Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 4668550, Japan
Received on February 23, 2000; revised on May 1, 2000; accepted on May 17, 2000.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: alternative splicing/ß-1,6-N-acetylglucosaminyl-transferase/gene structure/I antigen/poly-N-acetyllactosamine
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Branched poly-N-acetyllactosamines are good scaffolds for cell-surface antigens and recognition markers, and polyvalent epitopes on them are expected to have strong affinity to the antibodies or to the receptors. Indeed, the multivalent sialyl LeX epitope on branched poly-N-acetyllactosamines is a potent antagonist of L-selectin (Turunen et al., 1995). Furthermore, erythrocyte poly-N-acetyllactosamines carry ABH blood group antigens (Krusius et al., 1978
; Fukuda et al., 1984a
,b), and embryonal poly-N-lactosamines carry LeX antigen (Ozawa et al., 1985
; Kamada et al., 1987
).
Embryonal poly-N-acetyllactosamines are expected to carry carbohydrate recognition markers necessary for embryogenesis (Muramatsu, 1988). Actually, a LeX oligosaccharide or its derivative has been reported to inhibit tight cell adhesion of preimplantation embryos, a phenomenon known as compaction (Bird and Kimber, 1984
; Fenderson et al., 1984
). Furthermore, poly-N-acetyllactosamine branching is developmentally regulated (Fukuda et al., 1979
; Muramatsu, 1988
), and branched poly-N-acetyllactosamine levels progressively decrease during embryogenesis (Muramatsu et al., 1978
, 1979; Kapadia et al., 1981
). However, the physiological significance of branching in poly-N-acetyllactosamines has not yet been proven genetically.
Due to the potential biological significance of poly-N-acetyllactosamine branching, various enzymological and molecular biological studies have been carried out on the branching enzymes. They are a class of specific ß-1,6-N-acetylglucosaminyltransferases and are collectively known as I N-acetylglucosaminyltransferase (IGnT) based on the fact that the branched poly-N-acetyllactosamine structure is the epitope of the blood group I antigen (Leppänen et al., 1991; Gu et al., 1992
; Bierhuizen et al., 1993
; Helin et al., 1997
; Sakamoto et al., 1998
; Leppänen et al., 1998
; Mattila et al., 1998
). One IGnT has been cloned from human embryonal carcinoma cells (Bierhuizen et al., 1993
), and its mouse homologue has been obtained (Magnet and Fukuda, 1997
). In addition, a cloned ß-1,6-N-acetylglucosaminyltransferase, C2GnT-M, acts on core 2 mucin-type chains and also forms poly-N-acetyllactosamine branches (Yeh et al., 1999
). Whether there are further molecular species of poly-N-acetyllactosamine branching enzymes is of interest from the viewpoint of regulation of the biosynthesis of the branched structure, and as a basis for genetic manipulation of the transferase.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Southern blotting analyses of mouse genomic DNA with a DNA probe for Exon 3 showed a simple pattern of hybridizing bands, indicating that the mouse IGnT gene is a single-copy gene (Figure 2A). In contrast to the single hybridizing band revealed in each restriction enzyme-digested lane with a probe for Exon 1A, multiple band patterns were observed with a probe for Exon 1B (Figure 2A). Since the probe for Exon 1A and that for Exon 1B did not cross-hybridize, there appeared to be at least one more exon or pseudo-exon related to Exon 1B in the mouse genome. We also noted an additional band in PstI-digested DNA hybridized with Exon 1B probe and BamHI-digested DNA hybridized with Exon 3 probe. Since there is no PstI or BamHI site in either of the exon, those bands may be the result of cross-reactive hybridization.
|
Enzymatic activity of IGnT B
We expressed IGnT A and B as transmembrane forms or as soluble forms fused to protein A. The activities of the proteins were determined using lacto-N-neotetraose (Galß14GlcNAcß13Galß14Glc) as an acceptor and UDP-[14C]-GlcNAc as the sugar donor. Both the transmembrane and soluble forms of IGnT A or B transferred N-acetylglucosamine to lacto-N-neotetraose (Figure 3A). The transfer efficiency of IGnT B was found to be better than that of IGnT A in both the transmembrane form (Figure 3A) and soluble form fused to protein A (Figure 3B).
|
|
|
|
|
|
When para-lacto-N-neohexaose (Galß14GlcNAcß13Gal-ß14GlcNAcß13Galß14Glc) was used as the substrate, two product bands were observed on TLC (Figure 5, lane 4). The two products were separated by Bio-Gel P-4 column chromatography. The species that migrated faster on TLC was eluted from the Bio-Gel P-4 column about 45 hexose units earlier than GlcNAcß13(GlcNAcß16)Galß14Glc (Figure 4B, open diamonds and shaded circles). Since one N-acetylglucosamine contributes to the mobility in the column as 1.82.0 hexose units (Kobata et al., 1987), the mobility of the product corresponded to that of the mono-branched species. On the other hand, the slow migrating band was larger (Figure 4B, solid squares), indicating that it was a di-branched product. When the purified mono-branched products (Figure 4A, lane 7) were digested with endo-ß-galactosidase, the major product migrated as a hexasaccharide (Figure 4A, lane 8) on TLC, and the minor product migrated as a tetrasaccharide (Figure 4A, lane 8). The enzyme cleaves internal galactosyl residues substituted by GlcNAcß13 linkage, but a branched oligosaccharide, Galß14GlcNAß13(GlcNAcß16)Galß14Glc-NAc, was shown to be resistant (Fukuda et al., 1984a
,b; Scudder et al., 1984
). Taking the specificity of the endoglycosidase into account, the major product was concluded to be Galß14Glc-NAcß13(GlcNAcß16)Galß14GlcNAcß13Galß14Glc, while the minor product was concluded to be Galß14Glc-NAc- ß13Galß14GlcNAcß13(GlcNAcß16)Galß14Glc. The di-branched product (Figure 4A, lane 10) was resistant to endo-ß-galactosidase (lane 11), but was cleaved by ß-galactosidase (lane 12), indicating that the product was Galß14Glc-NAcß13(GlcNAcß16)Galß14GlcNAcß13(GlcNAcß16)-Gal-ß14Glc.
IGnT B preferentially formed the mono-branched products, especially when the acceptor oligosaccharide concentration was higher than that of UDP-GlcNAc (Figure 6A). During prolonged incubation, the di-branched product increased (Figure 6B). Furthermore, when the isolated mono-branched products were incubated with IGnT B, the majority was converted to the di-branched product (Figure 6C). These results indicated that the number of branches is dependent on the relative amounts of the enzyme, UDP-GlcNAc and acceptor sites. A proposed pathway for the formation of the di-branched structure by IGnT B is shown in Figure 7.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The substrate specificity of the enzyme studied herein was generally in agreement with that of the cloned IGnT with cIGnT activity. Thus, the enzyme reported here acted on Galß14GlcNAcß13Galß14Glc and transferred an N-acetylglucosamine residue to the underlined central galactose, but did not act on GlcNAcß13Galß14Glc. When a hexasaccharide Galß14GlcNAcß13Galß14GlcNAcß13Galß14Glc was used as the substrate, it acted on both intrachain galactosyl residues, with higher activity toward the doubly underlined galactosyl residue, and during prolonged incubation branches were formed in both galactosyl residues. This is precisely what was found for IGnT A (Ujita et al., 1999a,b). Fucose linked to N-acetylglucosamine by
13 linkage abolished the activity as reported for human serum IGnT (Leppänen et al., 1997
). However, our study of the substrate specificity was more comprehensive than those reported previously. Furthermore, NeuAc
23Galß14GlcNAcß13Galß14Glc does not serve as an acceptor of the rat intestinal enzyme (Gu et al., 1992
).
Interestingly, the C-terminal 1/4 portion of IGnT B shows sequence identity to murine IGnT A. Genomic analysis revealed that IGnT A and IGnT B are formed by alternative splicing. Although the sequences of IGnT A and IGnT B are different from their N-termini, the closely related modes of expression of IGnT A and IGnT B suggested that they share some of the 5' untranslated region and the promoter sequence. Alternatively, the first exons may be different between IGnT A and B, but they share almost identical promoter sequences. In any event, IGnT A and IGnT B appear to have been generated by duplication of some portion of the IGnT gene.
Although apparent levels of COS cell-expressed soluble forms of IGnT A and IGnT B were similar, expressed activity of IGnT was stronger in IGnT B than in IGnT A (Figure 3B). The splicing variant reported here appeared to be the more important form in constructing branched poly-N-acetyllactosamines both in early embryos and in adult tissues. The present results are also important in view of knockout of the IGnT gene. If the exon encoding the N-terminal portion of the protein is to be deleted as in usual gene knockout strategies, significant IGnT activity will remain especially when IGnT A is deleted. Thus, constructs for use in knockout experiments should delete the exon encoding the C-terminal portion of the enzyme.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of cDNA and genomic clones of IGnT
The EST data base was screened for sequences related to known ß-1,6-N-acetylglucosaminyltransferases. One potentially novel ß-1,6-N-acetylglucosaminyltransferase-like EST (GenBank accession number AI528293) was identified and the corresponding cDNA was obtained from ICR mouse liver cDNA by RT-PCR. The entire coding sequence of the gene was obtained by 5'- and 3'-RACE, performed on a cDNA library derived from the ICR mouse embryonic brain using gene-specific primers. The primers used were, 5'-TAAATGGCCCTGAAGAGTCTC-3' (nt. 863843 Figure 1A) and 5'-TCGGGAAATGATAGTTCTTCC-3' (nt. 619599) for 5'-RACE, and 5'-CAGCCTATCTCTCATTGCTGC-3' (nt. 542573) and 5'-TCTCTGAAGAAGAAGCCCG-3' (nt. 773791) for 3'-RACE. Further BLAST searches identified one EST clone (GenBank accession number AV024162) that extended the 5'-untranslated region of IGnT B.
The entire coding regions of mouse IGnT A and B were obtained by RT-PCR. Primers and cDNAs used were as follows: 5'-AGAGAGCTCGAGTTAGGCCGGAGCTGCTGCGGGTC-3' (nt. 14601438, underlined nucleotides were added for restriction enzyme digestion; nucleotide no. refers to GenBank accession No. U68182), 5'-AGAGCTCGAGCATGCCTCTGTCCGTGCGTTACTTC-3' (nt. 235260) and F9 cDNA as a template for IGnT A; 5'-GCCAAGGAGCTTTGCTCATCAGAGCC-3' (nt. 367392), 5'-GAGACTCGAGCCGGAGCTGCTGCGGGTCAGA-3' (nt. 17381718) and ICR mouse liver cDNA as a template for IGnT B. The cloned IGnT A showed some nucleotide sequence substitutions and insertions compared to a previously cloned IGnT obtained from PCC4 mouse embryonal carcinoma cells (Magnet and Fukuda, 1997). Briefly, nucleotide substitutions at nt 244, T
C; nt 278, G
C; nt 292, G
T; nt 294, G
A; nt 301, C
T; nt 508 and 509, GT
TC; nt 679, T
C; nt 1247, A
T; nt 1411, G
T and three nucleotide (CAT) insertions between nt 11681169 (nucleotide numbers are based on Magnet and Fukuda, 1997
; GenBank accession no. U68182). The nucleotide sequences of the open reading frame of IGnT A obtained from ICR mouse liver cDNA, F9 cell cDNA and a 129 SV genomic library were identical, indicating that the above nucleotide differences were not derived from PCR errors.
Genomic clones were obtained by screening of a 129SV EMBL3 genomic library with the full-length IGnT A and IGnT B cDNA fragments as described previously (Kurosawa et al., 1999). The localization of the exons was determined by LA-PCR (GeneAmp XL PCR kit, Perkin-Elmer). Sequence data were analyzed by accessing the databases at the National Center for Biotechnological Information (NCBI) and using Gene Works software (IntelliGenetics, USA).
Northern and genomic Southern blotting analyses
Northern and genomic Southern blotting analysis were performed as described previously (Kurosawa et al., 1994). Poly(A) RNA (2 µg) was prepared from 129 SV mouse tissues, P19 embryonal carcinoma (EC) and D3 embryonic stem (ES) cells. Mouse multiple tissue Northern Blots (Clontech) were also used for hybridization analysis. The radioactive probes used were an IGnT A-specific probe (nt 324633), an IGnT B-specific probe (nt 5951035 in Figure 1) and an IGnT exon 3 probe (nt 15311743 in Figure 1).
Construction of expression plasmids
The putative catalytic domains of IGnT A and B were specifically amplified by PCR, followed by ligation into the mammalian expression vector pcDSA (Kojima et al., 1995), yielding expression plasmids pSA-IGnT A and pSA-IGnT B, respectively. The sense primer and cDNA template used for the amplification of IGnT A were 5-AGAGAGCTCGAGGGATCAAAGCTACCAGAAGCTG-3 (nt. 324345) and F9 cDNA. The sense primer and cDNA template used for the amplification of IGnT B were 5'-AGAGAGAATTCTTATGGAAGAACTATCATTTCCCGA-3' (nt. 595619) and ICR mouse liver cDNA. Antisense primers used were the same as those for construction of full-length IGnT A and B, respectively, as described above. Expression plasmids carrying the complete coding sequences of IGnT A (pCD-IGnT A) and IGnT B (pCD-IGnT B) were constructed by subcloning PCR-amplified cDNAs into the mammalian expression vector pcDSR
(Takebe et al., 1988
). The single insertion in the correct orientation was finally confirmed by restriction enzyme analysis and DNA sequencing.
COS-7 cells (2 x 106) were transiently transfected with 4 µg of pSA-IGnT A or pSA-IGnT B using Lipofectamine plus (Gibco BRL Life Technologies Inc, Rockville, MD). Forty-eight h after transfection, the culture medium was collected and the fusion protein secreted into the medium was adsorbed on IgG-Sepharose (25 µl/10ml medium) and used as the soluble form of the enzyme as described previously (Kojima et al., 1995). The medium from mock transfected COS cells was used as a negative control. [35S]-Methionine labeling of COS cell-expressed enzymes was performed as previously described (Kurosawa et al., 1994
). CHO cells (4 x 106) were transiently transfected with 4 µg of pCD-IGnT A, pCD-IGnT B, or pcDSR
as described above. Forty-eight hours after transfection, cells were harvested and suspended in 100 µl of lysis buffer (50 mM TrisHCl buffer, pH 7.0, containing 0.25 M sucrose, 1% Triton X-100), sonicated and centrifuged at 13,000 r.p.m. for 10 min. Aliquots of 1 µl of the supernatant containing the transmembrane form of IGnT were used as enzyme sources.
Acceptor substrate specificity of COS cell-expressed IGnT A and B
Enzyme assays were performed as described previously with some modifications (Ujita et al., 1999a,b). Each reaction mixture was comprised of 50 mM sodium cacodylate buffer, pH 7.0, 10 mM 2-acetamido-2-deoxy-D-glucono-1,5-lactone, 4 mM ATP, 1 mM EDTA, 0.1 mM acceptor substrate (lacto-N-neotetraose was used for determination of specific activity), 0.5 mM of UDP-[14C]GlcNAc (185 kBq/mmol) and enzyme, in a total volume of 25 µl. After incubation at 37 °C for the indicated periods, enzyme reaction was terminated by boiling, if necessary, followed by treatment with 1 mU of nucleotide pyrophosphatase (Sigma-Aldrich) for digestion of UDP-GlcNAc at 37°C for 4 h. The incubation mixtures were directly applied to TLC. The radioactive materials on TLC plates were visualized with a BAS2000 radioimage analyzer (Fuji Film, Tokyo, Japan), and the radioactivity incorporated into acceptor oligosaccharides was counted. TLC plates were developed with ethanol:pyridine:n-butanol:water:acetate = 100:10:10:30:3. Under these conditions, tetrasaccharides lacto-N-neotetraose and lacto-N-tetraose and hexasaccharides para-lacto-N-neohexaose and lacto-N-hexaose could be separated.
Product characterization by glycosidase digestion
Complete glycosylation of oligosaccharides was performed in 50 µl reaction mixtures as described above except that 250 nmol/h/ml of enzyme was used. After 12 h of incubation at 37°C, the reaction product was passed through a Dowex AG50 (H+)/AG1(AcO) column and subsequently lyophilized. The products were purified by Bio-Gel P-4 column chromatography (125x0.8 cm), equilibrated and eluted with water. Fractions of 625 µl were collected.
Endo-ß-galactosidase digestion was performed in 10 µl reaction mixtures consisting of purified radiolabeled oligosaccharides (1 x 104 c.p.m.), 5 mU endo-ß-galactosidase, 5 mM Na acetate buffer, pH 5.8, and 1 mM lacto-N-neotetraose as an internal control for digestion. Digestion with jack bean ß-galactosidase was performed by incubation of purified radiolabeled oligosaccharides (1 x 104 c.p.m.), 1 mU ß-galactosidase, 5 mM Na acetate buffer, pH 4.0, and 1 mM lacto-N-neotetraose in a total volume of 10 µl. After incubation for 12 h at 37°C, reaction mixtures were applied to TLC plates. Oligosaccharides were visualized using orsinol-H2SO4.
NMR-spectroscopy
Complete glycosylation of lacto-N-neotetraose by soluble IGnT B was performed in a reaction mixture consisting of 0.2 mM lacto-N-neotetraose, 0.2 mM UDP-GlcNAc, 50 mM Na cacodylate buffer, pH 7.0, 10 mM 2-acetamido-2-deoxy-D-glucono-1,5-lactone, 4 mM ATP, 1 mM EDTA, and IGnT B (900 nmol/h/ml) in a final volume of 1.5 ml. After 12 h of incubation at 37°C, reaction products were desalted and purified by Bio-Gel P-4 column chromatography (125 x 0.8 cm). One-dimensional proton nuclear magnetic resonance spectra of oligosaccharides were recorded at 23 °C in D2O at 600 MHz on a Unity Inova-600 spectrometer.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bird,J.M. and Kimber,S.-J. (1984) Oligosaccharides containing fucose linked (13) and
(14) to N-acetylglucosamine cause decompaction of mouse morulae. Dev. Biol., 104, 449460.[ISI][Medline]
Fenderson,B.A., Zahavi,U. and Hakomori,S. (1984) A multivalent lacto-N-fucopentaose III-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective. J. Exp. Med., 160, 15911596.[Abstract]
Fukuda,M., Fukuda,M.N. and Hakomori,S. (1979) Developmental change and genetic defect in the carbohydrate strucutre of band 3 glycoprotein of human erythrocyte membrane. J. Biol. Chem., 254, 37003703.
Fukuda,M., Dell,A. and Fukuda,M.N. (1984a) Structure of fetal lactosaminoglycan. The carbohydrate moiety of band 3 isolated from human umbilical cord erythrocytes. J. Biol. Chem., 259, 47824791.
Fukuda,M., Dell,A., Oates,J.E. and Fukuda,M.N. (1984b) Structure of branched lactosaminoglycan. The carbohydrate moiety of band 3 isolated from adult human erythrocytes. J. Biol. Chem., 260, 82608273.
Fukuda,M.N., Dell,A., Oates,J.E. and Fukuda,M. (1985) Embryonal lactosaminoglycan. The structure of branched lactosaminoglycans with novel disialosyl (sialyl 2
9 sialyl) terminals isolated from PA1 human embryonal carcinoma cells. J. Biol. Chem., 260, 66236631.
Gu,J., Nishikawa,A., Fujii,S., Gasa,S. and Taniguchi,N. (1992) Biosynthesis of blood group I and i antigens in rat tissues. Identification of a novel ß16-N-acetylglucosaminyltransferase. J. Biol. Chem., 267, 29942999.
Helin,J., Penttilä,L., Leppänen,A., Maaheimo,H., Lauri S., Costello,C.E. and Renkonen,O. (1997) The ß1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone. FEBS Lett., 412, 637642.[ISI][Medline]
Järnefelt,J., Rush,J., Li,Y.-T. and Laine,R.A. (1978) Erythroglycan, a high molecular weight glycopeptide with the repeating structure [galactosyl- (14)-2-deoxy-2-acetamido-glucosyl (1
3)] comprising more than one-third of the protein-bound carbohydrate of human erythrocyte stroma. J. Biol. Chem. 253, 80068009.[Abstract]
Kamada,Y., Arita,Y., Ogata,S., Muramatsu,H. and Muramatsu,T. (1987) Receptors for fucose-binding proteins of Lotus tetragonolobus isolated from mouse embryonal carcinoma cells: structural characteristics of the poly (N-acetyllactosamine)-type glycan, Eur. J. Biochem., 163, 497502.[Abstract]
Kapadia,A., Feizi,T. and Evans,M.J. (1981) Changes in the expression and polarization of blood group I and i antigens in post-implantation embryos and teratocarcinomas of mouse associated with cell differentiation. Exp. Cell Res., 131, 185195.[ISI][Medline]
Kobata,A., Yamashita,K. and Takasaki,S. (1987) Bio-Gel P-4 column chromatography of oligosaccharides: effective size of oligosaccharides expressed in glucose units. Methods Enzymol., 138, 8494.[ISI][Medline]
Kojima,N., Yoshida,Y., Kurosawa,N., Lee,Y.C. and Tsuji,S. (1995) Enzymatic activity of a developmentally regulated member of the sialyltransferase family (STX): evidence for 2,8-sialyltransferase activity toward N-linked oligosaccharides. FEBS Lett., 360, 14.[ISI][Medline]
Krusius,T., Finne,J. and Rauvala,H. (1978) The poly (glycosyl) chains of glycoproteins. Characterisation of a novel type of glycoprotein saccharides from human erythrocyte membrane. Eur. J. Biochem., 92, 289300.[ISI]
Kurosawa,N., Kojima,N., Inoue,M., Hamamoto,T. and Tsuji,S. (1994) Cloning and expression of Galß1,3GalNAc-specific GalNAc2,6-sialyltransferase. J. Biol. Chem., 269, 1904819053.
Kurosawa,N., Kanemitsu,Y., Masui,T., Shimada,K., Ishihara,H. and Muramatsu,T. (1999) Genomic analysis of a murine cell-surface sialomucin, MGC-24/CD164. Eur. J. Biochem., 265, 466472.
Leppänen,A., Niemelä,R. and Renkonen,O. (1997) Enzymatic midchain branching of polylactosamine backbones is restricted in a site-specific manner in 1,3-fucosylated chains. Biochemistry, 36, 1372913735.[ISI][Medline]
Leppänen,A., Penttilä,L., Niemelä,R., Helin,J., Seppo,A., Lusa,S. and Renkonen,O. (1991) Human serum contains a novel ß1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo (N-acetyllactosaminoglycans). Biochemistry, 30, 92879296.[ISI][Medline]
Leppänen,A., Zhu,Y., Maaheimo,H., Helin,J., Lehtonen,E. and Renkonen,O. (1998) Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain ß1,6-N-acetylgulcosaminyltransferase activity that generates branches to preformed linear backbones. J. Biol. Chem., 273, 1739917405.
Magnet,A.D. and Fukuda,M. (1997) Expression of the large I antigen forming ß-1,6-N-acetylglucosaminyltransferase in various tissues of adult mice. Glycobiology, 7, 285295.[Abstract]
Mattila,P., Salminen,H., Hirvas,L., Niittymäki,J., Salo,H., Niemelä,R., Fukuda,M., Renkonen,O. and Renkonen,R. (1998) The centrally acting ß1,6-N-acetylglucosaminyltransferase (GlcNAc to Gal). Functional expression, purification and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines. J. Biol. Chem., 273, 2763327639.
Muramatsu,T. (1988) Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis. J. Cell. Biochem., 36, 114.[Medline]
Muramatsu,T., Gachelin,G., Nicolas,J.F., Condamine,H., Jakob,H. and Jacob,F. (1978) Carbohydrate structure and cell differentiation: unique properties of fucosyl glycopeptides isolated from embryonal carcinoma cells. Proc. Natl Acad. Sci. USA, 75, 23152319.[Abstract]
Muramatsu,T., Gachelin,G., Damonneville,M., Delarbre,C. and Jacob,F. (1979) Cell surface carbohydrates of embryonal carcinoma cells: polysacchaidic side chains of F9 antigens and receptors to two lectins, FBP and PNA. Cell, 18, 183191.[ISI][Medline]
Ozawa,M., Muramatsu,T. and Solter,D. (1985) SSEA-1, a stage-specific embryonic antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells. Cell Differ., 16, 169173.[ISI][Medline]
Sakamoto,Y., Taguchi,T., Tano,Y., Ogawa,T., Leppänen,A., Kinnunen,M., Aitio,O., Parmanne,P., Renkonen,O. and Taniguchi,N. (1998) Purification and characterization of UDP-GlcNAc: Galß14GlcNAcß13Gal*ß14Glc(NAc)-R(GlcNAc to *Gal) ß1,6-N-acetylglucosaminyltransferase from hog small intestine. J. Biol. Chem., 273, 2762527632.
Sasaki,K., Kurata-Miura,K., Ujita,M., Angata,K., Nakagawa,S., Sekine,S., Nishi,T.and Fukuda,M. (1997) Expression cloning of cDNA encoding a human ß-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis. Proc. Natl. Acad. Sci. USA, 94, 1429414299.
Scudder,P., Hanfland,P., Uemura,K. and Feizi,T. (1984) Endo-ß-D-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series. J. Biol. Chem., 259, 65866592.
Takebe,Y., Seiki,M., Fujisawa,J., Hoy,P., Yokota,K., Arai,K., Yoshida,M. and Arai,N. (1988) SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol., 8, 466472.[ISI][Medline]
Turunen,J.P., Majuri,M.-L., Seppo,A., Tiisala,S., Paavonen,T., Miyasaka,M., Lemström,K., Penttilä,L., Renkonen,O. and Renkonen,R. (1995) De novo expression of endothelial sialyl Lewis a and sialyl Lewis x during cardiac transplant rejection: superior capacity of a tetravalent sialyl Lewis x oligosaccharide in inhibiting L-selectin-dependent lymphocyte adhesion. J. Exp. Med., 182, 11331142.[Abstract]
Ujita,M., McAuliffe,J., Hindsgaul,O., Sagaki,K., Fukuda, MN., Fukuda,M. (1999a) Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and ß 1,4-galactosyltransferase I. J. Biol. Chem., 274, 1671716726.
Ujita,M., McAuliffe,J., Suzuki,M., Hindsgaul,O., Clausen,H., Fukuda, MN., Fukuda,M. (1999b) Regulation of I-branched poly-N-acetyllactosamine synthesis, Concerted actions by I-extension enzyme, I-branching enzyme and ß 1,4-galactosyltransferase I. J. Biol. Chem., 274, 92969304.
Watanabe,K., Hakomori,S.I., Childs,R.A. and Feizi,T. (1979) Characterization of a blood group I-active ganglioside. Structural requirements for I and i specificities. J. Biol. Chem., 254, 32213228.[ISI][Medline]
Yeh,J.-C., Ong,E. and Fukuda,M. (1999) Molecular cloning and expression of a novel ß-1,6-N-acetylglucosaminyltransferase that forms core 2, core 4,and I branches. J. Biol. Chem., 274, 32153221.