTeS3    Ceramide  Theoretical  Observed  Theoretical  Observed  Theoretical  Observed    h20:0-d14:1    1265.73  c:1265.2  1265.73  c:1265.2  1224.71  c:1224.2  h21:0-d14:0    1281.77  d:1281.2  1281.77  1281.2  1240.74  d:1240.2  h22:0-d14:1  h20:0-d16:1  1293.77  e:1293.2  1293.77  e:1293.2  1252.74  e:1252.2  h23:0-d14:0  h21:0-d16:0  1309.80  f:1309.2  1309.80  1309.2  1268.77  f:1268.2        CPS  CHS        Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1468.81  c:1468.3  1671.89  c:1671.4      h21:0-d14:0    1484.85  d:1484.3  1687.93  d:1687.4      h22:0-d14:1  h20:0-d16:1  1496.85  e:1496.3  1699.93  e:1699.5      h23:0-d14:0  h21:0-d16:0  1512.88  f:1512.3  1715.96  f:1715.5        h22:0-d16:0  1526.89  g:1526.3  1729.97  g:1729.5            CHpS  COS      Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1874.97  c:1874.6  2078.05  c:2078.4      h21:0-d14:0    1891.00  d:1890.7  2094.08        h22:0-d14:1  h20:0-d16:1  1903.00  e:1902.6  2106.08  e:2106.4      h23:0-d14:0  h21:0-d16:0  1919.04  f:1918.7  2122.12          h22:0-d16:0  1933.05  g:1932.7  2136.13        Ceramide    CMS  CDS      Theoretical  Observed  Theoretical  Observed      C20:0-d14:1    722.56  722.8  884.61  884.9      C21:0-d14:1    736.57  736.8  898.62        C22:0-d14:1  C20:0-d16:1  750.59  750.8  912.64  912.9      C22:0-d14:0  C20:0-d16:0  752.60  752.8  914.65        C23:0-d14:1  C21:0-d16:1  764.60  764.8  926.65        h20:0-d14:1    738.55  738.8  900.60  900.9      h21:0-d14:0    754.58  754.7  916.63        h22:0-d14:1  h20:0-d16:1  766.58  766.8  928.63  928.9      h23:0-d14:0  h21:0-d16:0  782.61  782.8  944.67              CTS1  CTS2        Ceramide  Theoretical  Observed  Theoretical  Observed        C20:0-d14:1    1087.69  a:1087.9  1046.66        C22:0-d14:1  C20:0-d16:1  1115.72  b:1115.9  1074.69        h20:0-d14:1    1103.68    1062.66  1062.9      h22:0-d14:1  h20:0-d16:1  1131.71    1090.69  1090.9            CTeS1  CTeS2  CTeS3    Ceramide  Theoretical  Observed  Theoretical  Observed  Theoretical  Observed    h20:0-d14:1    1265.73  c:1265.2  1265.73  c:1265.2  1224.71  c:1224.2  h21:0-d14:0    1281.77  d:1281.2  1281.77  1281.2  1240.74  d:1240.2  h22:0-d14:1  h20:0-d16:1  1293.77  e:1293.2  1293.77  e:1293.2  1252.74  e:1252.2  h23:0-d14:0  h21:0-d16:0  1309.80  f:1309.2  1309.80  1309.2  1268.77  f:1268.2        CPS  CHS        Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1468.81  c:1468.3  1671.89  c:1671.4      h21:0-d14:0    1484.85  d:1484.3  1687.93  d:1687.4      h22:0-d14:1  h20:0-d16:1  1496.85  e:1496.3  1699.93  e:1699.5      h23:0-d14:0  h21:0-d16:0  1512.88  f:1512.3  1715.96  f:1715.5        h22:0-d16:0  1526.89  g:1526.3  1729.97  g:1729.5            CHpS  COS      Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1874.97  c:1874.6  2078.05  c:2078.4      h21:0-d14:0    1891.00  d:1890.7  2094.08        h22:0-d14:1  h20:0-d16:1  1903.00  e:1902.6  2106.08  e:2106.4      h23:0-d14:0  h21:0-d16:0  1919.04  f:1918.7  2122.12          h22:0-d16:0  1933.05  g:1932.7  2136.13        [M + Na]+ ions were calculated using the monoisotropic mass. Fig. 3 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSLs. Multiple [M + Na]+ peaks indicate different ceramide moieties. Peaks a–g correspond to those in Table IV. Symbol nomenclature was described according to the format of Consortium for Functional Glycomics. Fig. 3 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSLs. Multiple [M + Na]+ peaks indicate different ceramide moieties. Peaks a–g correspond to those in Table IV. Symbol nomenclature was described according to the format of Consortium for Functional Glycomics. The sugar sequences of each CTeS were determined in the PSD mode of MALDI-TOF MS (Fig. 4). Fragment ions were sequentially observed from the predominant [M + Na]+ ion to the ceramide monosaccharide sodium adduct ion species. In the PSD mass spectra of CTeS1 and CTeS2, sequential ions differing by m/z 203, 162 and 162 were observed, which was consistent with a linear HexNAc–Hex–Hex–Hex–Cer sequence (precursor ion m/z 1293.3 and fragment ion m/z 1090.4, 928.3 and 766.2). In the PSD mass spectrum of CTeS3, sequential ions differing by m/z 162 were observed, which was consistent with a linear Hex–Hex–Hex–Hex–Cer sequence (precursor ion m/z 1252.3 and fragment ion m/z 1090.4, 928.5 and 766.3). Fig. 4 View largeDownload slide Positive-ion PSD mode MALDI-TOF MS spectra of neutral GSLs. For the precursor masses of CTeS1 (1293.3), CTeS3 (1252.3) and COS (2105.7), ion gate masses were set to 1292.7–1295.7, 1251.5–1255.2 and 2103.9–2107.0, respectively. Symbol nomenclature was the same as that in Fig. 3. Fig. 4 View largeDownload slide Positive-ion PSD mode MALDI-TOF MS spectra of neutral GSLs. For the precursor masses of CTeS1 (1293.3), CTeS3 (1252.3) and COS (2105.7), ion gate masses were set to 1292.7–1295.7, 1251.5–1255.2 and 2103.9–2107.0, respectively. Symbol nomenclature was the same as that in Fig. 3. Characterization of ceramide penta-, hexa-, hepta- and octasaccharides from dried pupae GSLs containing major long sugar chains were separated by Iatrobeads column chromatography utilizing the chloroform:methanol:water solvent system based on their sugar components. Some of these fractions were contaminated with slower migrating minor components as revealed by TLC using the propanol:water:ammonium hydroxide solvent system. Purified CPS (4.3 mg), CHS (4.2 mg), CHpS (5.2 mg) and COS (9.5 mg) were the major components of the neutral GSL fraction (120 mg total) from dried pupae. Similar to CTeS1, these four GSLs contained Man, Gal, Glc and GalNAc as common sugar components (Table I and Supplementary Fig. S2). An increased GalNAc content indicated GalNAc elongation of CTeS1. In methylation analysis, common PMAA derivatives corresponding to 3-Man + 4-Glc (single peak), 4-Gal, t-GalNAc and 4-linked GalNAc (1,4,5-tri-O-acetyl-3,6-di-O-methyl-N-acetylgalactosaminitol, 4-GalNAc) were detected in all four long sugar chain GSLs. The ratios of t-GalNAc to 4-GalNAc intensity were 1:0.7 for CPS, 1:1.2 for CHS, 1:1.8 for CHpS and 1:2.8 for COS, which was consistent with the addition of 4-GalNAc units started at the non-reducing end of CTeS1. The increase in GalNAc residues was confirmed by sugar composition analysis using GC (Supplementary Fig. S2). The 1 H NMR spectra of these four long sugar chain-containing GSLs (Fig. 2) exhibited three major H-1 resonances with chemical shifts and J1,2 coupling constants for β-Glc (4.19 ppm; J1,2 = 7.8 or 8.2 Hz), β-Gal (4.32 ppm; J1,2 = 6.4–7.3 Hz) and β-Man (4.55 ppm; J1,2 = ∼1 Hz). Chemical shifts and J1,2 coupling constants for α-GalNAc were 4.86 or 4.87 ppm and J1,2 = 3.2 or 3.7 Hz for the terminal sugar, 4.89 or 4.90 ppm and J1,2 = 3.2 or 3.7 Hz for the second sugar from the terminal, and 4.93 or 4.94 ppm and J1,2 = 3.7 Hz or not determined for the internal sugar (Table II). The ratio of integral intensity for terminal and second α-GalNAcs in CHS was 1.00:1.00, and those of integral intensity for the terminal, secondary, and internal α-GalNAc moieties of the longer forms were 1.00:1.11:0.81 for CHS, 1.02:1.00:1.77 for CHpS and 1.01:1.00:2.64 for COS. The integral intensities were the sums of the individual units, for example 5× α-GalNAcα for COS. The presence of characteristic coupled vinyl H-4 and H-5 multiplets indicates that these GSLs possess an (E)-4-sphingenine base as the common ceramide composition. The ceramide constituent (Table III) of four of these GSLs contained d14:1 as the major sphingoid (∼70% of the total content) and h20:0 and h22:0 acids as the major fatty acids (∼60% of the total content) according to GC. The putative structures of these four GSLs were confirmed by the positive-ion reflectron mode of MALDI-TOF MS analysis as shown in Fig. 3 and the observed masses listed in Table IV. Two major peaks differing by 28 U were observed in all four GSL spectra, reflecting the presence of the same ceramide species as in shorter GSLs. The mass spectra of the four longer sugar chain GSLs had sodium adduct ion species, [M + Na]+, detected at m/z 1468.3 and 1496.3 for CPS, m/z 1671.4 and 1699.5 for CHS, m/z 1874.6 and 1902.6 for CHpS, and m/z 2078.4 and 2106.4 for COS, coinciding with HexNAc elongation starting from HexNAc1Hex3Cer (CTeS1) and mainly h20:0-d14:1 or h22:0-d14:1 ceramides. In the PSD mass spectra of COS (Fig. 4), fragment ions included all shorter sugar chain GSLs, each differing by m/z 203 and 162, which is consistent with a linear HexNAc–HexNAc–HexNAc–HexNAc–HexNAc–Hex–Hex–Hex–Cer sequence (precursor ion m/z 2105.7 and fragment ion m/z 1901.8, 1699.6, 1496.1, 1293.1 and 1090.1). Confirmation of arthro-series ceramide trisaccharide CTS1 in dried pupae The CTS fraction was separated into CTS1 (0.7 mg) and CTS2 (2.3 mg) by Iatrobeads column chromatography using the propanol:water:ammonium hydroxide solvent system. Fraction CTS1 containing GlcNAc eluted faster than CTS2. The sugar components of these CTSs, as determined by GC, GC-MS and 1 H NMR, were as follows: GlcNAcβ3Manβ4Glcβ1- (arthro-triaosyl) for CTS1 and Galβ3Manβ4Glcβ1- for CTS2 (Fig. 2 and Table II). GC indicated that the ceramide constituents of CTS1 were d14:1 as the major sphingoid (∼70% of the total content) and normal C20:0 and C22:0 acids as the major fatty acids (∼80% of the total content), whereas the ceramide constituents of CTS2 mainly consisted of d14:1 and 2-hydroxy C20:0 and C22:0 fatty acids. Comparison of GSL expression patterns between larvae and pupae GSL fractions from whole tissues were separated by column chromatography with ion-exchange Sephadex (DEAE- and QAE-Sephadex) and magnesium silicate (Florisil). A comparison revealed differing acidic (DEAE resin adsorbed), polar (QAE resin adsorbed) and neutral fractions between larvae and pupae, whereas the zwitterionic fraction was similar according to qualitative TLC results (Fig. 5). In the neutral GSL fraction, CDS, CTS and CTeS levels were lower in larvae than in pupae; however, similar amounts of the sample were added on the TLC plate, while CMS, CPS, CHS, CHpS and COS levels in larvae were similar to those in pupae. Fig. 5 View largeDownload slide Thin-layer chromatogram of GSL fractions from silkworm larvae and pupae. Lane S, neutral GSL fraction from the green bottle fly; lanes 1 and 2, sphingolipid fractions (150 and 300 μg added for TLC, respectively); lanes 3 and 4, acidic sphingolipid fractions (24 and 120 μg); lanes 5 and 6, polar sphingolipid fractions (75 and 210 μg); lanes 7 and 8, neutral sphingolipid fractions (12 and 15 μg); lanes 9 and 10, zwitterionic sphingolipid fractions (30 and 150 μg). Odd number lanes, lipid fractions from larvae; even number lanes, those from pupae. The plate was developed with chloroform:methanol:water (60:40:10, v/v/v) and the spots were visualized by the orcinol:H2SO4 reagent. Fig. 5 View largeDownload slide Thin-layer chromatogram of GSL fractions from silkworm larvae and pupae. Lane S, neutral GSL fraction from the green bottle fly; lanes 1 and 2, sphingolipid fractions (150 and 300 μg added for TLC, respectively); lanes 3 and 4, acidic sphingolipid fractions (24 and 120 μg); lanes 5 and 6, polar sphingolipid fractions (75 and 210 μg); lanes 7 and 8, neutral sphingolipid fractions (12 and 15 μg); lanes 9 and 10, zwitterionic sphingolipid fractions (30 and 150 μg). Odd number lanes, lipid fractions from larvae; even number lanes, those from pupae. The plate was developed with chloroform:methanol:water (60:40:10, v/v/v) and the spots were visualized by the orcinol:H2SO4 reagent. Comparison of ceramide composition in larval and pupal GSLs It is difficult to directly compare the amount of neutral GSLs in different preparations by the peak abundance in MALDI-TOF MS (Fig. 6). Differences in the ceramide composition of GSLs are reflected by numerous separated peaks, but the total peak abundance cannot be compared among preparations (such as those from larvae and pupae). However, the relative ratios of GSL species with different ceramide compositions were comparable within a given GSL fraction by peak abundance. In this experiment, the only exception was observed when contaminants disturbed the detection of the CDS molecular weight area at m/z 850–950. In the dried pupae used for structural analysis, the ceramide species in GSLs were predominantly c: h20:0-d14:1 (∼55%) and e: h22:0-d14:1 (∼45%). In bred pupae, the predominant ceramide species were d: h21:0-d14:0 (∼30%) and f: h23:0-d14:0 (∼70%), which were also minor components of dried pupae. Larvae contained all four of these ceramide species at proportions of ∼15% (c), ∼25% (d), ∼25% (e) and ∼35% (f), in addition to a minute amount of h22:0-d14:0 as a stage-specific species. Fig. 6 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSL fractions from larvae, pupae and dried pupae. Peaks c–f correspond to different ceramide compositions of GSL; c, h20:0-d14:1; d, h21:0-d14:0; e, h22:0-d14:1; and f, h23:0-d14:0. Insets: magnified molecular weight area of each CHS from larvae, pupae and dried pupae. Fig. 6 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSL fractions from larvae, pupae and dried pupae. Peaks c–f correspond to different ceramide compositions of GSL; c, h20:0-d14:1; d, h21:0-d14:0; e, h22:0-d14:1; and f, h23:0-d14:0. Insets: magnified molecular weight area of each CHS from larvae, pupae and dried pupae. Discussion GSLs containing HexNAc (CTeS1 and CTeS2) eluted earlier than Hex (CTeS3) by Iatrobeads column chromatography using the propanol:water:ammonium hydroxide solvent system. Similar separation was observed for the separation of CTSs from the marine crab Erimacrus isenbeckii (25). In this experiment, GlcNAc-containing GSL (CTS1) was also separated from Gal-containing GSL (CTS2) because of its faster elution. Furthermore, GlcNAc-containing GSL (CTeS1) eluted faster than its constituent GalNAc (CTeS2). This separation is caused by a dimensional influence, such as linkage position at the secondary sugar (e.g. third or fourth position on galactose). Using silica gel chromatography with the propanol:water:ammonium hydroxide solvent system, GlcNAc containing-GSLs could also be separated, such as CTS1 (At3Cer: GlcNAcβ3Manβ4Glcβ1Cer). At3Cer has been identified from several animal species, such as fly (17, 18), moth (19), krill, shrimp (26), brine shrimp (20), crab (25), millipede (27) (all Arthropoda), parasitic Nematodes (28–31) and C. elegans (32, 33) (another Nematoda). Contrarily, no arthro-series GSLs have been detected in other animal species studied in our laboratory, such as Annelida (34–36) or Mollusca (37–40). This suggests that arthro-series CTS At3Cer functions as a common GSL in molting animals (Ecdysozoa) belonging to the phyla Arthropoda and Nematoda. Both CTS2 (Galβ3Manβ4Glcβ1Cer) and CTeS2 (GalNAcα4Galβ3Manβ4Glcβ1Cer) have been reported as non-arthro-series mannose-containing GSL in the cell line High-Five™ established from the ovarian cells of the cabbage lopper T. ni (order Lepidoptera, class Insecta, phylum Arthropoda) (16). In addition, CTS2 has been reported in the marine crab E. isenbeckii (class Crustacean, phylum Arthropoda) (25). The long sugar chain-containing neutral GSLs found in this study were characterized as novel silkworm-specific structures with GalNAc polymeric elongation started from CTS2. A characteristic sugar sequence (GalNAcα4Galβ3) has been characterized in the O-linked oligosaccharide of the jelly coat surrounding the eggs of frog (41), and a similar structure has been found as GalNAcα4GalNAcβ in the arthro-series GSLs (At5Cer: GalNAcα4GalNAcβ4GlcNAcβ3Manβ4Glcβ1Cer) of fly (42, 43). Most GalNAcs are observed as β-linked moieties in oligosaccharide structures. Among GSLs, a characteristic sugar sequence (GalNAcα4Gal) appears to be a common structure in Lepidoptera. Another characteristic feature is the presence of mono sugar polymeric sequences. Heptamer-GalNAcα1-4 has not been identified in any animal GSL, whereas tetramer-Galβ1-6 sequence has been characterized in GSLs of the tapeworm Taenia crassiceps (44). Most GSL structures are composed of hetero sugars or repeating units such as LacNAc (20, 45–47). The main molecular species composing ceramide in each purified GSL from dried pupae were h20:0 and h22:0-d14:1 (fatty acid-sphingoid). These hydroxy fatty acid components (h20:0 and h22:0) are characteristic features of GSLs found in silkworms. The ceramides of a large number of GSLs in most invertebrate species consist of saturated fatty acids and dihydroxy-sphingoid. Hydroxy fatty acid was the major fatty acid constituent only in krill (h22:1 and h24:1) (48) and roundworm (h24:0) (30, 32). The length of the fatty acid chain in B. mori (20:0 and 22:0) is similar to that found in other invertebrate animals. The GlcNAcβ3Manβ4Glcβ1- sequence containing arthro-series CTS1 was composed of normal C20:0 and C22:0 acids, whereas the Galβ3Manβ4Glcβ1- sequence containing GSLs was composed of h20:0 and h22:0 acids. Ceramide mono- and disaccharides possess both fatty acid components as biosynthesis precursors. The structure of Golgi apparatus is slightly different in mammalian cells and insect cells, such as Drosophila, which is a well-studied invertebrate model (49). The Golgi found in insect is not present as a flattened compartment observed in mammalian cells. The Golgi organization in Drosophila cells are not interconnected to form a single-copy organelle. The fly’s Golgi stacks remain dispersed throughout the cytoplasm and are found in close association to tER sites to form ‘tER-Golgi units’ (50). This may explain why we observed such changes in ceramide composition in CTS1 and CTS2 as each tER-Golgi units may be able to preferentially synthesize specific GSLs with specific ceramide compositions. In contrast, two types of GSLs had been purified: Galβ3Manβ4Glcβ1Cer as major GSL and At3Cer as a minor component from the cell line High-Five™ (16), though the ceramide composition has not been particularly described. It seems that the cell can biosynthesize different sugars of GSLs with the same ceramide pool by regulating glycosyltransferase expression or activity. It is possible that this particular ceramide composition is derived from the glycosyltransferase specificities or biosynthesis of GSLs within organs with distinct ceramide pools. After the characterization of major GSLs from the whole body of silkworms in this study, we should perform detailed analysis in the future on GSL expression patterns within ceramide composition in different organs or several stages in silkworms. However, this difference appears to derive from our selected purification techniques, which can give rise to only highly purified fractions from several column chromatograms. In particular, the minor GSL components eluted from the combination of column chromatograms with solvent systems of chloroform: methanol:water and 1-propanol:water:28% ammonium hydroxide were not investigated for insufficient yields to chemical structural analysis. Tetradeca- and hexadeca-4-sphingenines detected in silkworms have also been reported as major sphingoid species in arthropods:fly (d14:1 and d16:1) (51, 52), crab (d14:1) (25) and brine shrimp (d16:1) (20). Although the hexadecasphinganine content (∼10%) in silkworms is similar to that in crabs, the tetradecasphinganine content (also ∼10%) is higher than that in any other examined species. In Monduca sexta, another moth, predominant sphingoids are the same as those in flies and silkworms (d14:1 and d16:1), specifically doubly unsaturated sphingoids such as tetradecasphiga-4,6-diene and hexadecasphiga-4,6-diene (14:2 and 16:2) and d14:0 and d16:0 in minute amounts (19). In silkworms, we could not detect doubly unsaturated sphingoids by GC analysis. Several molecular masses consistent with odd-numbered fatty acids (h21:0 or h23:0) and sphinganine (d14:0 or d16:0) were detected by MALDI-TOF MS analysis. Other analytically noteworthy results in NMR spectra were the presence of two doublet anomeric signals of Glc with chemical shifts of 4.14 and 4.13 ppm (CMS) and 4.18 and 4.15 ppm (CDS) (Fig. 2 and Table II). Similar to phosphonocerebroside from the Antarctic krill (48), the division of anomeric signals was reflected by the contents of hydroxy and nonhydroxy fatty acids in GSLs. Compositions and expression patterns of fats and proteins vary during silkworm development and transformation, particularly body weight, water content and lipid content. After starvation, the larval body is rebuilt during pupation with the excretion of water and storage of fat (as accumulated triacylglycerides). During lipid extraction, bulk amounts of simple lipids within the body of pupa contaminate the total lipid fraction and it is difficult to separate a group of simple lipid contaminants from the sphingolipid fraction even after alkaline treatment. Further, a positive spot with the orcinol reagent at the origin on TLC plates contaminates each preparation except the neutral fraction. We attempted to compare five fractions between larval and pupal components by TLC, but several contaminants disturbed the quantitative analysis. In Fig. 5, pupal GSLs were spotted on the TLC plate with 2–5-fold greater amount than for larval GSLs, whereas the neutral fractions of larval and pupal GSLs were spotted at almost same amounts. The comparison of GSL expression patterns on TLC revealed that expression levels of several neutral GSLs were lower in larvae than in pupae. These GSLs were defined as CDS, CTS and CTeS by qualitative analysis after separation and chemical structural analysis (Supplementary Fig. S3), whereas TLC showed similar expression levels of acidic, polar and zwitterionic fractions. Further studies of differences in minor components of ionic fractions are still required. The biosynthesis of GSL in insects, particularly Drosophila, has been well studied due to the identification of two mutants, egghead and brainiac, which are glycosyltransferases responsible for GSL synthesis (47, 53–56). Egghead and brainiac have a Notch-like neuronal hypertrophic phenotype and defective EGF-R signalling in oocytes (57–59). The β4GalNAcT A enzyme catalyzes the elongation of GSLs, and the mutation causes defects in neuromuscular junction innervation (60, 61). In contrast, a β4GalNAcT B mutant exhibited ventralizing ovarian follicles, similar to egghead, brainiac and EGF-R knockouts (62). Besides, the overexpression of α4GalNAcT1/2, which is responsible for the biosynthesis of long sugar chains attached to GSLs, suppresses the mindbomb Notch-like phenotype and inhibit apoptosis in eye disks (63). The change in MacCer expression between larvae and pupae suggests that the egghead gene is involved in the biosynthesis of GSLs in silkworms. Moreover, At3Cer, which is a common GSL in Arthropoda, exists as only a minor component in silkworms, whereas αGalNAc-rich GSLs predominate. Brainiac or α4GalNAcT1/2 expression in silkworms may regulate the expression patterns and consequently the function of GSLs. In Fig. 6, differences in the ceramide composition of GSLs were observed among larvae, pupae and dried pupae. The biosynthesis of odd fatty acid-saturated sphingoid components are accumulated in pupa or conserved, whereas the ratio of ceramide with even fatty acid-unsaturated sphingoid components is reduced during development. The histolysis and remodelling of the organism during transformation from larvae and pupae may require selective changes in the biosynthesis of GSLs. The ceramide composition of dried pupae significantly differed from that in bred pupae, but all components found in dried and bred pupae were found in larvae. There are several possibilities for the difference in ceramide composition between bred pupae and dried pupae: (i) sample preparation, (ii) difference in the number of days after pupation or (iii) difference in subspecies. We speculate that the differences arose from the processing of dried pupae purchased from a silk string manufacturer. These pupae were obtained after cocoon removal following treatment with boiling water, and they were then dried under the sun. During boiling and drying, some decomposition of unstable ceramide components may have occurred. In addition, larvae molt in the cocoon to form pupae within ∼2 days after cocoon spinning, and imagoes hatch from the cocoon after ∼2 weeks. At silk factories, pupae are collected on different days after spinning off the silk. In this experiment, pupae were collected 5 days after cocoon spinning, which is a relatively early pupal stage. It will be necessary to study the compositional change between pupae and imagoes. Further, there are three silkworm subspecies bred by Japanese silk companies. The difference is at the subspecies level lies mainly during seasonal breeding. Dried pupae we obtained from the company in bulk and contained a mixture of subspecies, whereas all bred silkworms used for the comparison belonged to the same species. Therefore, it is unlikely that differences in ceramide composition are due to differences in subspecies. In light of the differential expression of GSLs between silkworm larvae and pupae observed in this experiment, GSL expression changes across developmental stages from the larva to the imago by an in-depth analysis of glycan sequence and ceramide composition should be investigated. In this study, we profiled silkworm GSLs in different biological forms to study how the biosynthesis of GSLs is regulated during transformation, which involves various glycan-associated enzymes as well as ceramide synthases. GSL expression is spatially and temporally regulated across developmental stages and organs. This has been particularly well examined for the ganglioside expression pattern in the nervous system. For instance, alterations in frog GSL expression patterns in the nervous system have been reported during metamorphosis (64), but there is no significant difference in the ceramide composition of frog GSLs. GSL composition also changes during the differentiation of human leukemic granulocytes and the promyelocytic leukemic cell line HL-60 (65). With respect to the composition and content of GSLs, there are no significant differences between normal and leukemic mature neutrophils, and they synthesize the same ceramide species (66). In contrast, changes in the ceramide composition of bovine milk gangliosides with the stage of lactation have been reported (67). A significant decrease in saturated and long-chain fatty acids with concomitant increases in C18:1 and C18:2 have been observed from colostrum to milk. Thus, altered ceramide composition reflects developmental changes and possibly associated functional changes. In this study, we applied high-resolution MALDI-TOF MS to analyse silkworm GSLs and observed alteration in ceramide composition during transformation; changes were not detectable using traditional TLC analysis. Our sequential GSL enrichment protocol using magnesium silicate column chromatography (Florisil) and silica gel chromatography (Iatrobeads) is capable of producing highly purified GSLs from a crude lipid mixture and can be applied for any lipid extract from various biological samples (20, 25, 68). Highly purified GSL preparations are crucial to profile the heterogeneity of ceramide composition by in-depth glycolipidomics analysis. Supplementary data Supplementary data are available at JB Online. Acknowledgements We acknowledge the valuable comments provided by Dr. Kazuhiro Aoki of the University of Georgia. The authors would like to thank Enago (www.enago.jp) for the English language review. Funding This study was supported in part by the Grant-in-Aid for Scientific Research (C) (22500276) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Shiga University Research Support Fund from Shiga University. Conflict of Interest None declared. References 1 Gunsalus K.C., Piano F. ( 2005) RNAi as a tool to study cell biology: building the genome-phenome bridge. Curr. Opin. 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( 2013) Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids. Glycoconj J . 30, 257– 268 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations At3Cer GlcNAcβ3Manβ4Glcβ1Cer At5Cer GalNAcα4GalNAcβ4GlcNAcβ3Manβ4Glcβ1Cer CDS ceramide disaccharide CMS ceramide monosaccharide CPS ceramide pentasaccharide CHpS ceramide heptasaccharide CHS ceramide hexasaccharide COS ceramide octasaccharide CTeS ceramide tetrasaccharide CTS ceramide trisaccharide FID flame ionization detector GlcCer glucosylceramide GSL glycosphingolipid MacCer mactosylceramide (Manβ4Glcβ1Cer) PMAA partially methylated alditol acetate PSD post-source decay © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Structural analysis of neutral glycosphingolipids from the silkworm Bombyx mori and the difference in ceramide composition between larvae and pupae

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Oxford University Press
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© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvx072
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Abstract

Abstract Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcβ1Cer, Manβ4Glcβ1Cer, GlcNAcβ3Manβ4Glcβ1Cer, Galβ3Manβ4Glcβ1Cer, GalNAcα4Galβ3Manβ4Glcβ1Cer, GlcNAcβ3Galβ3Manβ4Glcβ1Cer, Galα4Galβ3Manβ4Glcβ1Cer and (GalNAcα4)1–4 GalNAcα4Galβ3Manβ4Glcβ1Cer. Lin‐ear elongation of α4-GalNAc was observed at the non-reducing end of Galβ3Manβ4Glcβ1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcβ3Manβ4Glcβ1Cer, a characteristic GSL–glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis. development, fatty acid, glycolipid, mass spectrometry, sphingolipids Glycosphingolipids (GSLs) contribute to various biological processes and functions. They are biosynthesized by several glycosyltransferases in a systematic sequential process. The analysis of the correlation between the glycosyltransferase gene and GSL expression patterns is important to study the functions of GSLs. Chemical structural analysis and expression patterns of GSL are major determinants of the phenotype of an organism. Such analyses are aided by detailed knowledge of the genome, proteome and phenome of organisms (1) such as Drosophila melanogaster (2–4) and Caenorhabditis elegans (5–8). In Drosophila, the expression and biosynthesis of GSLs have been studied in well-known developmental pathways and the nervous system, revealing essential functions of GSLs (9). The silkworm Bombyx mori, which has been domesticated for silk production since ancient times, is a well-established lepidopteran animal model for which substantial research on its developmental biology, genetics, molecular biology and immunology has been conducted. The silkworm B. mori (order Lepidoptera, class Insecta, phylum Arthropoda) exhibits complete (holometabolous) metamorphosis in the stages of the ovum, larva (fifth instar), pupa (in cocoon) and imago. Various studies on innate immunity in invertebrates have utilized muscle contraction in silkworm larvae to demonstrate the activation of insect cytokines by humoral and cellular components (10). Several databases on silkworms provide genome-wide sequences, cDNAs and expressed sequence tags (11–14). Therefore, silkworms are a valuable model for comparing DNA sequences within the same genus (e.g. versus flies) or with other genera such as roundworms. In addition, an established transgenic silkworm strain has been developed for the in vivo evaluation of human protein–protein interactions (15). Furthermore, the body size of silkworms is an advantage in GSL research. Extracted GSLs cannot be amplified; therefore, experiments require many animal models to obtain sufficient amounts of GSLs for analyses. A fifth instar silkworm larva is approximately 5 cm in body length, 1 cm in diameter and over 4 g in weight. Therefore, silkworms are advantageous for conducting comparative studies of GSL expression, structure and function across different stages in Arthropod development. In this study, we described the common GSLs extracted from the larvae and pupae of silkworms and compared them with GSLs of the cell line High-Five™ established from ovarian cells of the cabbage lopper Trichoplusia ni (order Lepidoptera, class Insecta, phylum Arthropoda) (16), the fly Lucilia caesar (17), the fly Calliphora vicina (18) and the moth Manduca sexta (19), which have characteristic arthro-series sugar chains (At3Cer: GlcNAcβ3Manβ4Glcβ1Cer). The comparison of larval and pupal GSLs revealed developmental differences in ceramide compositions. Materials and Methods Bulk extraction and isolation of neutral GSLs from dried pupae for structural characterization Dried pupae (2.0 kg) without cocoons were purchased from a silk string manufacturing corporation (Usui-seishi, Annaka, Gunma, Japan). Whole lipids were extracted from a sample using a mixture of chloroform: methanol (2:1 and 1:1, v/v) and chloroform:methanol:water (1:1:0.2, v/v/v). The extracts were combined and dried, and the dried lipids were subjected to mild alkaline hydrolysis to prepare the sphingolipid fraction as previously reported (20). Alkaline-stable products were added to a diethylaminoethyl (DEAE)-Sephadex A-25 column (CH3COO− form; GE Healthcare UK Led., Buckinghamshire, UK) to remove acidic compounds. Neutral and polar compounds were eluted with a mixture of chloroform:methanol:water (30:60:8, v/v/v). The mixture was then subjected to QAE-Sephadex A-25 chromatography (OH− form; GE Healthcare Co.) for the removal of contaminant polar compounds. Neutral compounds were eluted with five column volumes of chloroform:methanol:water (30:60:8, v/v/v). The eluates were completely dried and acetylated by pyridine:acetic anhydride (3:2, v/v) for further purification of neutral GSLs by magnesium silicate column chromatography (Florisil, 60–100 mesh; Nakalai Tesque, Kyoto) (21). The columns were successively eluted with six column volumes of n-hexane: dichloroethane (1:4, v/v), 1 vol of pure dichloroethane, 3 vol of dichloroethane: acetone (1:1, v/v), 3 vol of dichloroethane:methanol (3:1, v/v), 3 vol of dichloroethane:methanol:water (2:8:1, v/v/v), and 3 vol of chloroform:methanol:water (6:4:1, v/v/v). The fractions of acetylated GSLs eluted by dichloroethane:acetone (1:1) and dichloroethane:methanol (3:1) were combined, evaporated to dryness and deacetylated with 0.5 M KOH in methanol at 37°C for 6 h. The reaction mixture was dialyzed against tap water overnight and evaporated to dryness (yield of 173 mg). The neutral GSL fraction (120 mg) was added to a column of porous silica gel (1.8 × 90 cm, Iatrobeads 6RS-8060; Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) pre-equilibrated with chloroform:methanol:water (80:20:1, v/v/v). This column was treated with two consecutive linear gradient systems of chloroform:methanol:water (80:20:1, v/v/v 665 ml–50:50:5, 755 ml and 50:50:5, 680 ml–20:80:10, 755 ml). The effluent was collected in 3-ml fractions, and an aliquot from each tube was assayed by thin-layer chromatography (TLC) with a solvent system of chloroform:methanol:water (60:40:10, v/v/v) and orcinol:H2SO4 reagent for detecting sugars. On the basis of the TLC appearance, neutral GSLs expressed in substantial quantities were fractionated into 16 fractions. Several fractions (CTeSs, CPSs, CHpSs and COSs) required further purification using a high-performance liquid chromatography (HPLC) system (Shimadzu LC-9A) with an Iatrobeads column (1.0 × 30 cm) and solvent systems of 1-propanol:water:28% ammonium hydroxide (75:8.5:5 v/v/v for CTSs, 75:12.5:5 v/v/v for CTeSs and CPSs, 75:18.5:5 v/v/v for CHpS and 75:17.5:5 v/v/v for COS) at a flow rate of 1 ml/min. Extraction and fractionation of neutral GSLs for the comparison of GSL expression patterns Third instar larvae were purchased from a silkworm breeding company (Kougensha Co. Ltd, Matsumoto, Nagano, Japan) and bred to fifth instar larvae and pupae. Starting materials used for were 429 larvae (total: 1825 g) and 200 pupae (total: 239 g). Whole lipids were extracted from lyophilized samples (431.9 g for larvae and 57.3 g for pupae). Neutral GSLs were isolated using a similar method utilized for the bulk extraction and isolation of neutral GSLs from dried pupae for structural characterization, except if starting from lyophilized material. The neutral GSL fractions (yield: 192 mg for larvae and 30 mg for pupae) were analysed by TLC as described below. Solvents for TLC analysis TLC was performed using silica gel 60 (Merck KGaA) and visualized by spraying the chromatogram with the orcinol–H2SO4 reagent followed by heating at 110°C. The following solvent systems were utilized for development: chloroform:methanol:water (60:40:10, v/v/v) and 1-propanol:water:28% ammonium hydroxide (75:25:5, v/v/v). Compositional sugar analysis by gas chromatography (GC) Compositional sugar analysis of each purified GSL was conducted following derivatization to trimethylsilyl (TMSi) methyl glycosides using GC (Shimadzu GC-18A). The GC system included a capillary column that was chemically bonded (Shimadzu HiCap-CBP 5, 0.22 mm × 25 m) with temperature programmed at 2°C/min from 140 to 230 °C. The ceramide compositions of GSLs were determined by analysing fatty acid methyl ester and TMSi-sphingoids (22) on the same GC capillary column programmed at 2 °C/min from 170 to 240 °C for fatty acids and at 2 °C/min from 200 to 240 °C for sphingoids (23). Sugar linkage analysis Samples (∼200 μg) of purified GSLs were permethylated with NaOH and CH3I in dimethyl sulfoxide (24). Partially methylated GSLs were hydrolyzed with a 0.3 ml mixture of HCl:water:acetic acid (8.0:0.5:1.5, v/v/v) at 80 °C for 18 h. Hydrolysates were dried under an N2 stream and were reduced with 1% NaBH4 in 0.01 M NaOH overnight. Reaction products were dried and then acetylated with a mixture of acetic anhydride:pyridine (1:1, v/v) at 100 °C for 10 min (25). Partially methylated alditol acetates (PMAAs) obtained were analysed by GC and GC-mass spectrometry (MS) using the same Shimadzu HiCap-CBP 5 capillary column as described above. Electron ionization mass spectra were acquired using a Shimadzu GCMS-QP 5050 gas chromatograph-mass spectrometer under the following conditions: oven temperature, 80 °C (2 min) to 180 °C (20 °C/min) to 240 °C (4 °C/min); interface temperature, 250 °C; injection port temperature, 240 °C; helium gas pressure, 100 kPa; ionizing voltage, 70 eV; and ionizing current, 60 μA. Peaks on gas chromatograms were identified by comparing them with retention times and physicochemical data from NIST/EPA/NIH Mass Spectral Library NIST 11 (Shimadzu, Tokyo, Japan). Proton nuclear magnetic resonance spectroscopy (1 H-NMR) 1 H NMR spectra of purified GSLs were obtained using a JEOL JNM-ECS-400 400 MHz 1 H NMR spectrometer at an operating temperature of 60 °C. GSL samples (1 mg) were dissolved in 0.5 ml of dimethyl sulfoxide-d6 containing 2% D2O. Chemical shifts were referenced to solvent signals (δH 2.49 ppm) in d6-demethylsulfoxide. Matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS) MALDI-TOF MS analysis of purified GSLs was performed using a Shimadzu AXIMA Confidence MALDI-TOF mass spectrometer that was operating in the positive-ion reflectron mode for full MS analysis and post-source decay (PSD) mode for MS/MS analysis. Data were acquired in both modes using a UV nitrogen laser (337 nm) and in the automatic measurement mode at an acceleration voltage of 20 kV. Positive-ion mass spectra were acquired with 100 profiles/run at ∼80 power for the reflectron mode and with 100 shots/run at ∼90 power in a curved field reflectron in the PSD mode. Precursor ions were selected using an ion gate width of ∼3 m/z units in the PSD mode. Purified GSL (4–10 μg) dissolved in chloroform:methanol (2:1, v/v) solution was spotted on a sample plate and air dried. Totally, 0.5 μl of matrix solution was spotted on sample wells followed by air drying. The matrix solution used was α-cyano-4-hydroxycinnamic acid (Shimadzu GLC, Kyoto, Japan) at a saturated concentration [5 mg in 1 mL of ethanol:water (1:1, v/v)]. External mass calibration was provided by the [M + H]+ ions of bradykinin fragment 1–7 (757.40 Da; Sigma Chemical Co.) and ACTH fragment 18–39 (2465.20 Da; Sigma Chemical Co). Results Three starting materials were used: dried pupae, which were purchased from a silk string manufacturing corporation, and larvae and pupae, which were bred in our laboratory. For the structural characterization of GSLs, besides the yield of GSL, dried pupae were used as they can be easily obtained in bulk amounts for lipid extraction. (Bred) larvae and (bred) pupae were used for the comparison of GSL expression patterns. Purification and fractionation of neutral GSLs from dried pupae Neutral GSLs from dried pupae were separated into 16 fractions by silica gel chromatography using a gradient solvent system of chloroform:methanol:water (Fig. 1A). Several fractions of neutral GSLs were further separated by TLC into two or three GSL species using a propanol:water:ammonium hydroxide solvent system (Fig. 1B). After purification and separation (Supplementary Fig. S1), fraction (Fr.) 3 (Fr. 3, lane 3 in Fig. 1) yielded ceramide trisaccharide (CTS) 1 (CTS1, Fr. 3-1) and CTS2 (Fr. 3-5); Fr. 6 (lane 6) yielded ceramide tetrasaccharide 1 (CTeS1, Fr. 6-3), CTeS2 (Fr. 6-5) and CTeS3 (Fr. 6-7); Fr. 7 (lane 7) yielded ceramide pentasaccharide (CPS, Fr. 7-4); Fr. 11 (lane 11) yielded ceramide heptasaccharide (CHpS, Fr. 11-2-3); and Fr. 13 (lane 13) yielded ceramide octasaccharide (COS, Fr. 13-4). Ceramide mono- (CMS), di- (CDS) and hexasaccharide (CHS) were obtained by first-step silica gel chromatography. In addition, fractions containing minor components were observed by TLC; however, the yields were insufficient for chemical structural analysis. Fig. 1 View largeDownload slide Thin-layer chromatograms of neutral GSL fractions from the silkworm B. mori. Lane T, neutral GSL fraction obtained by Florisil column chromatography through acetylation and deacetylation; lanes 1–16, neutral GSL fractions separated by Iatrobeads column chromatography using a gradient elution system with chloroform:methanol:water. The plate in A was developed with chloroform:methanol:water (60:40:10, v/v/v) and that in B by propanol:water:28% ammonium hydroxide (75:25:5, v/v/v). The spots were visualized by the orcinol–H2SO4 reagent. Fig. 1 View largeDownload slide Thin-layer chromatograms of neutral GSL fractions from the silkworm B. mori. Lane T, neutral GSL fraction obtained by Florisil column chromatography through acetylation and deacetylation; lanes 1–16, neutral GSL fractions separated by Iatrobeads column chromatography using a gradient elution system with chloroform:methanol:water. The plate in A was developed with chloroform:methanol:water (60:40:10, v/v/v) and that in B by propanol:water:28% ammonium hydroxide (75:25:5, v/v/v). The spots were visualized by the orcinol–H2SO4 reagent. Characterization of the ceramide tetrasaccharides CTeS1, CTeS2 and CTeS3 from dried pupae GSLs containing HexNAc (CTeS1 and CTeS2) were eluted faster than constituent Hex (CTeS3) by Iatrobeads column chromatography using the propanol:water:ammonium hydroxide solvent system. The GSL containing GlcNAc (CTeS1) eluted faster than the GSL containing GalNAc (CTeS2). Purified CTeS2 (2.1 mg) was the major CTeS component, whereas CTeS1 (1.0 mg) and CTeS3 (0.9 mg) were minor components of the 120 mg dried neutral GSL mixture in pupae; however, crude side-fractions also contained these GSLs. Sugar analyses indicated that all three CTeSs contained Man, Gal and Glc as common components, whereas CTeS1 contained only GlcNAc and CTeS2 contained only GalNAc. In methylation analysis (Table I and Supplementary Fig. S2), common PMAA derivatives corresponding to 3-linked Man (1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannnitol, 3-Man) and 4-linked Glc (1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgluctitol, 4-Glc) were detected in all three CTeSs as indicated by a 2-fold higher single peak using a low polar capillary column (Supplementary Fig. S2). PMAAs from each monosaccharide at the non-reducing end and the penultimate Gal differed among CTeSs, with terminal-GlcNAc (1,5-di-O-acetyl-3,4,6-tri-O-methyl-N-acetylglucosaminitol, t-GlcNAc) and 3-linked Gal (1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol, 3-Gal) in CTeS1, terminal-GalNAc (1,5-di-O-acetyl-3,4,6-tri-O-methyl-N-acetylgalactosaminitol, t-GalNAc) and 4-linked Gal (1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol, 4-Gal) in CTeS2, and terminal-Gal (1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol, t-Gal) and 4-Gal in CTeS3. Table I. PMAA analysis of neutral glycosphingolipids (GSLs) from the silkworm B. mori CMS  t-Glc                    CDS    t-Man    4-Glc              CTS1            3-Man + 4-Glc    t-GlcNAc      CTS2      t-Gal      3-Man + 4-Glc          CTeS1            3-Man + 4-Glc  3-Gal  t-GlcNAc      CTeS2          4-Gal  3-Man + 4-Glc      t-GalNAc    CTeS3      t-Gal    4-Gal  3-Man + 4-Glc          CPS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHpS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  COS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CMS  t-Glc                    CDS    t-Man    4-Glc              CTS1            3-Man + 4-Glc    t-GlcNAc      CTS2      t-Gal      3-Man + 4-Glc          CTeS1            3-Man + 4-Glc  3-Gal  t-GlcNAc      CTeS2          4-Gal  3-Man + 4-Glc      t-GalNAc    CTeS3      t-Gal    4-Gal  3-Man + 4-Glc          CPS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  CHpS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  COS          4-Gal  3-Man + 4-Glc      t-GalNAc  4-GalNAc  Results summarized as detectable peaks according to the order of retention using a HiCap-CBP 5 capillary column for GC (Supplementary Fig. S2). The abbreviations indicate the following: t-Glc; terminal Glc, t-Man; terminal Man, 4-Glc; 4-linked Glc, 3-Man; 3-linked Man, t-GlcNAc; terminal GlcNAc, t-Gal; terminal Gal, 4-Gal; 4-linked Gal, and 4-GalNAc; 4-linked GalNAc. PMAA of 3-Man and 4-Glc could not be separated by this column. The 1 H NMR spectra of the three CTeSs (Fig. 2) exhibited three typical major H-1 resonances with chemical shifts and J1,2 coupling constants for β-Glc (4.19 ppm; J1,2 = 7.3–7.8 Hz), β-Gal (4.31 or 4.32 ppm; J1,2 = 6.9–7.8 Hz) and β-Man (4.53 or 4.55 ppm; J1,2 = ∼1 Hz), whereas the terminal sugar residue exhibited distinct shifts and J1,2 coupling constants as follows (Table II): β-GlcNAc (4.64 ppm; J1,2 = 7.8 Hz) for CTeS1, α-GalNAc (4.84 ppm; J1,2 = 3.7 Hz) for CTeS2, and α-Gal (4.82 ppm; J1,2 = 3.7 Hz) for CTeS3. Table II. Chemical shifts and J1,2 coupling constants of protons in neutral GSLs at 60 °C CMS            Glc1-  Chemical shifts (ppm)            4.14  Coupling constants (Hz)            7.8  Chemical shifts (ppm)            4.13  Coupling constants (Hz)            7.8  CDS          Man1-  4Glc1-  Chemical shifts (ppm)          4.50  4.18  Coupling constants (Hz)          a  8.2  Chemical shifts (ppm)            4.15  Coupling constants (Hz)            7.8  CTS1        GlcNAc1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.54  4.52  4.16  Coupling constants (Hz)        7.3  a  7.8  CTS2        Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.26  4.54  4.19  Coupling constants (Hz)        7.3  a  7.8  CTeS1      GlcNAc1-  3Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.64  4.31  4.54  4.19  Coupling constants (Hz)      7.8  7.8  a  7.8  CTeS2      GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.84  4.32  4.55  4.19  Coupling constants (Hz)      3.7  6.9  a  7.8  CTeS3      Gal1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.82  4.31  4.53  4.19  Coupling constants (Hz)      3.7  7.3  a  7.3  CPS    GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)    4.87  4.90  4.32  4.55  4.19  Coupling constants (Hz)    3.7  3.7  7.3  a  7.8  CHS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.89  4.93  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  3.7  6.9  a  7.8  CHpS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.7  3.7  b  6.4  a  7.8  COS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  b  6.9  a  8.2  CMS            Glc1-  Chemical shifts (ppm)            4.14  Coupling constants (Hz)            7.8  Chemical shifts (ppm)            4.13  Coupling constants (Hz)            7.8  CDS          Man1-  4Glc1-  Chemical shifts (ppm)          4.50  4.18  Coupling constants (Hz)          a  8.2  Chemical shifts (ppm)            4.15  Coupling constants (Hz)            7.8  CTS1        GlcNAc1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.54  4.52  4.16  Coupling constants (Hz)        7.3  a  7.8  CTS2        Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)        4.26  4.54  4.19  Coupling constants (Hz)        7.3  a  7.8  CTeS1      GlcNAc1-  3Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.64  4.31  4.54  4.19  Coupling constants (Hz)      7.8  7.8  a  7.8  CTeS2      GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.84  4.32  4.55  4.19  Coupling constants (Hz)      3.7  6.9  a  7.8  CTeS3      Gal1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)      4.82  4.31  4.53  4.19  Coupling constants (Hz)      3.7  7.3  a  7.3  CPS    GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)    4.87  4.90  4.32  4.55  4.19  Coupling constants (Hz)    3.7  3.7  7.3  a  7.8  CHS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.89  4.93  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  3.7  6.9  a  7.8  CHpS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.7  3.7  b  6.4  a  7.8  COS  GalNAc1-  4GalNAc1-  4GalNAc1-  4Gal1-  3Man1-  4Glc1-  Chemical shifts (ppm)  4.86  4.90  4.94  4.32  4.55  4.19  Coupling constants (Hz)  3.2  3.2  b  6.9  a  8.2  aSinglet. bBroad singlet. Fig. 2 View largeDownload slide Anomeric proton regions of 1 H NMR spectra for neutral GSLs. Spectra obtained in DMSO-d6 containing 2% D2O at an operating temperature of 60 °C. Chemical shifts and J1,2 coupling constants are summarized in Table II. Sph-4 and -5 indicate vinyl H-4 and H-5 multiplets, respectively, from the (E)-4-sphinganine base. Fig. 2 View largeDownload slide Anomeric proton regions of 1 H NMR spectra for neutral GSLs. Spectra obtained in DMSO-d6 containing 2% D2O at an operating temperature of 60 °C. Chemical shifts and J1,2 coupling constants are summarized in Table II. Sph-4 and -5 indicate vinyl H-4 and H-5 multiplets, respectively, from the (E)-4-sphinganine base. Gas chromatography and GC-MS revealed that the ceramide constituents (Table III) of the three CTeSs were composed of tetradecasphingenine (14:1) as the major sphingoid and 2-hydroxy C20:0 and C22:0 acids (h20:0 and h22:0) as the major fatty acids. Furthermore, from the 1 H NMR spectra, characteristic coupled vinyl H-4 and H-5 multiplets at ∼5.38 and 5.58 ppm, respectively, indicated that these ceramides contained an (E)-4-sphingenine base. Table III. Ceramide composition of neutral GSLs Fatty acid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS  C14:0  –  3.1  tr.  3.3  1.1  tr.  –  1.8  –  tr.  0.7  C15:0  –  –  tr.  1.7  –  –  –  tr.  –  –  –  C16:0  tr.  3.5  3.6  4.5  2.9  2.5  tr.  5.3  tr.  1.3  3.1  C17:0  –  –  2.1  3.0  –  –  –  tr.  –  –  –  C18:0  1.9  6.8  6.2  8.0  2.4  1.8  1.6  1.6  2.5  2.0  4.0  C19:0  tr.  tr.  tr.  1.4  –  –  –  tr.  1.1  tr.  1.8  C20:0  12.1  22.3  33.9  4.0  2.8  1.3  1.2  1.9  7.3  7.9  11.6  C21:0  tr.  tr.  1.0  tr.  –  –  –  tr.  1.4  tr.  1.6  C22:0  15.0  26.0  46.3  4.6  4.0  tr.  1.0  3.1  13.6  12.2  10.7  C23:0  1.0  tr.  1.3  tr.  –  –  –  tr.  1.4  tr.  1.8  C24:0  3.6  1.6  3.3  tr.  –  –  7.4  –  –  1.2  1.9  h16:0  tr.  2.6  –  –  –  –  –  –  –  –  –  h18:0  2.1  3.0  –  –  –  1.6  1.4  –  –  1.1  1.3  h19:0  1.0  tr.  –  –  –  1.4  1.4  –  –  1.3  2.9  h20:0  24.3  12.2  1.0  16.9  17.3  26.2  28.5  20.9  19.7  23.3  20.6  h21:0  2.6  tr.  –  3.2  3.6  4.4  4.6  3.9  –  3.7  4.0  h22:0  30.5  17.3  1.3  38.0  48.2  49.5  48.0  47.7  41.8  37.8  28.0  h23:0  1.7  tr.  –  6.3  9.3  6.7  –  7.8  6.5  4.9  3.6  h24:0  4.2  1.6  –  5.1  8.4  4.6  4.9  6.0  4.7  3.3  2.4  Total  100  100  100  100  100  100  100  100  100  100  100    Sphingoid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS    d14:1  59.0  76.4  72.9  61.9  60.2  75.1  81.2  68.1  64.5  66.5  66.7  d14:0  16.4  6.8  5.6  9.3  14.5  11.2  10.1  12.1  11.7  13.9  3.7  d15:1  –  –  2.7  3.1  –  –  –  –  3.6  –  –  d15:0  –  –  –  2.0  –  –  –  –  –  –  –  d16:1  12.4  11.2  11.1  9.5  11.2  7.8  5.5  10.5  11.1  9.8  7.4  d16:0  12.2  5.6  7.7  14.2  14.1  5.9  3.2  9.3  9.1  9.8  22.2  Total  100  100  100  100  100  100  100  100  100  100  100  Fatty acid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS  C14:0  –  3.1  tr.  3.3  1.1  tr.  –  1.8  –  tr.  0.7  C15:0  –  –  tr.  1.7  –  –  –  tr.  –  –  –  C16:0  tr.  3.5  3.6  4.5  2.9  2.5  tr.  5.3  tr.  1.3  3.1  C17:0  –  –  2.1  3.0  –  –  –  tr.  –  –  –  C18:0  1.9  6.8  6.2  8.0  2.4  1.8  1.6  1.6  2.5  2.0  4.0  C19:0  tr.  tr.  tr.  1.4  –  –  –  tr.  1.1  tr.  1.8  C20:0  12.1  22.3  33.9  4.0  2.8  1.3  1.2  1.9  7.3  7.9  11.6  C21:0  tr.  tr.  1.0  tr.  –  –  –  tr.  1.4  tr.  1.6  C22:0  15.0  26.0  46.3  4.6  4.0  tr.  1.0  3.1  13.6  12.2  10.7  C23:0  1.0  tr.  1.3  tr.  –  –  –  tr.  1.4  tr.  1.8  C24:0  3.6  1.6  3.3  tr.  –  –  7.4  –  –  1.2  1.9  h16:0  tr.  2.6  –  –  –  –  –  –  –  –  –  h18:0  2.1  3.0  –  –  –  1.6  1.4  –  –  1.1  1.3  h19:0  1.0  tr.  –  –  –  1.4  1.4  –  –  1.3  2.9  h20:0  24.3  12.2  1.0  16.9  17.3  26.2  28.5  20.9  19.7  23.3  20.6  h21:0  2.6  tr.  –  3.2  3.6  4.4  4.6  3.9  –  3.7  4.0  h22:0  30.5  17.3  1.3  38.0  48.2  49.5  48.0  47.7  41.8  37.8  28.0  h23:0  1.7  tr.  –  6.3  9.3  6.7  –  7.8  6.5  4.9  3.6  h24:0  4.2  1.6  –  5.1  8.4  4.6  4.9  6.0  4.7  3.3  2.4  Total  100  100  100  100  100  100  100  100  100  100  100    Sphingoid (%)  CMS  CDS  CTS1  CTS2  CTeS1  CTeS2  CTeS3  CPS  CHS  CHpS  COS    d14:1  59.0  76.4  72.9  61.9  60.2  75.1  81.2  68.1  64.5  66.5  66.7  d14:0  16.4  6.8  5.6  9.3  14.5  11.2  10.1  12.1  11.7  13.9  3.7  d15:1  –  –  2.7  3.1  –  –  –  –  3.6  –  –  d15:0  –  –  –  2.0  –  –  –  –  –  –  –  d16:1  12.4  11.2  11.1  9.5  11.2  7.8  5.5  10.5  11.1  9.8  7.4  d16:0  12.2  5.6  7.7  14.2  14.1  5.9  3.2  9.3  9.1  9.8  22.2  Total  100  100  100  100  100  100  100  100  100  100  100  Results were calculated from the peak area of GC as detected by FID. h, 2-hydroxy fatty acid; d, dihydroxysphingoid; tr., trace; -, not detected. The putative structures of the three CTeSs were confirmed by the positive-ion reflectron mode of MALDI-TOF MS analysis, as shown in Fig. 3 and the detected peaks listed in Table IV. Two major peaks differing by 28 U were observed in all three CTeS spectra, with shifts reflecting the presence of distinct ceramide species consisting of d14:1 sphingoid and h20:0 and h22:0 fatty acids. The mass spectra of CTeS1 and CTeS2 exhibited sodium adduct ion species, [M + Na]+, at m/z 1265.2 and 1293.2, coinciding with HexNAc1Hex3Cer containing mainly h20:0-d14:1 and h22:0-d14:1 ceramides. The [M + Na]+ ions of CTeS3 at m/z 1224.2 and 1252.2 coincided with the mass values of Hex4Cer containing mainly h20:0-d14:1 and h22:0-d14:1 ceramides. The [M + Na]+ ions of CTeS1 at m/z 1309.2 and of CTeS3 at m/z 1268.2 coincided with a d14:0-h23:0 ceramide (peak f in Fig. 3). Table IV. Summary of MALDI-TOF MS analysis of major positive ions from neutral GSLs Ceramide    CMS  CDS      Theoretical  Observed  Theoretical  Observed      C20:0-d14:1    722.56  722.8  884.61  884.9      C21:0-d14:1    736.57  736.8  898.62        C22:0-d14:1  C20:0-d16:1  750.59  750.8  912.64  912.9      C22:0-d14:0  C20:0-d16:0  752.60  752.8  914.65        C23:0-d14:1  C21:0-d16:1  764.60  764.8  926.65        h20:0-d14:1    738.55  738.8  900.60  900.9      h21:0-d14:0    754.58  754.7  916.63        h22:0-d14:1  h20:0-d16:1  766.58  766.8  928.63  928.9      h23:0-d14:0  h21:0-d16:0  782.61  782.8  944.67              CTS1  CTS2        Ceramide  Theoretical  Observed  Theoretical  Observed        C20:0-d14:1    1087.69  a:1087.9  1046.66        C22:0-d14:1  C20:0-d16:1  1115.72  b:1115.9  1074.69        h20:0-d14:1    1103.68    1062.66  1062.9      h22:0-d14:1  h20:0-d16:1  1131.71    1090.69  1090.9            CTeS1  CTeS2  CTeS3    Ceramide  Theoretical  Observed  Theoretical  Observed  Theoretical  Observed    h20:0-d14:1    1265.73  c:1265.2  1265.73  c:1265.2  1224.71  c:1224.2  h21:0-d14:0    1281.77  d:1281.2  1281.77  1281.2  1240.74  d:1240.2  h22:0-d14:1  h20:0-d16:1  1293.77  e:1293.2  1293.77  e:1293.2  1252.74  e:1252.2  h23:0-d14:0  h21:0-d16:0  1309.80  f:1309.2  1309.80  1309.2  1268.77  f:1268.2        CPS  CHS        Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1468.81  c:1468.3  1671.89  c:1671.4      h21:0-d14:0    1484.85  d:1484.3  1687.93  d:1687.4      h22:0-d14:1  h20:0-d16:1  1496.85  e:1496.3  1699.93  e:1699.5      h23:0-d14:0  h21:0-d16:0  1512.88  f:1512.3  1715.96  f:1715.5        h22:0-d16:0  1526.89  g:1526.3  1729.97  g:1729.5            CHpS  COS      Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1874.97  c:1874.6  2078.05  c:2078.4      h21:0-d14:0    1891.00  d:1890.7  2094.08        h22:0-d14:1  h20:0-d16:1  1903.00  e:1902.6  2106.08  e:2106.4      h23:0-d14:0  h21:0-d16:0  1919.04  f:1918.7  2122.12          h22:0-d16:0  1933.05  g:1932.7  2136.13        Ceramide    CMS  CDS      Theoretical  Observed  Theoretical  Observed      C20:0-d14:1    722.56  722.8  884.61  884.9      C21:0-d14:1    736.57  736.8  898.62        C22:0-d14:1  C20:0-d16:1  750.59  750.8  912.64  912.9      C22:0-d14:0  C20:0-d16:0  752.60  752.8  914.65        C23:0-d14:1  C21:0-d16:1  764.60  764.8  926.65        h20:0-d14:1    738.55  738.8  900.60  900.9      h21:0-d14:0    754.58  754.7  916.63        h22:0-d14:1  h20:0-d16:1  766.58  766.8  928.63  928.9      h23:0-d14:0  h21:0-d16:0  782.61  782.8  944.67              CTS1  CTS2        Ceramide  Theoretical  Observed  Theoretical  Observed        C20:0-d14:1    1087.69  a:1087.9  1046.66        C22:0-d14:1  C20:0-d16:1  1115.72  b:1115.9  1074.69        h20:0-d14:1    1103.68    1062.66  1062.9      h22:0-d14:1  h20:0-d16:1  1131.71    1090.69  1090.9            CTeS1  CTeS2  CTeS3    Ceramide  Theoretical  Observed  Theoretical  Observed  Theoretical  Observed    h20:0-d14:1    1265.73  c:1265.2  1265.73  c:1265.2  1224.71  c:1224.2  h21:0-d14:0    1281.77  d:1281.2  1281.77  1281.2  1240.74  d:1240.2  h22:0-d14:1  h20:0-d16:1  1293.77  e:1293.2  1293.77  e:1293.2  1252.74  e:1252.2  h23:0-d14:0  h21:0-d16:0  1309.80  f:1309.2  1309.80  1309.2  1268.77  f:1268.2        CPS  CHS        Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1468.81  c:1468.3  1671.89  c:1671.4      h21:0-d14:0    1484.85  d:1484.3  1687.93  d:1687.4      h22:0-d14:1  h20:0-d16:1  1496.85  e:1496.3  1699.93  e:1699.5      h23:0-d14:0  h21:0-d16:0  1512.88  f:1512.3  1715.96  f:1715.5        h22:0-d16:0  1526.89  g:1526.3  1729.97  g:1729.5            CHpS  COS      Ceramide  Theoretical  Observed  Theoretical  Observed        h20:0-d14:1    1874.97  c:1874.6  2078.05  c:2078.4      h21:0-d14:0    1891.00  d:1890.7  2094.08        h22:0-d14:1  h20:0-d16:1  1903.00  e:1902.6  2106.08  e:2106.4      h23:0-d14:0  h21:0-d16:0  1919.04  f:1918.7  2122.12          h22:0-d16:0  1933.05  g:1932.7  2136.13        [M + Na]+ ions were calculated using the monoisotropic mass. Fig. 3 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSLs. Multiple [M + Na]+ peaks indicate different ceramide moieties. Peaks a–g correspond to those in Table IV. Symbol nomenclature was described according to the format of Consortium for Functional Glycomics. Fig. 3 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSLs. Multiple [M + Na]+ peaks indicate different ceramide moieties. Peaks a–g correspond to those in Table IV. Symbol nomenclature was described according to the format of Consortium for Functional Glycomics. The sugar sequences of each CTeS were determined in the PSD mode of MALDI-TOF MS (Fig. 4). Fragment ions were sequentially observed from the predominant [M + Na]+ ion to the ceramide monosaccharide sodium adduct ion species. In the PSD mass spectra of CTeS1 and CTeS2, sequential ions differing by m/z 203, 162 and 162 were observed, which was consistent with a linear HexNAc–Hex–Hex–Hex–Cer sequence (precursor ion m/z 1293.3 and fragment ion m/z 1090.4, 928.3 and 766.2). In the PSD mass spectrum of CTeS3, sequential ions differing by m/z 162 were observed, which was consistent with a linear Hex–Hex–Hex–Hex–Cer sequence (precursor ion m/z 1252.3 and fragment ion m/z 1090.4, 928.5 and 766.3). Fig. 4 View largeDownload slide Positive-ion PSD mode MALDI-TOF MS spectra of neutral GSLs. For the precursor masses of CTeS1 (1293.3), CTeS3 (1252.3) and COS (2105.7), ion gate masses were set to 1292.7–1295.7, 1251.5–1255.2 and 2103.9–2107.0, respectively. Symbol nomenclature was the same as that in Fig. 3. Fig. 4 View largeDownload slide Positive-ion PSD mode MALDI-TOF MS spectra of neutral GSLs. For the precursor masses of CTeS1 (1293.3), CTeS3 (1252.3) and COS (2105.7), ion gate masses were set to 1292.7–1295.7, 1251.5–1255.2 and 2103.9–2107.0, respectively. Symbol nomenclature was the same as that in Fig. 3. Characterization of ceramide penta-, hexa-, hepta- and octasaccharides from dried pupae GSLs containing major long sugar chains were separated by Iatrobeads column chromatography utilizing the chloroform:methanol:water solvent system based on their sugar components. Some of these fractions were contaminated with slower migrating minor components as revealed by TLC using the propanol:water:ammonium hydroxide solvent system. Purified CPS (4.3 mg), CHS (4.2 mg), CHpS (5.2 mg) and COS (9.5 mg) were the major components of the neutral GSL fraction (120 mg total) from dried pupae. Similar to CTeS1, these four GSLs contained Man, Gal, Glc and GalNAc as common sugar components (Table I and Supplementary Fig. S2). An increased GalNAc content indicated GalNAc elongation of CTeS1. In methylation analysis, common PMAA derivatives corresponding to 3-Man + 4-Glc (single peak), 4-Gal, t-GalNAc and 4-linked GalNAc (1,4,5-tri-O-acetyl-3,6-di-O-methyl-N-acetylgalactosaminitol, 4-GalNAc) were detected in all four long sugar chain GSLs. The ratios of t-GalNAc to 4-GalNAc intensity were 1:0.7 for CPS, 1:1.2 for CHS, 1:1.8 for CHpS and 1:2.8 for COS, which was consistent with the addition of 4-GalNAc units started at the non-reducing end of CTeS1. The increase in GalNAc residues was confirmed by sugar composition analysis using GC (Supplementary Fig. S2). The 1 H NMR spectra of these four long sugar chain-containing GSLs (Fig. 2) exhibited three major H-1 resonances with chemical shifts and J1,2 coupling constants for β-Glc (4.19 ppm; J1,2 = 7.8 or 8.2 Hz), β-Gal (4.32 ppm; J1,2 = 6.4–7.3 Hz) and β-Man (4.55 ppm; J1,2 = ∼1 Hz). Chemical shifts and J1,2 coupling constants for α-GalNAc were 4.86 or 4.87 ppm and J1,2 = 3.2 or 3.7 Hz for the terminal sugar, 4.89 or 4.90 ppm and J1,2 = 3.2 or 3.7 Hz for the second sugar from the terminal, and 4.93 or 4.94 ppm and J1,2 = 3.7 Hz or not determined for the internal sugar (Table II). The ratio of integral intensity for terminal and second α-GalNAcs in CHS was 1.00:1.00, and those of integral intensity for the terminal, secondary, and internal α-GalNAc moieties of the longer forms were 1.00:1.11:0.81 for CHS, 1.02:1.00:1.77 for CHpS and 1.01:1.00:2.64 for COS. The integral intensities were the sums of the individual units, for example 5× α-GalNAcα for COS. The presence of characteristic coupled vinyl H-4 and H-5 multiplets indicates that these GSLs possess an (E)-4-sphingenine base as the common ceramide composition. The ceramide constituent (Table III) of four of these GSLs contained d14:1 as the major sphingoid (∼70% of the total content) and h20:0 and h22:0 acids as the major fatty acids (∼60% of the total content) according to GC. The putative structures of these four GSLs were confirmed by the positive-ion reflectron mode of MALDI-TOF MS analysis as shown in Fig. 3 and the observed masses listed in Table IV. Two major peaks differing by 28 U were observed in all four GSL spectra, reflecting the presence of the same ceramide species as in shorter GSLs. The mass spectra of the four longer sugar chain GSLs had sodium adduct ion species, [M + Na]+, detected at m/z 1468.3 and 1496.3 for CPS, m/z 1671.4 and 1699.5 for CHS, m/z 1874.6 and 1902.6 for CHpS, and m/z 2078.4 and 2106.4 for COS, coinciding with HexNAc elongation starting from HexNAc1Hex3Cer (CTeS1) and mainly h20:0-d14:1 or h22:0-d14:1 ceramides. In the PSD mass spectra of COS (Fig. 4), fragment ions included all shorter sugar chain GSLs, each differing by m/z 203 and 162, which is consistent with a linear HexNAc–HexNAc–HexNAc–HexNAc–HexNAc–Hex–Hex–Hex–Cer sequence (precursor ion m/z 2105.7 and fragment ion m/z 1901.8, 1699.6, 1496.1, 1293.1 and 1090.1). Confirmation of arthro-series ceramide trisaccharide CTS1 in dried pupae The CTS fraction was separated into CTS1 (0.7 mg) and CTS2 (2.3 mg) by Iatrobeads column chromatography using the propanol:water:ammonium hydroxide solvent system. Fraction CTS1 containing GlcNAc eluted faster than CTS2. The sugar components of these CTSs, as determined by GC, GC-MS and 1 H NMR, were as follows: GlcNAcβ3Manβ4Glcβ1- (arthro-triaosyl) for CTS1 and Galβ3Manβ4Glcβ1- for CTS2 (Fig. 2 and Table II). GC indicated that the ceramide constituents of CTS1 were d14:1 as the major sphingoid (∼70% of the total content) and normal C20:0 and C22:0 acids as the major fatty acids (∼80% of the total content), whereas the ceramide constituents of CTS2 mainly consisted of d14:1 and 2-hydroxy C20:0 and C22:0 fatty acids. Comparison of GSL expression patterns between larvae and pupae GSL fractions from whole tissues were separated by column chromatography with ion-exchange Sephadex (DEAE- and QAE-Sephadex) and magnesium silicate (Florisil). A comparison revealed differing acidic (DEAE resin adsorbed), polar (QAE resin adsorbed) and neutral fractions between larvae and pupae, whereas the zwitterionic fraction was similar according to qualitative TLC results (Fig. 5). In the neutral GSL fraction, CDS, CTS and CTeS levels were lower in larvae than in pupae; however, similar amounts of the sample were added on the TLC plate, while CMS, CPS, CHS, CHpS and COS levels in larvae were similar to those in pupae. Fig. 5 View largeDownload slide Thin-layer chromatogram of GSL fractions from silkworm larvae and pupae. Lane S, neutral GSL fraction from the green bottle fly; lanes 1 and 2, sphingolipid fractions (150 and 300 μg added for TLC, respectively); lanes 3 and 4, acidic sphingolipid fractions (24 and 120 μg); lanes 5 and 6, polar sphingolipid fractions (75 and 210 μg); lanes 7 and 8, neutral sphingolipid fractions (12 and 15 μg); lanes 9 and 10, zwitterionic sphingolipid fractions (30 and 150 μg). Odd number lanes, lipid fractions from larvae; even number lanes, those from pupae. The plate was developed with chloroform:methanol:water (60:40:10, v/v/v) and the spots were visualized by the orcinol:H2SO4 reagent. Fig. 5 View largeDownload slide Thin-layer chromatogram of GSL fractions from silkworm larvae and pupae. Lane S, neutral GSL fraction from the green bottle fly; lanes 1 and 2, sphingolipid fractions (150 and 300 μg added for TLC, respectively); lanes 3 and 4, acidic sphingolipid fractions (24 and 120 μg); lanes 5 and 6, polar sphingolipid fractions (75 and 210 μg); lanes 7 and 8, neutral sphingolipid fractions (12 and 15 μg); lanes 9 and 10, zwitterionic sphingolipid fractions (30 and 150 μg). Odd number lanes, lipid fractions from larvae; even number lanes, those from pupae. The plate was developed with chloroform:methanol:water (60:40:10, v/v/v) and the spots were visualized by the orcinol:H2SO4 reagent. Comparison of ceramide composition in larval and pupal GSLs It is difficult to directly compare the amount of neutral GSLs in different preparations by the peak abundance in MALDI-TOF MS (Fig. 6). Differences in the ceramide composition of GSLs are reflected by numerous separated peaks, but the total peak abundance cannot be compared among preparations (such as those from larvae and pupae). However, the relative ratios of GSL species with different ceramide compositions were comparable within a given GSL fraction by peak abundance. In this experiment, the only exception was observed when contaminants disturbed the detection of the CDS molecular weight area at m/z 850–950. In the dried pupae used for structural analysis, the ceramide species in GSLs were predominantly c: h20:0-d14:1 (∼55%) and e: h22:0-d14:1 (∼45%). In bred pupae, the predominant ceramide species were d: h21:0-d14:0 (∼30%) and f: h23:0-d14:0 (∼70%), which were also minor components of dried pupae. Larvae contained all four of these ceramide species at proportions of ∼15% (c), ∼25% (d), ∼25% (e) and ∼35% (f), in addition to a minute amount of h22:0-d14:0 as a stage-specific species. Fig. 6 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSL fractions from larvae, pupae and dried pupae. Peaks c–f correspond to different ceramide compositions of GSL; c, h20:0-d14:1; d, h21:0-d14:0; e, h22:0-d14:1; and f, h23:0-d14:0. Insets: magnified molecular weight area of each CHS from larvae, pupae and dried pupae. Fig. 6 View largeDownload slide Positive-ion reflectron mode MALDI-TOF MS spectra of neutral GSL fractions from larvae, pupae and dried pupae. Peaks c–f correspond to different ceramide compositions of GSL; c, h20:0-d14:1; d, h21:0-d14:0; e, h22:0-d14:1; and f, h23:0-d14:0. Insets: magnified molecular weight area of each CHS from larvae, pupae and dried pupae. Discussion GSLs containing HexNAc (CTeS1 and CTeS2) eluted earlier than Hex (CTeS3) by Iatrobeads column chromatography using the propanol:water:ammonium hydroxide solvent system. Similar separation was observed for the separation of CTSs from the marine crab Erimacrus isenbeckii (25). In this experiment, GlcNAc-containing GSL (CTS1) was also separated from Gal-containing GSL (CTS2) because of its faster elution. Furthermore, GlcNAc-containing GSL (CTeS1) eluted faster than its constituent GalNAc (CTeS2). This separation is caused by a dimensional influence, such as linkage position at the secondary sugar (e.g. third or fourth position on galactose). Using silica gel chromatography with the propanol:water:ammonium hydroxide solvent system, GlcNAc containing-GSLs could also be separated, such as CTS1 (At3Cer: GlcNAcβ3Manβ4Glcβ1Cer). At3Cer has been identified from several animal species, such as fly (17, 18), moth (19), krill, shrimp (26), brine shrimp (20), crab (25), millipede (27) (all Arthropoda), parasitic Nematodes (28–31) and C. elegans (32, 33) (another Nematoda). Contrarily, no arthro-series GSLs have been detected in other animal species studied in our laboratory, such as Annelida (34–36) or Mollusca (37–40). This suggests that arthro-series CTS At3Cer functions as a common GSL in molting animals (Ecdysozoa) belonging to the phyla Arthropoda and Nematoda. Both CTS2 (Galβ3Manβ4Glcβ1Cer) and CTeS2 (GalNAcα4Galβ3Manβ4Glcβ1Cer) have been reported as non-arthro-series mannose-containing GSL in the cell line High-Five™ established from the ovarian cells of the cabbage lopper T. ni (order Lepidoptera, class Insecta, phylum Arthropoda) (16). In addition, CTS2 has been reported in the marine crab E. isenbeckii (class Crustacean, phylum Arthropoda) (25). The long sugar chain-containing neutral GSLs found in this study were characterized as novel silkworm-specific structures with GalNAc polymeric elongation started from CTS2. A characteristic sugar sequence (GalNAcα4Galβ3) has been characterized in the O-linked oligosaccharide of the jelly coat surrounding the eggs of frog (41), and a similar structure has been found as GalNAcα4GalNAcβ in the arthro-series GSLs (At5Cer: GalNAcα4GalNAcβ4GlcNAcβ3Manβ4Glcβ1Cer) of fly (42, 43). Most GalNAcs are observed as β-linked moieties in oligosaccharide structures. Among GSLs, a characteristic sugar sequence (GalNAcα4Gal) appears to be a common structure in Lepidoptera. Another characteristic feature is the presence of mono sugar polymeric sequences. Heptamer-GalNAcα1-4 has not been identified in any animal GSL, whereas tetramer-Galβ1-6 sequence has been characterized in GSLs of the tapeworm Taenia crassiceps (44). Most GSL structures are composed of hetero sugars or repeating units such as LacNAc (20, 45–47). The main molecular species composing ceramide in each purified GSL from dried pupae were h20:0 and h22:0-d14:1 (fatty acid-sphingoid). These hydroxy fatty acid components (h20:0 and h22:0) are characteristic features of GSLs found in silkworms. The ceramides of a large number of GSLs in most invertebrate species consist of saturated fatty acids and dihydroxy-sphingoid. Hydroxy fatty acid was the major fatty acid constituent only in krill (h22:1 and h24:1) (48) and roundworm (h24:0) (30, 32). The length of the fatty acid chain in B. mori (20:0 and 22:0) is similar to that found in other invertebrate animals. The GlcNAcβ3Manβ4Glcβ1- sequence containing arthro-series CTS1 was composed of normal C20:0 and C22:0 acids, whereas the Galβ3Manβ4Glcβ1- sequence containing GSLs was composed of h20:0 and h22:0 acids. Ceramide mono- and disaccharides possess both fatty acid components as biosynthesis precursors. The structure of Golgi apparatus is slightly different in mammalian cells and insect cells, such as Drosophila, which is a well-studied invertebrate model (49). The Golgi found in insect is not present as a flattened compartment observed in mammalian cells. The Golgi organization in Drosophila cells are not interconnected to form a single-copy organelle. The fly’s Golgi stacks remain dispersed throughout the cytoplasm and are found in close association to tER sites to form ‘tER-Golgi units’ (50). This may explain why we observed such changes in ceramide composition in CTS1 and CTS2 as each tER-Golgi units may be able to preferentially synthesize specific GSLs with specific ceramide compositions. In contrast, two types of GSLs had been purified: Galβ3Manβ4Glcβ1Cer as major GSL and At3Cer as a minor component from the cell line High-Five™ (16), though the ceramide composition has not been particularly described. It seems that the cell can biosynthesize different sugars of GSLs with the same ceramide pool by regulating glycosyltransferase expression or activity. It is possible that this particular ceramide composition is derived from the glycosyltransferase specificities or biosynthesis of GSLs within organs with distinct ceramide pools. After the characterization of major GSLs from the whole body of silkworms in this study, we should perform detailed analysis in the future on GSL expression patterns within ceramide composition in different organs or several stages in silkworms. However, this difference appears to derive from our selected purification techniques, which can give rise to only highly purified fractions from several column chromatograms. In particular, the minor GSL components eluted from the combination of column chromatograms with solvent systems of chloroform: methanol:water and 1-propanol:water:28% ammonium hydroxide were not investigated for insufficient yields to chemical structural analysis. Tetradeca- and hexadeca-4-sphingenines detected in silkworms have also been reported as major sphingoid species in arthropods:fly (d14:1 and d16:1) (51, 52), crab (d14:1) (25) and brine shrimp (d16:1) (20). Although the hexadecasphinganine content (∼10%) in silkworms is similar to that in crabs, the tetradecasphinganine content (also ∼10%) is higher than that in any other examined species. In Monduca sexta, another moth, predominant sphingoids are the same as those in flies and silkworms (d14:1 and d16:1), specifically doubly unsaturated sphingoids such as tetradecasphiga-4,6-diene and hexadecasphiga-4,6-diene (14:2 and 16:2) and d14:0 and d16:0 in minute amounts (19). In silkworms, we could not detect doubly unsaturated sphingoids by GC analysis. Several molecular masses consistent with odd-numbered fatty acids (h21:0 or h23:0) and sphinganine (d14:0 or d16:0) were detected by MALDI-TOF MS analysis. Other analytically noteworthy results in NMR spectra were the presence of two doublet anomeric signals of Glc with chemical shifts of 4.14 and 4.13 ppm (CMS) and 4.18 and 4.15 ppm (CDS) (Fig. 2 and Table II). Similar to phosphonocerebroside from the Antarctic krill (48), the division of anomeric signals was reflected by the contents of hydroxy and nonhydroxy fatty acids in GSLs. Compositions and expression patterns of fats and proteins vary during silkworm development and transformation, particularly body weight, water content and lipid content. After starvation, the larval body is rebuilt during pupation with the excretion of water and storage of fat (as accumulated triacylglycerides). During lipid extraction, bulk amounts of simple lipids within the body of pupa contaminate the total lipid fraction and it is difficult to separate a group of simple lipid contaminants from the sphingolipid fraction even after alkaline treatment. Further, a positive spot with the orcinol reagent at the origin on TLC plates contaminates each preparation except the neutral fraction. We attempted to compare five fractions between larval and pupal components by TLC, but several contaminants disturbed the quantitative analysis. In Fig. 5, pupal GSLs were spotted on the TLC plate with 2–5-fold greater amount than for larval GSLs, whereas the neutral fractions of larval and pupal GSLs were spotted at almost same amounts. The comparison of GSL expression patterns on TLC revealed that expression levels of several neutral GSLs were lower in larvae than in pupae. These GSLs were defined as CDS, CTS and CTeS by qualitative analysis after separation and chemical structural analysis (Supplementary Fig. S3), whereas TLC showed similar expression levels of acidic, polar and zwitterionic fractions. Further studies of differences in minor components of ionic fractions are still required. The biosynthesis of GSL in insects, particularly Drosophila, has been well studied due to the identification of two mutants, egghead and brainiac, which are glycosyltransferases responsible for GSL synthesis (47, 53–56). Egghead and brainiac have a Notch-like neuronal hypertrophic phenotype and defective EGF-R signalling in oocytes (57–59). The β4GalNAcT A enzyme catalyzes the elongation of GSLs, and the mutation causes defects in neuromuscular junction innervation (60, 61). In contrast, a β4GalNAcT B mutant exhibited ventralizing ovarian follicles, similar to egghead, brainiac and EGF-R knockouts (62). Besides, the overexpression of α4GalNAcT1/2, which is responsible for the biosynthesis of long sugar chains attached to GSLs, suppresses the mindbomb Notch-like phenotype and inhibit apoptosis in eye disks (63). The change in MacCer expression between larvae and pupae suggests that the egghead gene is involved in the biosynthesis of GSLs in silkworms. Moreover, At3Cer, which is a common GSL in Arthropoda, exists as only a minor component in silkworms, whereas αGalNAc-rich GSLs predominate. Brainiac or α4GalNAcT1/2 expression in silkworms may regulate the expression patterns and consequently the function of GSLs. In Fig. 6, differences in the ceramide composition of GSLs were observed among larvae, pupae and dried pupae. The biosynthesis of odd fatty acid-saturated sphingoid components are accumulated in pupa or conserved, whereas the ratio of ceramide with even fatty acid-unsaturated sphingoid components is reduced during development. The histolysis and remodelling of the organism during transformation from larvae and pupae may require selective changes in the biosynthesis of GSLs. The ceramide composition of dried pupae significantly differed from that in bred pupae, but all components found in dried and bred pupae were found in larvae. There are several possibilities for the difference in ceramide composition between bred pupae and dried pupae: (i) sample preparation, (ii) difference in the number of days after pupation or (iii) difference in subspecies. We speculate that the differences arose from the processing of dried pupae purchased from a silk string manufacturer. These pupae were obtained after cocoon removal following treatment with boiling water, and they were then dried under the sun. During boiling and drying, some decomposition of unstable ceramide components may have occurred. In addition, larvae molt in the cocoon to form pupae within ∼2 days after cocoon spinning, and imagoes hatch from the cocoon after ∼2 weeks. At silk factories, pupae are collected on different days after spinning off the silk. In this experiment, pupae were collected 5 days after cocoon spinning, which is a relatively early pupal stage. It will be necessary to study the compositional change between pupae and imagoes. Further, there are three silkworm subspecies bred by Japanese silk companies. The difference is at the subspecies level lies mainly during seasonal breeding. Dried pupae we obtained from the company in bulk and contained a mixture of subspecies, whereas all bred silkworms used for the comparison belonged to the same species. Therefore, it is unlikely that differences in ceramide composition are due to differences in subspecies. In light of the differential expression of GSLs between silkworm larvae and pupae observed in this experiment, GSL expression changes across developmental stages from the larva to the imago by an in-depth analysis of glycan sequence and ceramide composition should be investigated. In this study, we profiled silkworm GSLs in different biological forms to study how the biosynthesis of GSLs is regulated during transformation, which involves various glycan-associated enzymes as well as ceramide synthases. GSL expression is spatially and temporally regulated across developmental stages and organs. This has been particularly well examined for the ganglioside expression pattern in the nervous system. For instance, alterations in frog GSL expression patterns in the nervous system have been reported during metamorphosis (64), but there is no significant difference in the ceramide composition of frog GSLs. GSL composition also changes during the differentiation of human leukemic granulocytes and the promyelocytic leukemic cell line HL-60 (65). With respect to the composition and content of GSLs, there are no significant differences between normal and leukemic mature neutrophils, and they synthesize the same ceramide species (66). In contrast, changes in the ceramide composition of bovine milk gangliosides with the stage of lactation have been reported (67). A significant decrease in saturated and long-chain fatty acids with concomitant increases in C18:1 and C18:2 have been observed from colostrum to milk. Thus, altered ceramide composition reflects developmental changes and possibly associated functional changes. In this study, we applied high-resolution MALDI-TOF MS to analyse silkworm GSLs and observed alteration in ceramide composition during transformation; changes were not detectable using traditional TLC analysis. Our sequential GSL enrichment protocol using magnesium silicate column chromatography (Florisil) and silica gel chromatography (Iatrobeads) is capable of producing highly purified GSLs from a crude lipid mixture and can be applied for any lipid extract from various biological samples (20, 25, 68). Highly purified GSL preparations are crucial to profile the heterogeneity of ceramide composition by in-depth glycolipidomics analysis. Supplementary data Supplementary data are available at JB Online. Acknowledgements We acknowledge the valuable comments provided by Dr. Kazuhiro Aoki of the University of Georgia. The authors would like to thank Enago (www.enago.jp) for the English language review. Funding This study was supported in part by the Grant-in-Aid for Scientific Research (C) (22500276) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Shiga University Research Support Fund from Shiga University. Conflict of Interest None declared. References 1 Gunsalus K.C., Piano F. ( 2005) RNAi as a tool to study cell biology: building the genome-phenome bridge. Curr. Opin. 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The Journal of BiochemistryOxford University Press

Published: Mar 1, 2018

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