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Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase

Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:β-D-Galactoside... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 28, Issue of July 12, pp. 16975–16981, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:b-D-Galactoside 2-a-L-Fucosyltransferase* (Received for publication, October 4, 1995, and in revised form, April 15, 1996) ¶ i Seiji Hitoshi‡§ , Susumu Kusunoki§, Ichiro Kanazawa§, and Shuichi Tsuji‡ From ‡Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Saitama 351-01, Japan and the §Department of Neurology, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan Recent molecular investigation revealed that two fluids of mammals that had several different kinetic character- closely related structural genes encode distinct GDP-L- istics (1–5). In humans, genetic and biochemical studies have fucose:b-D-galactoside 2-a-L-fucosyltransferases (a1,2-fu- indicated that two distinct but closely linked structural genes cosyltransferases). Some human cancer cells or tissues (H and Se) code a1,2-fucosyltransferases with tissue-specific may express an aberrant a1,2-fucosyltransferase other patterns (1, 6). The human H gene controls the expression of H than H- and Secretor-type a1,2-fucosyltransferase. How- (Fuca1,2Galb) antigens (along with A or B antigens or both) on ever, definite evidence of the existence of a third type of erythrocytes, whereas the Se gene determines the soluble A, B, a1,2-fucosyltransferase has not been demonstrated. and H antigens in secretory glands, and Lewis blood group Here we report the molecular cloning of a third type of antigens on red cells (for review, see Ref. 7). Homozygosity for rabbit a1,2-fucosyltransferase (RFT-III) from a rabbit null alleles for the H and Se genes yields the rare Bombay blood genomic DNA library. The DNA sequence included an type and non-Secretor phenotype, respectively. Recent molecu- open reading frame coding for 347 amino acids, and the lar cloning of the H and Se genes provided the molecular basis deduced amino acid sequence of RFT-III showed 59 and for the Bombay and para-Bombay blood types and the non- 80% identity with those of the previously reported two Secretor phenotype, respectively, revealing point mutations types of rabbit a1,2-fucosyltransferase, RFT-I and RFT- II, respectively. COS-7 cells transfected with the RFT-III within the coding regions that abolish the a1,2-fucosyltrans- gene exhibited a1,2-fucosyltransferase activity toward ferase activity (8–10). On the other hand, the Lewis phenotyp- phenyl-b-Gal as a substrate. Neuro2a (a murine neuro- ing of erythrocytes and secretory glands revealed the Le(a1b1) blastoma cell line) cells transfected with the RFT-III and partial Secretor phenotypes in selected Polynesian and gene expressed fucosyl G (type 3 H) but not Ulex eu- M1 Asian individuals (11, 12). These phenotypes, which are virtu- ropaeus agglutinin-1 lectin reactive antigens (type 2 H). ally absent in Caucasians, are thought to be caused by weak Kinetic studies revealed that RFT-III exhibits higher Se-type a1,2-fucosyltransferase activity. The molecular basis of affinity to types 1 (Galb1, 3GlcNAc) and 3 (Galb1, 3Gal- weak Secretor phenotypes, whether weak Se-type a1,2-fucosyl- NAc) than to type 2 (Galb1, 4GlcNAc) oligosaccharides, transferase is encoded by an altered Se gene or a gene other which suggests that RFT-III as well as RFT-II is a Secre- than H and Se, has yet to be determined. tor-type a1,2-fucosyltransferase. RFT-III was expressed Recently, aberrant a1,2-fucosyltransferase activity, which in the adult gastrointestinal tract. The RFT-I, -II, and b a y x synthesized Le from Le or Le from the Le determinant, or -III genes were assigned within 90 kilobases on pulsed both, was found in cancer cells and tissues, suggesting the field gel electrophoresis analysis. These results consti- possibility of a third distinct a1,2-fucosyltransferase gene (13, tute direct evidence that, at least in one mammalian 14). In the rabbit, the possibility of a third type of a1,2-fuco- species, three active a1,2-fucosyltransferases exist. syltransferase was suggested by immunohistochemical studies on DRG neurons. We recently cloned two types of rabbit a1,2- GDP-L-fucose:b-D-galactoside 2-a-L-fucosyltransferase (a1,2- fucosyltransferase, RFT-I and RFT-II, showing that RFT-I but fucosyltransferase) catalyzes the fucosylation of terminal b-D- not RFT-II is expressed in postnatal rabbit brain (15). RFT-I Gal residues and synthesizes H antigens. The activity of a1,2- shows comparable affinities to types 1, 2, and 3 acceptors, fucosyltransferase was detected in various tissues and body which suggests that the binding specificity of RFT-I is primar- ily restricted to the terminal b-D-Gal residues of acceptors. In rabbit DRG neurons, fucosyl G (type 3 H) is readily detected M1 * This work was supported by Grants-in-aid for Scientific Research immunohistochemically on embryonic day 25, followed by the on Priority Areas 05274103, 06254213, 07279249, and 07273274 and appearance of UEA-1 lectin-reactive antigens (type 2 H) post- Grant-in-aid for Scientific Research (C) 07680860 from the Ministry of Education, Japan. The costs of publication of this article were defrayed natally (16, 17). UEA-1 lectin-reactive antigens of DRG neu- in part by the payment of page charges. This article must therefore be rons in postnatal rabbits could be formed through fucosylation hereby marked “advertisement” in accordance with 18 U.S.C. Section catalyzed by RFT-I. In contrast, fucosyl G observed in DRG M1 1734 solely to indicate this fact. The nucleotide sequences reported in this paper have been submitted neurons of embryonic day 25 rabbits might not be the product TM to the GenBank /EMBL Data Bank under accession number 91269. of RFT-I because UEA-1 lectin-reactive antigens are not de- ¶ Research Fellow of the Japan Society for the Promotion of Science. tected at that stage. This observation suggests the existence of To whom correspondence should be addressed. Tel.: 81-48-462-1111 another type of a1,2-fucosyltransferase that catalyzes prefer- (ext. 6521); Fax: 81-48-462-4692. The abbreviations used are: a1,2-fucosyltransferase, GDP-L-fucose: ential fucosylation to type 3 rather than type 2 glycochains. b-D-galactoside 2-a-L-fucosyltransferase; Se, Secretor; Le, Lewis; DRG, Here we report the molecular cloning of a third type of rabbit dorsal root ganglia; UEA-1, Ulex europaeus agglutinin-1; FITC, fluores- a1,2-fucosyltransferase, which could synthesize fucosyl G . M1 cein isothiocyanate; kb, kilobase(s); UTR, untranslated region; PBS, This is the first direct evidence that, at least in one mammalian phosphate-buffered saline. The nomenclature for gangliosides and gly- colipids follows the system of Svennerholm (25). species, three active a1,2-fucosyltransferases exist. This is an open access article under the CC BY license. 16975 16976 Third Type of Rabbit a1,2-Fucosyltransferase FIG.1. Restriction maps for RG193 and the sequence analysis strategy. The coding region is depicted as a shaded box and the non-coding region as a solid line. The arrows indicate the direction and extent of sequencing. EXPERIMENTAL PROCEDURES fragment from 59-UTR and the coding region of RFT-III for Northern blotting. Materials—GDP-Fuc, phenyl-b-D-Gal, Galb1,3GlcNAc, Galb1, Expression of a1,2-Fucosyltransferase (RFT-III)—A 1.3-kb PstI frag- 4GlcNAc, Galb1,3GalNAc, lacto-N-tetraose, lacto-N-neotetraose, lacto- ment of RG193 DNA containing the full open reading frame of RFT-III N-fucopentaoses II and III, G , and FITC-labeled UEA-1 lectin were M1 14 32 was ligated into mammalian expression vector pcD-SRa (19), yielding from Sigma. GDP-[ C]Fuc (10.5 GBq/mmol) and [a- P]dCTP (111 pcD-SRa-RFT-III. The single insertion in the correct orientation was TBq/mmol) were from DuPont NEN. lipofectAMINE was from Life finally analyzed with restriction enzymes. Technologies, Inc. Paragloboside was from Dia-Iatron (Tokyo, Japan). Neuro2a (a murine neuroblastoma cell line) cells were transiently The anti-fucosyl G mouse monoclonal antibody was developed in our M1 transfected with pcD-SRa-RFT-III by means of LipofectAMINE accord- laboratory (17). FITC-labeled anti-mouse Ig (G and M) was from Tago, ing to the manufacturer’s instructions. The cells were trypsinized and Inc. (Burlingame, CA). Restriction endonucleases were from Takara divided into several dishes at 24 h post-transfection. The cells from one (Kyoto, Japan). dish were stained with FITC-labeled UEA-1 lectin or anti-fucosyl G M1 Cloning of a Third Type of Rabbit a1,2-Fucosyltransferase—The antibody at 72 h post-transfection. After washing with PBS, the cells standard molecular cloning techniques described by Maniatis and co- were fixed with formaldehyde for 3 min, washed, and then incubated in workers were used (18). The construction of a rabbit genomic DNA 1% bovine serum albumin/PBS. After washing briefly with PBS, the library and subcloning of polymerase chain reaction fragments (229 cells were incubated in 2 ng/ml FITC-labeled UEA-1 lectin in 1% bovine base pairs) used as probes were described previously (15). We used the serum albumin/PBS for 1.5 h or in anti-fucosyl G monoclonal anti- M1 polymerase chain reaction fragments as probes for further cloning of body for 1.5 h and then washed with PBS, followed by incubation with a1,2-fucosyltransferase, because these probes hybridized to more than 6 FITC-labeled anti-mouse Ig (G and M) for 1 h. After washing three two bands on rabbit genomic Southern blotting. Another 2 3 10 times with PBS, the cells were observed under a fluorescence micro- plaques of the rabbit genomic DNA library were screened and a scope. The cells from another dish were trypsinized, fixed with formal- positive plaque containing a 16.8-kb (RG193) insert was isolated to dehyde, and stained as above for flow cytometry analysis using FACS- homogeneity. Flow (Becton Dickinson). Transfection of the gene was verified by The RG193 DNA fragments were digested with appropriate restric- measuring fucosyltransferase activity using cell extract from another tion enzymes and then subcloned into vector plasmid pUC 119. The dish. DNA sequences were determined by the dideoxynucleotide chain-ter- COS-7 cells (60-mm culture dish) were transiently transfected with 5 mination method using an Autocycle DNA sequencing kit and an A.L.F. mg of pcD-SRa-RFT-III using the DEAE-dextran procedure (20). The DNA sequencer (Pharmacia Biotech Inc.). The sequences were analyzed cells were washed with PBS, washed with 25 mM 4-morpholineethane- using PC/Gene (Teijin System Technology, Osaka, Japan). sulfonic acid for 10 min, and then collected with a rubber policeman and Pulsed Field Gel Electrophoresis and Northern Hybridization—Rab- pelleted by centrifugation. The pellets were resuspended in 100 mlof bit lymphocytes were prepared by density gradient (Lympholyte-Rab- cold 1% Triton X-100 and then sonicated briefly. bit; Cedarlane, Hornby, Canada), suspended in agarose blocks, and Fucosyltransferase Assay—The fucosyltransferase assays were per- then digested with 0.2 mg/ml proteinase K and 1% Sarkosyl. Digestion formed according to previous reports (15, 21) in a mixture of 25 mM of DNA suspended in agarose blocks was carried out using 10 units of sodium phosphate (pH 6.1), 5 mM ATP, 30 mM GDP-fucose, 3 mM GDP- restriction endonucleases in appropriate restriction buffers at 37 °C for [ C]fucose (10.5 Bq/pmol), the enzyme solution, and substrates in a 3 h. Pulsed field gel electrophoresis was performed using pulse times of final volume of 10 ml. Each reaction mixture for oligosaccharide accep- 0.2–18 s linearly ramped at 200 V for 18 h. After electrophoresis, the tors was incubated at 37 °C for 2 h and then applied to a Silica Gel 60 gels were stained with ethidium bromide to visualize size markers high performance thin layer chromatography plate (Merck). The plate followed by alkali transfer onto nylon membranes (Nytran; Schleicher was developed with ethanol/pyridine/1-butanol/water/acetic acid (100: & Schuell). Hybridization was performed in 6 3 SSC, 5 3 Denhardt’s, 10:10:30:3). When glycolipids were used as substrates, the reaction 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA at 65 °C. mixture was applied to a C-18 Sep-Pak cartridge (Waters-Millipore, Total RNA was prepared by the guanidium thiocyanate method and Milford, MA) after2hof incubation at 37 °C, washed with 2 ml of water, purified by ultracentrifugation through 5.7 M CsCl. Poly(A)-rich RNA and then eluted with 1 ml of methanol. The eluate was then applied to was purified with Oligotex-dT30 (Takara). The poly(A)-rich RNA (5 mg) a high performance thin layer chromatography plate, which was devel- was fractionated on a denaturing formaldehyde-agarose gel (1.2%) and oped with chloroform/methanol/0.5% CaCl (55:45:10). For glycoprotein then transferred onto a nylon membrane. Northern filters were hybrid- acceptors, the reaction was terminated by the addition of 10 mlof ized in 50% formamide, 5 3 saline/sodium/phosphate/EDTA, 5 3 Den- SDS-polyacrylamide gel electrophoresis loading buffer after2hofin- hardt’s, 0.5% SDS, 0.25% sodium lauryl Sarkosyl, and 100 mg/ml dena- cubation at 37 °C, and the incubation mixtures were directly subjected tured salmon sperm DNA at 37 °C. To quantify the RNA loading, the to SDS-polyacrylamide gel electrophoresis. The radioactivity on each Northern filters were rehybridized with a labeled cDNA fragment of plate and gel was visualized and determined with a BAS2000 radioim- rabbit glyceraldehyde-3-phosphate dehydrogenase. 32 age analyzer (Fuji Film, Tokyo, Japan). All probes were labeled with [a- P]dCTP, using the random priming method. The gene probes used for hybridizations were a 1.8-kb SalI- RESULTS SacI fragment from 39-UTR of RFT-I, a 0.7-kb SacI-PstI fragment from Cloning and Nucleotide Sequence of a Third Type of Rabbit 59-UTR of RFT-II (15), a 0.6-kb SacI-EcoRI fragment from 39-UTR of RFT-III for pulsed field gel electrophoresis, and a 0.3-kb PstI-NaeI a1,2-Fucosyltransferase—On screening of a rabbit genomic Third Type of Rabbit a1,2-Fucosyltransferase 16977 FIG.2. Nucleotide and deduced amino acid sequences of rabbit RFT-III (A), and comparison of the deduced amino acid sequences of rabbit and human a1,2-fucosyltransferases (B). A, nucleotide and amino acid sequences are numbered from the presumed start codon and initiation methionine, respectively. The boxed amino acids correspond to a putative signal-anchor domain. The asterisks indicate potential N-glycosylation sites (Asn-X-Ser/Thr). B, each pair of amino acid sequences is aligned by the PALIGN software of PC/Gene and adjusted by eye. A putative signal-anchor domain is boxed. Potential N-glycosylation sites are circled. The asterisks indicate that all the aligned residues of active a1,2-fucosyltransferases (RFT-I, -II, and -III and human H and Se genes) are identical. observed among rabbit and human DNA library with polymerase chain reaction fragments (229 a1,2-fucosyltransferases. base pairs) as probes, one positive clone, RG193, was obtained Comparison of the primary structure of RFT-III revealed sig- (Fig. 1). Sequence analysis revealed that RG193 contained an nificant (59 and 80%) amino acid identity with those of RFT-I entire open reading frame with three closely located in-frame and RFT-II, respectively. The putative extracellular region methionine codons in the starting region (Fig. 2A). The last of composed of the glycine residue at position 66 to the alanine the three methionine codons was a good candidate for an ini- residue at position 339 of RFT-III was highly conserved among tiator, accompanied by the Kozak consensus translation initi- not only rabbit but also human a1,2-fucosyltransferases (Fig. ation sequence. The open reading frame beginning with the last 2B), suggesting that this region is an active domain. In partic- methionine codon encodes 347 amino acids with a predicted ular, this region was highly conserved among RFT-II and RFT- molecular mass of 39 kDa (RFT-III). The deduced amino acid III, containing a 515-base-pair stretch of identical bases with sequence contains a putative transmembrane region in the two exceptions. The deduced amino acid identities between NH -terminal region, indicating RFT-III is a type 2 protein, as rabbit and human a1,2-fucosyltransferases are summarized in is usual with glycosyltransferases. RFT-III contains three po- Table I. In this case, the amino acids were translated from the tential N-linked glycosylation sites, two of which are commonly Sec1 DNA sequence (10), correcting the frameshift that dis- 16978 Third Type of Rabbit a1,2-Fucosyltransferase TABLE I rupted the reading frame of Sec1 and yielding a long reading Comparison of deduced amino acids between rabbit and frame appropriately corresponding to other a1,2-fucosyltrans- human a1,2-fucosyltransferases ferases. The deduced amino acid identities of the entire sequence, accompa- Pulsed Field Gel Electrophoresis and Northern Hybridiza- nied by the putative cytoplasmic, transmembrane, and stem/active tion—By analyzing human chromosome 19 cosmid libraries, domains in parentheses, are shown. Amino acid length and calculated molecular weight of polypeptide deduced from each gene are also the physical relationship of human a1,2-fucosyltransferases shown. The deduced amino acids for Sec1 were translated, correcting and the a1,2-fucosyltransferase-related genes was defined, re- the frameshift that disrupts the reading frame of Sec1 and yielding a vealing that all genes are located within 80 kb—the Se (Sec2) long reading frame appropriately corresponding to other a1,2-fucosyl- gene is 35.5 and 12 kb apart from the H (FUT1) and Sec1 genes, transferases. respectively (22). To determine the physical relationship of the Human RFT-I, -II, and -III genes, pulsed field gel electrophoresis was a b b Rabbit M H (FUT1) Se (Sec2) Sec1 performed. All probes recognized a common 90-kb SalI frag- (365 aa) (343 aa) (347 aa) 41,249 39,017 39,099 ment, and the RFT-II and -III probes recognized a common 100-kb NotI fragment and a common 105-kb MluI fragment %%% (Fig. 3). These results suggested that maximum distance RFT-I (373 aa) 42,098 80 (59/88) 59 (10/70) 53 (17/64) encompassing these genes to be 90 kb and that both RFT-II RFT-II (354 aa) 40,082 54 (12/68) 74 (28/86) 76 (70/79) RFT-III (347 aa) 39,469 59 (12/70) 83 (54/89) 67 (22/79) and -III genes are located in 39 region of RFT-I, because the a RFT-I probe locates 39 side of the SalI restriction site in DNA sequence from Ref. 8. DNA sequence from Ref. 10. RG11. DNA sequence from Ref. 15. aa, amino acid. FIG.3. Pulsed field gel electrophoresis analysis. Sequential hy- bridization with RFT-I, -II, and -III gene probes on pulsed field gel electrophoresis blots separating DNA fragments. All probes recognized a common SalI (90 kb) restriction fragment, and the RFT-II and -III gene probes recognized common NotI (100 kb) and common MluI (105 kb) restriction fragments. FIG.5. Expression of fucosyl G on RFT-III transfected M1 Neuro2a cells. A, Neuro2a cells were transiently transfected with 5 mg of pcD-SRa or pcD-SRa-RFT-I, -II, or -III using LipofectAMINE. The cells were stained with FITC-labeled UEA-1 lectin or anti-fucosyl G M1 FIG.4. Northern blot analysis of RFT-III. Poly(A)-rich rabbit monoclonal antibody followed by FITC-labeled anti-mouse Ig (G and M) RNAs (5 mg) were electrophoresed on a denaturing formaldehyde-aga- at 72 h post-transfection and observed under fluorescent microscopy. B, rose gel, transferred onto a nylon membrane, and hybridized with the transfected Neuro2a cells from another dish were trypsinized, stained 0.3-kb PstI-NaeI fragment of RFT-III (upper panel). The lower panel as above, and subjected to flow cytometry. Transfection of RFT-I, -II, or shows the same blot rehybridized with a labeled rabbit glyceraldehyde- -III was verified by measuring fucosyltransferase activity using the cell 3-phosphate dehydrogenase (GAPDH) cDNA probe. extract from another dish. Third Type of Rabbit a1,2-Fucosyltransferase 16979 TABLE II Acceptor specificities of RFT-I, -II, and -III The apparent K and V /K values of RFT-III and those of RFT-I and RFT-II reported previously are shown. The relative a1,2-fucosyltrans- m max m ferase activities of the incorporation of fucose into phenyl-b-D-Gal as a substrate are also shown. The actual activities of phenyl-b-D-Gal as an acceptor were measured with cell extracts prepared at the same time and under the same conditions. a a RFT-III RFT-I RFT-II Acceptor 211 211 211 K V /K (310 l/min) K V /K (310 l/min) K V /K (310 l/min) m max m m max m m max m mM mM mM Phenyl-b-D-Gal 5.7 0.82 4.0 2.3 17.1 0.13 Galb1,3GlcNAc 1.5 0.49 3.1 11.4 2.2 0.36 Galb1,4GlcNAc 6.7 0.65 4.2 8.9 12.5 0.21 Galb1,3GalNAc 1.0 0.75 3.5 5.8 4.6 0.50 Lacto-N-tetraose 1.6 0.30 3.5 3.0 4.8 0.51 Lacto-N-neotetraose 4.2 0.23 5.5 1.2 12.5 0.29 Relative activity RFT-III RFT-I RFT-II %%% b b b Phenyl-b-D-Gal (25 mM) 100 (1.70) 100 (3.38) 100 (0.21) Asialofetuin (1 mg/ml) 2.7 2.4 ND Asialo-a1-acid glycoprotein (1 mg/ml) 0.6 0.9 ND G (2 mM) 31.0 19.7 15.7 M1 Paragloboside (2 mM) 5.6 23.0 ND Lacto-N-fucopentaose II (2 mM) 3.0 2.3 13.8 Lacto-N-fucopentaose III (2 mM) 2.0 1.1 ND From Ref. 15. The actual activities (pmol/h/ml of enzyme) of fucosyltransferases are shown in parentheses. ND, not detected. FIG.6. Lineweaver-Burk plots. Lin- eweaver-Burk plots used to calculate the K and V /K values are shown. Exper- m max m iments were performed in triplicate and typical plots are shown. Northern blot analysis revealed that a 1.8-kb mRNA of RFT- Expression of RFT-III and Enzyme Assaying—Neuro2a cells III was expressed in the adult gastrointestinal tract (Fig. 4). transiently transfected with pcD-SRa-RFT-I, -II, and -III The expression of minor 5.0- and 6.4-kb mRNAs of RFT-III was showed a1,2-fucosyltransferase activity of 1.93, 0.12, and 0.52 also observed in the adult colon. Other secretory glands such as pmol/h/ml of enzyme, respectively, whereas parent Neuro2a salivary and mammary glands did not express RFT-III. cells contained no activity. Neuro2a cells transfected with RFT- 16980 Third Type of Rabbit a1,2-Fucosyltransferase III expressed fucosyl G but not UEA-1 lectin-reactive anti- Se-type a1,2-fucosyltransferase. The weak Se-type a1,2-fuco- M1 gens (Fig. 5, A and B), which suggested that the binding spec- syltransferase was postulated based on the results of Lewis ificity of RFT-III differed between type 2 and type 3 glycochains phenotype analysis in Polynesian people. The Lewis antigens or between glycoproteins and glycolipids. Neuro2a cells trans- on erythrocytes are regulated by two fucosyltransferases, Se- fected with RFT-I expressed both fucosyl G and UEA-1 lec- type a1,2- and Lewis a1,3/4-fucosyltransferases. With the con- M1 tin-reactive antigens, and Neuro2a cells transfected with ventional analysis method, three Lewis phenotypes of erythro- RFT-II expressed neither of them (Fig. 5, A and B). Cell ex- cytes were found in Caucasian adults, Le(a2b2), Le(a1b2), tracts of COS-7 cells transfected with pcD-SRa-RFT-III con- and Le(a2b1). When Se-type a1,2-fucosyltransferase is active tained a1,2-fucosyltransferase activity that transferred radio- (or in Secretor), most of the type 1 precursor is converted into labeled fucose to phenyl-b-D-Gal as a substrate. As shown in type 1 H, which can be transformed into Le by Lewis a1,3/4- Table II and Fig. 6, the acceptor specificity of RFT-III was fucosyltransferase. On the other hand, a fourth Lewis pheno- comparable with that of RFT-II. RFT-III showed higher affinity type, Le(a1b1), was found on erythrocytes from selected Pol- for types 1 and 3 acceptors than for type 2 acceptors and ynesian individuals. In addition, low levels of salivary ABH phenyl-b-D-Gal, like RFT-II and human Se-type a1,2-fucosyl- antigens, that is partial secretion, were found in saliva from transferase. RFT-III could transfer fucose to asialofetuin and Le(a1b2) and Le(a1b1) individuals, suggesting the presence asialo-a1-acid glycoproteins as well as G ganglioside. The of a weak Se-type a1,2-fucosyltransferase (11, 12). Molecular M1 relative activity of RFT-III toward G as to glycoproteins was analysis of the Se and Sec1 genes of Polynesian people, espe- M1 higher than that of RFT-I. RFT-III could also fucosylate lacto- cially of partial Secretor individuals, will facilitate determina- N-fucopentaoses II and III, a1,3- and a1,4-fucosylated oligosac- tion of whether or not RFT-II corresponds to the human Sec1 charides, respectively. gene. The fucosyltransferase assay showed that the relative activ- DISCUSSION ity of RFT-III toward G as to glycoproteins was higher than M1 In this and previous work (15) we reported the molecular that of RFT-I. RFT-III could also synthesize fucosyl G from M1 cloning of three types of rabbit a1,2-fucosyltransferase, RFT-I, G but not UEA-1 reactive antigens when expressed in M1 -II, and -III. These results constitute direct evidence that, at Neuro2a cells, where RFT-I could form both and RFT-II could least in one mammalian species, three active a1,2-fucosyltrans- synthesize neither under the same transfection conditions. ferases exist, one H type and two Se types, based on kinetic RFT-III is a good candidate for the enzyme that synthesizes analysis. fucosyl G expressed in a subpopulation of neurons of rabbit M1 RFT-I exhibits comparable kinetic properties and significant embryonic DRG (16, 17), although we could not detect the structural homology with human H-type a1,2-fucosyltrans- expression of RFT-III in embryonic brain. It is possible that the ferase, indicating that RFT-I is a counterpart of human H. expression of RFT-III is restricted to specific regions or specific RFT-II and -III show higher affinity to types 1 and 3 acceptors types of neurons. In situ hybridization analysis will provide than to type 2 acceptors and phenyl-b-D-Gal. The kinetic pa- further information. rameters of RFT-II and -III are comparable with those of hu- Recently, aberrant a1,2-fucosyltransferase activity that syn- b a y x man Se-type a1,2-fucosyltransferase (2, 3, 5). RFT-II and -III thesized Le from Le or Le from the Le determinant, or both, genes share remarkably conserved base pair sequence in the was found in cancer cells or tissues (13, 14). The classical b y putative active domain (95%) as compared with the RFT-I models assume that Le and Le determinants are synthesized gene. RFT-II and -III are thought to constitute Se-type a1,2- through the sequential actions of a1,2- and a1,3/4-fucosyltrans- fucosyltransferase family. A recent report (10) of the molecular ferases through H determinants. In this case, a1,2-fucosyl- cloning of human Se gene enables us to compare it with RFT-II transferase is not postulated to catalyze the fucosylation of Le and -III genes, as it was revealed that RFT-III exhibits higher or Le determinants. Accordingly, the a1,2-fucosyltransferase b a y x amino acid identity with human Se than RFT-II. These find- activity that formed Le from Le or Le from the Le determi- ings led us to conclude that RFT-III is a counterpart of the nant, or both, was supposed to represent an aberrant or new human Se. enzyme. In this study, however, we demonstrated that enzyme The RFT-I, -II, and -III genes are assigned within approxi- preparations from COS-7 cells transfected with rabbit a1,2- mately 90 kb, both RFT-II and -III genes being located in 39 fucosyltransferases contained activity that fucosylated Le or region of RFT-I, based on the results of pulsed field gel elec- Le or both determinants. Cancer cells or tissues of gastroin- trophoresis. The physical relationship of the RFT-I, -II, and -III testinal origin might, we think, express an unusually large genes are consistent with that of the human H, Se, and Sec1, amount of H or Se a1,2-fucosyltransferase but not express an a1,2-fucosyltransferase-related pseudogenes (22). These re- unusual a1,2-fucosyltransferase. sults suggest that RFT-II corresponds to the human Sec1 gene. Bombay individuals who lack active H and Se genes but who This idea is further supported by the structural analysis show- show no apparent abnormal phenotype cast doubt on the phys- ing that the putative cytoplasmic, transmembrane, and stem iological role of a1,2-fucosylation of glycoconjugates. However, domains of RFT-II and human Sec1 are well conserved as it remains possible that another a1,2-fucosyltransferase may compared with other a1,2-fucosyltransferases (Table I). In this operate at specific developmental stages or in restricted tissues case, an ancestral Se gene is thought to have been duplicated or regions. This possibility was increased by the present study, into two related genes, one of which was subsequently inacti- in which we showed that at least in one mammalian species vated by the frameshift mutations in humans. Site-directed three active a1,2-fucosyltransferases exist. point mutation analysis of the Sec1 gene, which corrects the REFERENCES frameshift, and kinetic studies on a1,2-fucosyltransferase ac- 1. Beyer, T. A., and Hill, R. L. (1980) J. Biol. Chem. 255, 5373–5379 tivity of the mutants will provide further information on the 2. Kumazaki, T., and Yoshida, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, relationship between RFT-II and Sec1. It is difficult to know 4193–4197 3. Le Pendu, J., Cartron, J. P., Lemieux, R. U., and Oriol, R. (1985) Am. J. Hum. the exact correspondence of recently cloned fragment of rat or Genet. 37, 749–760 porcine a1,2-fucosyltransferases (23, 24) to RFT-I, -II, or -III, 4. Sarnesto, A., Ko¨hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067–15075 because all a1,2-fucosyltransferases, including the human and 5. Sarnesto, A., Ko¨hlin, T., Hindsgaul, O., Thurin, J., and Blaszczyk-Thurin, M. rabbit H type and Se types, exhibit high homology. (1992) J. Biol. Chem. 267, 2737–2744 It is interesting to consider that RFT-II represents a weak 6. Le Pendu, J., Lemieux, R. U., Lambert, F., Dalix, A.-M., and Oriol, R. (1982) Third Type of Rabbit a1,2-Fucosyltransferase 16981 Am. J. Hum. Genet. 34, 402–415 I. (1992) Neurosci. Res. 15, 74–80 7. Watkins, W. M. (1980) Adv. Hum. Genet. 10, 1–136 17. Kusunoki, S., Chiba, A., Shimizu, T., and Kanazawa, I. (1994) Biochim. Bio- 8. Larsen, R. D., Ernst, L. K., Nair, R. P., and Lowe, J. B. (1990) Proc. Natl. Acad. phys. Acta 1214, 27–31 Sci. U. S. A. 87, 6674–6678 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A 9. Kelly, R. J., Ernst, L. K., Larsen, R. D., Bryant, J. G., Robinson, J. S., and Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Lowe, J. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5843–5847 Harbor, NY 10. Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. (1995) 19. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M., J. Biol. Chem. 270, 4640–4649 and Arai, N. (1988) Mol. Cell. Biol. 8, 466–472 11. Henry, S. M., Benny, A. G., and Woodfield, D. G. (1990) Vox Sang. 58, 61–66 20. McCutchan, J. H., and Pagano, J. S. (1968) J. Natl. Cancer Inst. 41, 351–357 12. Henry, S. M., Oriol, R., and Samuelsson, B. E. (1994) Glycoconj. J. 11, 593–599 21. Rajan, V. P., Larsen, R. D., Ajmera, S., Ernst, L. K., and Lowe, J. B. (1989) 13. Blaszczyk-Thurin, M., Sarnesto, A., Thurin, Y., Hindsgaul, O., and Koprowski, J. Biol. Chem. 264, 11158–11167 H. (1988) Biochem. Biophys. Res. Commun. 151, 100–108 22. Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, 14. Yazawa, S., Nakamura, J., Asao, T., Nagamachi, Y., Sagi, M., Matta, K. L., D. (1995) J. Biol. Chem. 270, 4632–4639 Tachikawa, T., and Akamatsu, M. (1993) Jpn. J. Cancer Res. 84, 989–995 23. Piau, J.-P., Labarriere, N., Dabouis, G., and Denis, M. G. (1994) Biochem. J. 15. Hitoshi, S., Kusunoki, S., Kanazawa, I., and Tsuji, S. (1995) J. Biol. Chem. 270, 300, 623–626 8844–8850 24. Thurin, J., and Blaszczyk-Thurin, M. (1995) J. Biol. Chem. 270, 26577–26580 16. Kusunoki, S., Inoue, K., Iwamori, M., Nagai, Y., Mannen, T., and Kanazawa, 25. Svennerholm, L. (1980) Adv. Exp. Biol. Med. 125, 533–544 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase

Hitoshi, Seiji; Kusunoki, Susumu; Kanazawa, Ichiro; Tsuji, Shuichi
Journal of Biological Chemistry – Jul 1, 1996
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 28, Issue of July 12, pp. 16975–16981, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:b-D-Galactoside 2-a-L-Fucosyltransferase* (Received for publication, October 4, 1995, and in revised form, April 15, 1996) ¶ i Seiji Hitoshi‡§ , Susumu Kusunoki§, Ichiro Kanazawa§, and Shuichi Tsuji‡ From ‡Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Saitama 351-01, Japan and the §Department of Neurology, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan Recent molecular investigation revealed that two fluids of mammals that had several different kinetic character- closely related structural genes encode distinct GDP-L- istics (1–5). In humans, genetic and biochemical studies have fucose:b-D-galactoside 2-a-L-fucosyltransferases (a1,2-fu- indicated that two distinct but closely linked structural genes cosyltransferases). Some human cancer cells or tissues (H and Se) code a1,2-fucosyltransferases with tissue-specific may express an aberrant a1,2-fucosyltransferase other patterns (1, 6). The human H gene controls the expression of H than H- and Secretor-type a1,2-fucosyltransferase. How- (Fuca1,2Galb) antigens (along with A or B antigens or both) on ever, definite evidence of the existence of a third type of erythrocytes, whereas the Se gene determines the soluble A, B, a1,2-fucosyltransferase has not been demonstrated. and H antigens in secretory glands, and Lewis blood group Here we report the molecular cloning of a third type of antigens on red cells (for review, see Ref. 7). Homozygosity for rabbit a1,2-fucosyltransferase (RFT-III) from a rabbit null alleles for the H and Se genes yields the rare Bombay blood genomic DNA library. The DNA sequence included an type and non-Secretor phenotype, respectively. Recent molecu- open reading frame coding for 347 amino acids, and the lar cloning of the H and Se genes provided the molecular basis deduced amino acid sequence of RFT-III showed 59 and for the Bombay and para-Bombay blood types and the non- 80% identity with those of the previously reported two Secretor phenotype, respectively, revealing point mutations types of rabbit a1,2-fucosyltransferase, RFT-I and RFT- II, respectively. COS-7 cells transfected with the RFT-III within the coding regions that abolish the a1,2-fucosyltrans- gene exhibited a1,2-fucosyltransferase activity toward ferase activity (8–10). On the other hand, the Lewis phenotyp- phenyl-b-Gal as a substrate. Neuro2a (a murine neuro- ing of erythrocytes and secretory glands revealed the Le(a1b1) blastoma cell line) cells transfected with the RFT-III and partial Secretor phenotypes in selected Polynesian and gene expressed fucosyl G (type 3 H) but not Ulex eu- M1 Asian individuals (11, 12). These phenotypes, which are virtu- ropaeus agglutinin-1 lectin reactive antigens (type 2 H). ally absent in Caucasians, are thought to be caused by weak Kinetic studies revealed that RFT-III exhibits higher Se-type a1,2-fucosyltransferase activity. The molecular basis of affinity to types 1 (Galb1, 3GlcNAc) and 3 (Galb1, 3Gal- weak Secretor phenotypes, whether weak Se-type a1,2-fucosyl- NAc) than to type 2 (Galb1, 4GlcNAc) oligosaccharides, transferase is encoded by an altered Se gene or a gene other which suggests that RFT-III as well as RFT-II is a Secre- than H and Se, has yet to be determined. tor-type a1,2-fucosyltransferase. RFT-III was expressed Recently, aberrant a1,2-fucosyltransferase activity, which in the adult gastrointestinal tract. The RFT-I, -II, and b a y x synthesized Le from Le or Le from the Le determinant, or -III genes were assigned within 90 kilobases on pulsed both, was found in cancer cells and tissues, suggesting the field gel electrophoresis analysis. These results consti- possibility of a third distinct a1,2-fucosyltransferase gene (13, tute direct evidence that, at least in one mammalian 14). In the rabbit, the possibility of a third type of a1,2-fuco- species, three active a1,2-fucosyltransferases exist. syltransferase was suggested by immunohistochemical studies on DRG neurons. We recently cloned two types of rabbit a1,2- GDP-L-fucose:b-D-galactoside 2-a-L-fucosyltransferase (a1,2- fucosyltransferase, RFT-I and RFT-II, showing that RFT-I but fucosyltransferase) catalyzes the fucosylation of terminal b-D- not RFT-II is expressed in postnatal rabbit brain (15). RFT-I Gal residues and synthesizes H antigens. The activity of a1,2- shows comparable affinities to types 1, 2, and 3 acceptors, fucosyltransferase was detected in various tissues and body which suggests that the binding specificity of RFT-I is primar- ily restricted to the terminal b-D-Gal residues of acceptors. In rabbit DRG neurons, fucosyl G (type 3 H) is readily detected M1 * This work was supported by Grants-in-aid for Scientific Research immunohistochemically on embryonic day 25, followed by the on Priority Areas 05274103, 06254213, 07279249, and 07273274 and appearance of UEA-1 lectin-reactive antigens (type 2 H) post- Grant-in-aid for Scientific Research (C) 07680860 from the Ministry of Education, Japan. The costs of publication of this article were defrayed natally (16, 17). UEA-1 lectin-reactive antigens of DRG neu- in part by the payment of page charges. This article must therefore be rons in postnatal rabbits could be formed through fucosylation hereby marked “advertisement” in accordance with 18 U.S.C. Section catalyzed by RFT-I. In contrast, fucosyl G observed in DRG M1 1734 solely to indicate this fact. The nucleotide sequences reported in this paper have been submitted neurons of embryonic day 25 rabbits might not be the product TM to the GenBank /EMBL Data Bank under accession number 91269. of RFT-I because UEA-1 lectin-reactive antigens are not de- ¶ Research Fellow of the Japan Society for the Promotion of Science. tected at that stage. This observation suggests the existence of To whom correspondence should be addressed. Tel.: 81-48-462-1111 another type of a1,2-fucosyltransferase that catalyzes prefer- (ext. 6521); Fax: 81-48-462-4692. The abbreviations used are: a1,2-fucosyltransferase, GDP-L-fucose: ential fucosylation to type 3 rather than type 2 glycochains. b-D-galactoside 2-a-L-fucosyltransferase; Se, Secretor; Le, Lewis; DRG, Here we report the molecular cloning of a third type of rabbit dorsal root ganglia; UEA-1, Ulex europaeus agglutinin-1; FITC, fluores- a1,2-fucosyltransferase, which could synthesize fucosyl G . M1 cein isothiocyanate; kb, kilobase(s); UTR, untranslated region; PBS, This is the first direct evidence that, at least in one mammalian phosphate-buffered saline. The nomenclature for gangliosides and gly- colipids follows the system of Svennerholm (25). species, three active a1,2-fucosyltransferases exist. This is an open access article under the CC BY license. 16975 16976 Third Type of Rabbit a1,2-Fucosyltransferase FIG.1. Restriction maps for RG193 and the sequence analysis strategy. The coding region is depicted as a shaded box and the non-coding region as a solid line. The arrows indicate the direction and extent of sequencing. EXPERIMENTAL PROCEDURES fragment from 59-UTR and the coding region of RFT-III for Northern blotting. Materials—GDP-Fuc, phenyl-b-D-Gal, Galb1,3GlcNAc, Galb1, Expression of a1,2-Fucosyltransferase (RFT-III)—A 1.3-kb PstI frag- 4GlcNAc, Galb1,3GalNAc, lacto-N-tetraose, lacto-N-neotetraose, lacto- ment of RG193 DNA containing the full open reading frame of RFT-III N-fucopentaoses II and III, G , and FITC-labeled UEA-1 lectin were M1 14 32 was ligated into mammalian expression vector pcD-SRa (19), yielding from Sigma. GDP-[ C]Fuc (10.5 GBq/mmol) and [a- P]dCTP (111 pcD-SRa-RFT-III. The single insertion in the correct orientation was TBq/mmol) were from DuPont NEN. lipofectAMINE was from Life finally analyzed with restriction enzymes. Technologies, Inc. Paragloboside was from Dia-Iatron (Tokyo, Japan). Neuro2a (a murine neuroblastoma cell line) cells were transiently The anti-fucosyl G mouse monoclonal antibody was developed in our M1 transfected with pcD-SRa-RFT-III by means of LipofectAMINE accord- laboratory (17). FITC-labeled anti-mouse Ig (G and M) was from Tago, ing to the manufacturer’s instructions. The cells were trypsinized and Inc. (Burlingame, CA). Restriction endonucleases were from Takara divided into several dishes at 24 h post-transfection. The cells from one (Kyoto, Japan). dish were stained with FITC-labeled UEA-1 lectin or anti-fucosyl G M1 Cloning of a Third Type of Rabbit a1,2-Fucosyltransferase—The antibody at 72 h post-transfection. After washing with PBS, the cells standard molecular cloning techniques described by Maniatis and co- were fixed with formaldehyde for 3 min, washed, and then incubated in workers were used (18). The construction of a rabbit genomic DNA 1% bovine serum albumin/PBS. After washing briefly with PBS, the library and subcloning of polymerase chain reaction fragments (229 cells were incubated in 2 ng/ml FITC-labeled UEA-1 lectin in 1% bovine base pairs) used as probes were described previously (15). We used the serum albumin/PBS for 1.5 h or in anti-fucosyl G monoclonal anti- M1 polymerase chain reaction fragments as probes for further cloning of body for 1.5 h and then washed with PBS, followed by incubation with a1,2-fucosyltransferase, because these probes hybridized to more than 6 FITC-labeled anti-mouse Ig (G and M) for 1 h. After washing three two bands on rabbit genomic Southern blotting. Another 2 3 10 times with PBS, the cells were observed under a fluorescence micro- plaques of the rabbit genomic DNA library were screened and a scope. The cells from another dish were trypsinized, fixed with formal- positive plaque containing a 16.8-kb (RG193) insert was isolated to dehyde, and stained as above for flow cytometry analysis using FACS- homogeneity. Flow (Becton Dickinson). Transfection of the gene was verified by The RG193 DNA fragments were digested with appropriate restric- measuring fucosyltransferase activity using cell extract from another tion enzymes and then subcloned into vector plasmid pUC 119. The dish. DNA sequences were determined by the dideoxynucleotide chain-ter- COS-7 cells (60-mm culture dish) were transiently transfected with 5 mination method using an Autocycle DNA sequencing kit and an A.L.F. mg of pcD-SRa-RFT-III using the DEAE-dextran procedure (20). The DNA sequencer (Pharmacia Biotech Inc.). The sequences were analyzed cells were washed with PBS, washed with 25 mM 4-morpholineethane- using PC/Gene (Teijin System Technology, Osaka, Japan). sulfonic acid for 10 min, and then collected with a rubber policeman and Pulsed Field Gel Electrophoresis and Northern Hybridization—Rab- pelleted by centrifugation. The pellets were resuspended in 100 mlof bit lymphocytes were prepared by density gradient (Lympholyte-Rab- cold 1% Triton X-100 and then sonicated briefly. bit; Cedarlane, Hornby, Canada), suspended in agarose blocks, and Fucosyltransferase Assay—The fucosyltransferase assays were per- then digested with 0.2 mg/ml proteinase K and 1% Sarkosyl. Digestion formed according to previous reports (15, 21) in a mixture of 25 mM of DNA suspended in agarose blocks was carried out using 10 units of sodium phosphate (pH 6.1), 5 mM ATP, 30 mM GDP-fucose, 3 mM GDP- restriction endonucleases in appropriate restriction buffers at 37 °C for [ C]fucose (10.5 Bq/pmol), the enzyme solution, and substrates in a 3 h. Pulsed field gel electrophoresis was performed using pulse times of final volume of 10 ml. Each reaction mixture for oligosaccharide accep- 0.2–18 s linearly ramped at 200 V for 18 h. After electrophoresis, the tors was incubated at 37 °C for 2 h and then applied to a Silica Gel 60 gels were stained with ethidium bromide to visualize size markers high performance thin layer chromatography plate (Merck). The plate followed by alkali transfer onto nylon membranes (Nytran; Schleicher was developed with ethanol/pyridine/1-butanol/water/acetic acid (100: & Schuell). Hybridization was performed in 6 3 SSC, 5 3 Denhardt’s, 10:10:30:3). When glycolipids were used as substrates, the reaction 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA at 65 °C. mixture was applied to a C-18 Sep-Pak cartridge (Waters-Millipore, Total RNA was prepared by the guanidium thiocyanate method and Milford, MA) after2hof incubation at 37 °C, washed with 2 ml of water, purified by ultracentrifugation through 5.7 M CsCl. Poly(A)-rich RNA and then eluted with 1 ml of methanol. The eluate was then applied to was purified with Oligotex-dT30 (Takara). The poly(A)-rich RNA (5 mg) a high performance thin layer chromatography plate, which was devel- was fractionated on a denaturing formaldehyde-agarose gel (1.2%) and oped with chloroform/methanol/0.5% CaCl (55:45:10). For glycoprotein then transferred onto a nylon membrane. Northern filters were hybrid- acceptors, the reaction was terminated by the addition of 10 mlof ized in 50% formamide, 5 3 saline/sodium/phosphate/EDTA, 5 3 Den- SDS-polyacrylamide gel electrophoresis loading buffer after2hofin- hardt’s, 0.5% SDS, 0.25% sodium lauryl Sarkosyl, and 100 mg/ml dena- cubation at 37 °C, and the incubation mixtures were directly subjected tured salmon sperm DNA at 37 °C. To quantify the RNA loading, the to SDS-polyacrylamide gel electrophoresis. The radioactivity on each Northern filters were rehybridized with a labeled cDNA fragment of plate and gel was visualized and determined with a BAS2000 radioim- rabbit glyceraldehyde-3-phosphate dehydrogenase. 32 age analyzer (Fuji Film, Tokyo, Japan). All probes were labeled with [a- P]dCTP, using the random priming method. The gene probes used for hybridizations were a 1.8-kb SalI- RESULTS SacI fragment from 39-UTR of RFT-I, a 0.7-kb SacI-PstI fragment from Cloning and Nucleotide Sequence of a Third Type of Rabbit 59-UTR of RFT-II (15), a 0.6-kb SacI-EcoRI fragment from 39-UTR of RFT-III for pulsed field gel electrophoresis, and a 0.3-kb PstI-NaeI a1,2-Fucosyltransferase—On screening of a rabbit genomic Third Type of Rabbit a1,2-Fucosyltransferase 16977 FIG.2. Nucleotide and deduced amino acid sequences of rabbit RFT-III (A), and comparison of the deduced amino acid sequences of rabbit and human a1,2-fucosyltransferases (B). A, nucleotide and amino acid sequences are numbered from the presumed start codon and initiation methionine, respectively. The boxed amino acids correspond to a putative signal-anchor domain. The asterisks indicate potential N-glycosylation sites (Asn-X-Ser/Thr). B, each pair of amino acid sequences is aligned by the PALIGN software of PC/Gene and adjusted by eye. A putative signal-anchor domain is boxed. Potential N-glycosylation sites are circled. The asterisks indicate that all the aligned residues of active a1,2-fucosyltransferases (RFT-I, -II, and -III and human H and Se genes) are identical. observed among rabbit and human DNA library with polymerase chain reaction fragments (229 a1,2-fucosyltransferases. base pairs) as probes, one positive clone, RG193, was obtained Comparison of the primary structure of RFT-III revealed sig- (Fig. 1). Sequence analysis revealed that RG193 contained an nificant (59 and 80%) amino acid identity with those of RFT-I entire open reading frame with three closely located in-frame and RFT-II, respectively. The putative extracellular region methionine codons in the starting region (Fig. 2A). The last of composed of the glycine residue at position 66 to the alanine the three methionine codons was a good candidate for an ini- residue at position 339 of RFT-III was highly conserved among tiator, accompanied by the Kozak consensus translation initi- not only rabbit but also human a1,2-fucosyltransferases (Fig. ation sequence. The open reading frame beginning with the last 2B), suggesting that this region is an active domain. In partic- methionine codon encodes 347 amino acids with a predicted ular, this region was highly conserved among RFT-II and RFT- molecular mass of 39 kDa (RFT-III). The deduced amino acid III, containing a 515-base-pair stretch of identical bases with sequence contains a putative transmembrane region in the two exceptions. The deduced amino acid identities between NH -terminal region, indicating RFT-III is a type 2 protein, as rabbit and human a1,2-fucosyltransferases are summarized in is usual with glycosyltransferases. RFT-III contains three po- Table I. In this case, the amino acids were translated from the tential N-linked glycosylation sites, two of which are commonly Sec1 DNA sequence (10), correcting the frameshift that dis- 16978 Third Type of Rabbit a1,2-Fucosyltransferase TABLE I rupted the reading frame of Sec1 and yielding a long reading Comparison of deduced amino acids between rabbit and frame appropriately corresponding to other a1,2-fucosyltrans- human a1,2-fucosyltransferases ferases. The deduced amino acid identities of the entire sequence, accompa- Pulsed Field Gel Electrophoresis and Northern Hybridiza- nied by the putative cytoplasmic, transmembrane, and stem/active tion—By analyzing human chromosome 19 cosmid libraries, domains in parentheses, are shown. Amino acid length and calculated molecular weight of polypeptide deduced from each gene are also the physical relationship of human a1,2-fucosyltransferases shown. The deduced amino acids for Sec1 were translated, correcting and the a1,2-fucosyltransferase-related genes was defined, re- the frameshift that disrupts the reading frame of Sec1 and yielding a vealing that all genes are located within 80 kb—the Se (Sec2) long reading frame appropriately corresponding to other a1,2-fucosyl- gene is 35.5 and 12 kb apart from the H (FUT1) and Sec1 genes, transferases. respectively (22). To determine the physical relationship of the Human RFT-I, -II, and -III genes, pulsed field gel electrophoresis was a b b Rabbit M H (FUT1) Se (Sec2) Sec1 performed. All probes recognized a common 90-kb SalI frag- (365 aa) (343 aa) (347 aa) 41,249 39,017 39,099 ment, and the RFT-II and -III probes recognized a common 100-kb NotI fragment and a common 105-kb MluI fragment %%% (Fig. 3). These results suggested that maximum distance RFT-I (373 aa) 42,098 80 (59/88) 59 (10/70) 53 (17/64) encompassing these genes to be 90 kb and that both RFT-II RFT-II (354 aa) 40,082 54 (12/68) 74 (28/86) 76 (70/79) RFT-III (347 aa) 39,469 59 (12/70) 83 (54/89) 67 (22/79) and -III genes are located in 39 region of RFT-I, because the a RFT-I probe locates 39 side of the SalI restriction site in DNA sequence from Ref. 8. DNA sequence from Ref. 10. RG11. DNA sequence from Ref. 15. aa, amino acid. FIG.3. Pulsed field gel electrophoresis analysis. Sequential hy- bridization with RFT-I, -II, and -III gene probes on pulsed field gel electrophoresis blots separating DNA fragments. All probes recognized a common SalI (90 kb) restriction fragment, and the RFT-II and -III gene probes recognized common NotI (100 kb) and common MluI (105 kb) restriction fragments. FIG.5. Expression of fucosyl G on RFT-III transfected M1 Neuro2a cells. A, Neuro2a cells were transiently transfected with 5 mg of pcD-SRa or pcD-SRa-RFT-I, -II, or -III using LipofectAMINE. The cells were stained with FITC-labeled UEA-1 lectin or anti-fucosyl G M1 FIG.4. Northern blot analysis of RFT-III. Poly(A)-rich rabbit monoclonal antibody followed by FITC-labeled anti-mouse Ig (G and M) RNAs (5 mg) were electrophoresed on a denaturing formaldehyde-aga- at 72 h post-transfection and observed under fluorescent microscopy. B, rose gel, transferred onto a nylon membrane, and hybridized with the transfected Neuro2a cells from another dish were trypsinized, stained 0.3-kb PstI-NaeI fragment of RFT-III (upper panel). The lower panel as above, and subjected to flow cytometry. Transfection of RFT-I, -II, or shows the same blot rehybridized with a labeled rabbit glyceraldehyde- -III was verified by measuring fucosyltransferase activity using the cell 3-phosphate dehydrogenase (GAPDH) cDNA probe. extract from another dish. Third Type of Rabbit a1,2-Fucosyltransferase 16979 TABLE II Acceptor specificities of RFT-I, -II, and -III The apparent K and V /K values of RFT-III and those of RFT-I and RFT-II reported previously are shown. The relative a1,2-fucosyltrans- m max m ferase activities of the incorporation of fucose into phenyl-b-D-Gal as a substrate are also shown. The actual activities of phenyl-b-D-Gal as an acceptor were measured with cell extracts prepared at the same time and under the same conditions. a a RFT-III RFT-I RFT-II Acceptor 211 211 211 K V /K (310 l/min) K V /K (310 l/min) K V /K (310 l/min) m max m m max m m max m mM mM mM Phenyl-b-D-Gal 5.7 0.82 4.0 2.3 17.1 0.13 Galb1,3GlcNAc 1.5 0.49 3.1 11.4 2.2 0.36 Galb1,4GlcNAc 6.7 0.65 4.2 8.9 12.5 0.21 Galb1,3GalNAc 1.0 0.75 3.5 5.8 4.6 0.50 Lacto-N-tetraose 1.6 0.30 3.5 3.0 4.8 0.51 Lacto-N-neotetraose 4.2 0.23 5.5 1.2 12.5 0.29 Relative activity RFT-III RFT-I RFT-II %%% b b b Phenyl-b-D-Gal (25 mM) 100 (1.70) 100 (3.38) 100 (0.21) Asialofetuin (1 mg/ml) 2.7 2.4 ND Asialo-a1-acid glycoprotein (1 mg/ml) 0.6 0.9 ND G (2 mM) 31.0 19.7 15.7 M1 Paragloboside (2 mM) 5.6 23.0 ND Lacto-N-fucopentaose II (2 mM) 3.0 2.3 13.8 Lacto-N-fucopentaose III (2 mM) 2.0 1.1 ND From Ref. 15. The actual activities (pmol/h/ml of enzyme) of fucosyltransferases are shown in parentheses. ND, not detected. FIG.6. Lineweaver-Burk plots. Lin- eweaver-Burk plots used to calculate the K and V /K values are shown. Exper- m max m iments were performed in triplicate and typical plots are shown. Northern blot analysis revealed that a 1.8-kb mRNA of RFT- Expression of RFT-III and Enzyme Assaying—Neuro2a cells III was expressed in the adult gastrointestinal tract (Fig. 4). transiently transfected with pcD-SRa-RFT-I, -II, and -III The expression of minor 5.0- and 6.4-kb mRNAs of RFT-III was showed a1,2-fucosyltransferase activity of 1.93, 0.12, and 0.52 also observed in the adult colon. Other secretory glands such as pmol/h/ml of enzyme, respectively, whereas parent Neuro2a salivary and mammary glands did not express RFT-III. cells contained no activity. Neuro2a cells transfected with RFT- 16980 Third Type of Rabbit a1,2-Fucosyltransferase III expressed fucosyl G but not UEA-1 lectin-reactive anti- Se-type a1,2-fucosyltransferase. The weak Se-type a1,2-fuco- M1 gens (Fig. 5, A and B), which suggested that the binding spec- syltransferase was postulated based on the results of Lewis ificity of RFT-III differed between type 2 and type 3 glycochains phenotype analysis in Polynesian people. The Lewis antigens or between glycoproteins and glycolipids. Neuro2a cells trans- on erythrocytes are regulated by two fucosyltransferases, Se- fected with RFT-I expressed both fucosyl G and UEA-1 lec- type a1,2- and Lewis a1,3/4-fucosyltransferases. With the con- M1 tin-reactive antigens, and Neuro2a cells transfected with ventional analysis method, three Lewis phenotypes of erythro- RFT-II expressed neither of them (Fig. 5, A and B). Cell ex- cytes were found in Caucasian adults, Le(a2b2), Le(a1b2), tracts of COS-7 cells transfected with pcD-SRa-RFT-III con- and Le(a2b1). When Se-type a1,2-fucosyltransferase is active tained a1,2-fucosyltransferase activity that transferred radio- (or in Secretor), most of the type 1 precursor is converted into labeled fucose to phenyl-b-D-Gal as a substrate. As shown in type 1 H, which can be transformed into Le by Lewis a1,3/4- Table II and Fig. 6, the acceptor specificity of RFT-III was fucosyltransferase. On the other hand, a fourth Lewis pheno- comparable with that of RFT-II. RFT-III showed higher affinity type, Le(a1b1), was found on erythrocytes from selected Pol- for types 1 and 3 acceptors than for type 2 acceptors and ynesian individuals. In addition, low levels of salivary ABH phenyl-b-D-Gal, like RFT-II and human Se-type a1,2-fucosyl- antigens, that is partial secretion, were found in saliva from transferase. RFT-III could transfer fucose to asialofetuin and Le(a1b2) and Le(a1b1) individuals, suggesting the presence asialo-a1-acid glycoproteins as well as G ganglioside. The of a weak Se-type a1,2-fucosyltransferase (11, 12). Molecular M1 relative activity of RFT-III toward G as to glycoproteins was analysis of the Se and Sec1 genes of Polynesian people, espe- M1 higher than that of RFT-I. RFT-III could also fucosylate lacto- cially of partial Secretor individuals, will facilitate determina- N-fucopentaoses II and III, a1,3- and a1,4-fucosylated oligosac- tion of whether or not RFT-II corresponds to the human Sec1 charides, respectively. gene. The fucosyltransferase assay showed that the relative activ- DISCUSSION ity of RFT-III toward G as to glycoproteins was higher than M1 In this and previous work (15) we reported the molecular that of RFT-I. RFT-III could also synthesize fucosyl G from M1 cloning of three types of rabbit a1,2-fucosyltransferase, RFT-I, G but not UEA-1 reactive antigens when expressed in M1 -II, and -III. These results constitute direct evidence that, at Neuro2a cells, where RFT-I could form both and RFT-II could least in one mammalian species, three active a1,2-fucosyltrans- synthesize neither under the same transfection conditions. ferases exist, one H type and two Se types, based on kinetic RFT-III is a good candidate for the enzyme that synthesizes analysis. fucosyl G expressed in a subpopulation of neurons of rabbit M1 RFT-I exhibits comparable kinetic properties and significant embryonic DRG (16, 17), although we could not detect the structural homology with human H-type a1,2-fucosyltrans- expression of RFT-III in embryonic brain. It is possible that the ferase, indicating that RFT-I is a counterpart of human H. expression of RFT-III is restricted to specific regions or specific RFT-II and -III show higher affinity to types 1 and 3 acceptors types of neurons. In situ hybridization analysis will provide than to type 2 acceptors and phenyl-b-D-Gal. The kinetic pa- further information. rameters of RFT-II and -III are comparable with those of hu- Recently, aberrant a1,2-fucosyltransferase activity that syn- b a y x man Se-type a1,2-fucosyltransferase (2, 3, 5). RFT-II and -III thesized Le from Le or Le from the Le determinant, or both, genes share remarkably conserved base pair sequence in the was found in cancer cells or tissues (13, 14). The classical b y putative active domain (95%) as compared with the RFT-I models assume that Le and Le determinants are synthesized gene. RFT-II and -III are thought to constitute Se-type a1,2- through the sequential actions of a1,2- and a1,3/4-fucosyltrans- fucosyltransferase family. A recent report (10) of the molecular ferases through H determinants. In this case, a1,2-fucosyl- cloning of human Se gene enables us to compare it with RFT-II transferase is not postulated to catalyze the fucosylation of Le and -III genes, as it was revealed that RFT-III exhibits higher or Le determinants. Accordingly, the a1,2-fucosyltransferase b a y x amino acid identity with human Se than RFT-II. These find- activity that formed Le from Le or Le from the Le determi- ings led us to conclude that RFT-III is a counterpart of the nant, or both, was supposed to represent an aberrant or new human Se. enzyme. In this study, however, we demonstrated that enzyme The RFT-I, -II, and -III genes are assigned within approxi- preparations from COS-7 cells transfected with rabbit a1,2- mately 90 kb, both RFT-II and -III genes being located in 39 fucosyltransferases contained activity that fucosylated Le or region of RFT-I, based on the results of pulsed field gel elec- Le or both determinants. Cancer cells or tissues of gastroin- trophoresis. The physical relationship of the RFT-I, -II, and -III testinal origin might, we think, express an unusually large genes are consistent with that of the human H, Se, and Sec1, amount of H or Se a1,2-fucosyltransferase but not express an a1,2-fucosyltransferase-related pseudogenes (22). These re- unusual a1,2-fucosyltransferase. sults suggest that RFT-II corresponds to the human Sec1 gene. Bombay individuals who lack active H and Se genes but who This idea is further supported by the structural analysis show- show no apparent abnormal phenotype cast doubt on the phys- ing that the putative cytoplasmic, transmembrane, and stem iological role of a1,2-fucosylation of glycoconjugates. However, domains of RFT-II and human Sec1 are well conserved as it remains possible that another a1,2-fucosyltransferase may compared with other a1,2-fucosyltransferases (Table I). In this operate at specific developmental stages or in restricted tissues case, an ancestral Se gene is thought to have been duplicated or regions. This possibility was increased by the present study, into two related genes, one of which was subsequently inacti- in which we showed that at least in one mammalian species vated by the frameshift mutations in humans. Site-directed three active a1,2-fucosyltransferases exist. point mutation analysis of the Sec1 gene, which corrects the REFERENCES frameshift, and kinetic studies on a1,2-fucosyltransferase ac- 1. Beyer, T. A., and Hill, R. L. (1980) J. Biol. Chem. 255, 5373–5379 tivity of the mutants will provide further information on the 2. Kumazaki, T., and Yoshida, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, relationship between RFT-II and Sec1. It is difficult to know 4193–4197 3. Le Pendu, J., Cartron, J. P., Lemieux, R. U., and Oriol, R. (1985) Am. J. 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Published: Jul 1, 1996

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