Glycobiology, 2000, Vol. 10, No. 10 1001-1011
© 2000 Oxford University Press
A novel variant form of murine ß-1,6-N-acetylglucosaminyltransferase forming branches in poly-N-acetyllactosamines
Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466–8550, Japan
Received on February 23, 2000; revised on May 1, 2000; accepted on May 17, 2000.
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Abstract |
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A novel form of murine ß-1,6-N-acetylglucosaminyltransferase that forms branches in poly-N-acetyllactosamines (designated as IGnT B) was cloned based on sequence homology to the known IGnT (designated as IGnT A). When expressed as proteins, IGnT B showed higher specific activity than IGnT A. The C-terminal 1/4 of IGnT B was identical to that of IGnT A, while the rest of the predicted sequences showed 63% identity. Genomic analysis indicated that IGnT A and IGnT B were derived by alternative splicing; the unique portion was encoded by exon 1, and the common portion was encoded by exons 2 and 3. IGnT B showed an expression profile closely related to that of IGnT A and was strongly expressed in the liver, kidney and intestine, and moderately in the mammary gland, submaxially gland, embryonic stem cells, and embryonal carcinoma cells. The specificity of IGnT B examined using various substrates was indistinguishable from that of IGnT A, which is classified as the central acting IGnT (cIGnT). Thus, IGnT B acted on Galß1–4GlcNAcß1–3Galß1–4Glc, but not on GlcNAcß1–3Galß1–4Glc. It formed branches in both of the internal galactosyl residues of Galß1–4Glc-NAcß1–3Galß1–4GlcNAcß1–3Galß1–4Glc, and prolonged incubation resulted in production of the di-branched oligosaccharide. Although addition of sialic acid to the terminal galactosyl residue did not abolish the acceptor activity,
2–6 sialylation was a preferred one as compared to 2–3 sialylation. Key words: alternative splicing/ß-1,6-N-acetylglucosaminyl-transferase/gene structure/I antigen/poly-N-acetyllactosamine
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Introduction |
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Poly-N-acetyllactosamines, which have repeated Galß- 1–4GlcNAcß1–3 units, are found in asparagine-linked and serine/threonine-linked glycans and glycolipids. Keratan sulfate is an extended and sulfated linear poly-N-acetyllactosamine. Occasionally, poly-N-lactosamine chains are branched by addition of an N-acetylglucosaminyl residue via ß1–6 linkage to a galactosyl residue. Linear poly-N-acetyllactosamines bear the i blood group antigen, while the branched molecules bear the I blood group antigen (Watanabe et al., 1979
). Branched poly-N-acetyllactosamines sometimes become high molecular mass, around 10,000 or even more; typical examples are poly-N-acetyllactosamines from human erythrocytes (Järnefelt et al., 1978; Krusius et al., 1978; Fukuda et al., 1984a,b) and those from early embryonic cells including embryonal carcinoma cells (Muramatsu et al., 1978, 1979; Fukuda et al., 1985).
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.
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Results |
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Molecular cloning and sequence analysis of a new variant form of ß-1,6-N-acetylglucosaminyltransferase (IGnT B)
In the mouse EST data base, we found a sequence homologous to mouse IGnT (Magnet and Fukuda, 1997
). Using the EST sequence as a probe, we screened mouse cDNA libraries and eventually obtained a sequence of 1890 nucleotides, encoding an open reading frame of 401 amino acids (Figure 1A). The predicted sequence has 69% identity to the known mouse IGnT (thereafter referred to as IGnT A). Interestingly, the C-terminal 1/4 of the newly cloned protein showed 100% amino acid sequence identity with IGnT A. Due to its high degree of protein sequence similarity to IGnT A, we called the new protein IGnT B. The protein was expected to have a type II transmembrane topology, while the putative transmembrane region did not show significant homology to IGnT A. We also noted that IGnT A sequence of our clone was different from that reported by Magnet and Fukuda (1997), while the difference is best explained by strain difference.
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Screening of a mouse genomic library with IGnT A and B cDNAs resulted in the isolation of three independent clones. DNA sequence analysis of these clones revealed that IGnT is produced as two splice variants. This differential splicing arises as a result of alternative 5' exon usage (Exons 1A and 1B), generating IGnT A and B, respectively (Figure 1B). Exons 1A and 1B both encode 307 amino acids, comprising a short cytoplasmic domain, a transmembrane domain and part of the putative catalytic domain. The common C-terminal 1/4 of the molecules were encoded by Exon 2 and Exon 3 (Figure 1B).
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.
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Northern blotting analysis using exon-specific probes revealed that IGnT B expression was closely related to that of IGnT A; both were strongly expressed in the liver, kidney and intestine, and moderately in the mammary gland, submaxillary gland, D3 embryonic stem cells and in P19 embryonal carcinoma cells (Figure 2B). Expression of IGnT A in the kidney, intestine and embryonal carcinoma cells was in agreement with the data published previously (Magnet and Fukuda, 1997
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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 (Galß1–4GlcNAcß1–3Galß1–4Glc) 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).
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Upon thin layer chromatography (TLC), the product of the enzymatic reaction by IGnT B migrated to a position corresponding to that of Galß1–4GlcNAcß1–3([14C]GlcNAcß1–6)-Galß1–4Glc produced by IGnT A (Figure 3A). The product was cleaved by ß-galactosidase, yielding a product that migrated at a position corresponding to that of GlcNAcß1–3-([14C]GlcNAcß1–6)Galß1–4Glc both upon TLC (Figure 4A, lane 3) and Bio gel P-4 column chromatography (Figure 4B, shaded circles). Therefore, the N-acetylglucosamine residue could not have been transferred to the terminal galactosyl residue; an example of terminal transfer is i enzyme, which forms a GlcNAcß1–3Gal structure (Sasaki et al., 1997
). Furthermore, the product of IGnT B was not cleaved by endo-ß-galactosidase (Figure 4A, lane 2); the product of i enzyme should be cleaved and release GlcNAcß1–3Gal disaccharide. To definitively identify the product, we determined 1H-NMR spectra of the pentasaccharide product. The 1H signal of 4.615 was in agreement with that of the GlcNAcß1–6 branch in Galß1–4-GlcNAcß1–3(GlcNAcß1–6)Galß1–4GlcNAc (Sakamoto et al., 1998; Leppänen et al., 1998), even though reducing end of the oligosaccharide was GlcNAc and that of our product was Glc. The result establishes the structure of the product as Galß1–4-GlcNAcß1–3(GlcNAcß1–6)Galß1–4Glc (Table I). Thus, IGnT B was verified to have IGnT activity.
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Specificity of IGnT B
The acceptor specificity of the enzyme was studied using various substrates (Figure 5) and the results are summarized in Table II. The enzyme did not act on GlcNAcß1–3Galß1–4Glc. Generally, oligosaccharides with the Galß1–4GlcNAcß1–3Gal-ß1–4Glc(NAc) sequence served as good substrates, except that Galß1–3GlcNAcß1–3Galß1–4GlcNAlß1–3Galß1–4Glc and Galß1–4(Fuc
1–3)GlcNAcß1–3Galß1–4Glc were poor substrates (Table II, Figure 5). Addition of Gal1–3 or sialic acid 2–6 to the non-reducing end galactose enhanced the acceptor activity, while sialic acid 2–3 linkage showed a repressive effect. The kinetic properties of IGnT B are summarized in Table III.
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When a pentasaccharide, GlcNAcß1–3Galß1–4GlcNAc-ß1–3Galß1–4Glc, was used as an acceptor, the product (Figure 4A, lane 4; B, shaded triangles) was completely cleaved by endo-ß-galactosidase and yielded an oligosaccharide with mobility corresponding to that of GlcNAc- ß1–3(GlcNAc-ß1–6)Galß1–4Glc (Figure 4A, lane 5). Therefore, of the two internal galactosyl residues (peridistal and central), only the central one served as an acceptor site.
When para-lacto-N-neohexaose (Galß1–4GlcNAcß1–3Gal-ß1–4GlcNAcß1–3Galß1–4Glc) 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 4–5 hexose units earlier than GlcNAcß1–3(GlcNAcß1–6)Galß1–4Glc (Figure 4B, open diamonds and shaded circles). Since one N-acetylglucosamine contributes to the mobility in the column as 1.8–2.0 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 GlcNAcß1–3 linkage, but a branched oligosaccharide, Galß1–4GlcNAß1–3(GlcNAcß1–6)Galß1–4Glc-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 Galß1–4Glc-NAcß1–3(GlcNAcß1–6)Galß1–4GlcNAcß1–3Galß1–4Glc, while the minor product was concluded to be Galß1–4Glc-NAc- ß1–3Galß1–4GlcNAcß1–3(GlcNAcß1–6)Galß1–4Glc. 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 Galß1–4Glc-NAcß1–3(GlcNAcß1–6)Galß1–4GlcNAcß1–3(GlcNAcß1–6)-Gal-ß1–4Glc.
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.
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Discussion |
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Recent studies have shown that IGnT activities can be classified into two types. The central acting activity (cIGnT) transfers N-acetylglucosamine to central galactose residues in Galß1–4GlcNAcß1–3Galß1–4GlcNAcß1–0R (Gu et al., 1992
; Leppänen et al., 1998; Mattila et al., 1998; Sakamoto et al., 1998), while the predistal acting activity (dIGnT) transfers N-acetylglucosamine to predistal galactosyl residues in GlcNAcß1–3Galß1–4GlcNAcß1–0R (Helin et al., 1997). IGnT cloned from human embryonal carcinoma cells (Bierhuizen et al., 1993) shows cIGnT activity, but not dIGnT activity, when linear oligo-N-acetyllactosamines are used as substrates (Mattila et al., 1998), while some dIGnT activity is observed using the oligosaccharides linked to a Man1–6 Man derivative (Yeh et al., 1999). In addition, ß-1,6-N-acetylglucosaminyltransferase preferentially acting on core 2 structures of mucin-type O-glycans (C2GnT-M) has some dIGnT and cIGnT activities (Yeh et al., 1999).
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 Galß1–4GlcNAcß1–3Galß1–4Glc and transferred an N-acetylglucosamine residue to the underlined central galactose, but did not act on GlcNAcß1–3Galß1–4Glc. When a hexasaccharide Galß1–4GlcNAcß1–3Galß1–4GlcNAcß1–3Galß1–4Glc 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 1–3 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, NeuAc2–3Galß1–4GlcNAcß1–3Galß1–4Glc 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.
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Materials and methods |
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Materials
The following chemicals were purchased from the sources indicated: [
-32P] dCTP (220 TBq/mmol), UDP-GlcNAc [glucosamine-U-14C] (11.1Gbq/mmol) were from Amersham Pharmacia Biotech. UDP-GlcNAc [glucosamine-6–3H] (1.4TBq/mmol) was from NEN Life Science (Boston). Lacto-N-neotetraose (Galß1–4GlcNAcß1–3Galß1–4Glc), Lacto-N-tetraose (Galß1–3GlcNAcß1–3Galß1–4Glc), para-lacto-N-hexaose (Galß1–3GlcNAcß1–3Galß1–4GlcNACß1–3Galß1–4Glc) and para-lacto-N-neohexaose (Galß1–4GlcNAcß1–3Galß1–4GlcNACß1–3Galß1–4Glc) were purchased from Sigma Aldrich. NeuAc2–6Galß1–4GlcNAcß1–3Galß1–4Glc, Galß1–4(Fuc1–3)Glc-NAcß1–3Galß1–4Glc, endo-ß-galactosidase (Escherichia freundii) and ß-galactosidase (jack bean) were from Seikagaku Corporation (Tokyo, Japan). Other oligosaccharides were from Toronto Research Chemicals Inc (North York, Canada). GlcNAcß1–3Galß1–4Glc and GlcNAcß1–3Galß1–4GlcNACß1–3Galß1–4Glc were prepared from lacto-N-neotetraose and para-lacto-N-neohexaose by ß-galactosidase digestion, respectively. NeuAc2–3Galß1–4-GlcNAcß1–3Galß1–4Glc was produced from lacto-N-neotetraose and CMP-sialic acid by incubation with –2,3-(N)-sialyltransferase (ST3Gal III) (Calbiochem-Novabiochem, CA). Galß1–4GlcNAcß1–3Galß1–4GlcNAcß1–3Galß1–4GlcNAc (L1L1L1) was a generous gift from Dr. Keiichi Yoshida, Seikagaku Corporation, Tokyo.
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. 863–843 Figure 1A) and 5'-TCGGGAAATGATAGTTCTTCC-3' (nt. 619–599) for 5'-RACE, and 5'-CAGCCTATCTCTCATTGCTGC-3' (nt. 542–573) and 5'-TCTCTGAAGAAGAAGCCCG-3' (nt. 773–791) 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. 1460–1438, underlined nucleotides were added for restriction enzyme digestion; nucleotide no. refers to GenBank accession No. U68182), 5'-AGAGCTCGAGCATGCCTCTGTCCGTGCGTTACTTC-3' (nt. 235–260) and F9 cDNA as a template for IGnT A; 5'-GCCAAGGAGCTTTGCTCATCAGAGCC-3' (nt. 367–392), 5'-GAGACTCGAGCCGGAGCTGCTGCGGGTCAGA-3' (nt. 1738–1718) 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, GC; nt 292, GT; nt 294, GA; nt 301, CT; nt 508 and 509, GTTC; nt 679, TC; nt 1247, AT; nt 1411, GT and three nucleotide (CAT) insertions between nt 1168–1169 (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 324–633), an IGnT B-specific probe (nt 595–1035 in Figure 1) and an IGnT exon 3 probe (nt 1531–1743 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. 324–345) and F9 cDNA. The sense primer and cDNA template used for the amplification of IGnT B were 5'-AGAGAGAATTCTTATGGAAGAACTATCATTTCCCGA-3' (nt. 595–619) 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 Tris–HCl 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.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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We thank Ms. M.Ishihara, H.Yoshida, and H.Inoue for secretarial assistance. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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CHO, Chinese hamster ovary; IgnT, I ß-1,6-N-acetylglucosaminyltransferase; dIGnT and cIGnT, distally acting and centrally acting I ß–1,6-N-acetylglucosaminyltransferase, respectively; EST, expressed sequence tag; lacto-N-neotetraose, Galß1–4GlcNAcß1–3Galß1–4Glc; para-lacto-N-neohexaose, Galß1–4GlcNAcß1–3Galß1–4GlcNACß1–3Gal-ß1–4Glc; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; TLC, thin layer chromatography.
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1 To whom correspondence should be addressed
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Bierhuizen,M.F.A., Mattei,M.-G. and Fukuda,M. (1993) Expression of the developmental I antigen by a cloned human cDNA encoding a member of a ß-1,6-N-acetylglucosaminyltransferase gene family. Genes Dev., 7, 468–478.
Bird,J.M. and Kimber,S.-J. (1984) Oligosaccharides containing fucose linked (1–3) and (1–4) to N-acetylglucosamine cause decompaction of mouse morulae. Dev. Biol., 104, 449–460.
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, 1591–1596.
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, 3700–3703.
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, 4782–4791.
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, 8260–8273.
Fukuda,M.N., Dell,A., Oates,J.E. and Fukuda,M. (1985) Embryonal lactosaminoglycan. The structure of branched lactosaminoglycans with novel disialosyl (sialyl 29 sialyl) terminals isolated from PA1 human embryonal carcinoma cells. J. Biol. Chem., 260, 6623–6631.
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 ß1–6-N-acetylglucosaminyltransferase. J. Biol. Chem., 267, 2994–2999.
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, 637–642.
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 (13)] comprising more than one-third of the protein-bound carbohydrate of human erythrocyte stroma. J. Biol. Chem. 253, 8006–8009.
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, 497–502.
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, 185–195.
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, 84–94.
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, 1–4.
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, 289–300.
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, 19048–19053.
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, 466–472.
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, 13729–13735.
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, 9287–9296.
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, 17399–17405.
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, 285–295.
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, 27633–27639.
Muramatsu,T. (1988) Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis. J. Cell. Biochem., 36, 1–14.
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, 2315–2319.
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, 183–191.
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, 169–173.
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: Galß1–4GlcNAcß1–3Gal*ß1–4Glc(NAc)-R(GlcNAc to *Gal) ß1,6-N-acetylglucosaminyltransferase from hog small intestine. J. Biol. Chem., 273, 27625–27632.
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, 14294–14299.
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, 6586–6592.
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, 466–472.
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, 1133–1142.
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, 16717–16726.
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, 9296–9304.
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, 3221–3228.
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, 3215–3221.
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