Deletion of Two Exons from the Lymnaea stagnalis β1→4-N-Acetylglucosaminyltransferase Gene Elevates the Kinetic Efficiency of the Encoded Enzyme for Both UDP-sugar Donor and Acceptor Substrates
1997; Elsevier BV; Volume: 272; Issue: 30 Linguagem: Inglês
10.1074/jbc.272.30.18580
ISSN1083-351X
AutoresHans Bakker, Angelique van Tetering, Marja Agterberg, August B. Smit, Dirk H. van den Eijnden, Irma van Die,
Tópico(s)Enzyme Production and Characterization
ResumoLymnaea stagnalisUDP-GlcNAc:GlcNAcβ-R β1→4-N-acetylglucosaminyltransferase (β4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal:GlcNAcβ-R β1→4-galactosyltransferase (β4-GalT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The β4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the β4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence similarity to part of the preceding exon (exon 6), suggesting that they have originated by exon duplication. The exon in the β4-GalT gene, corresponding to β4-GlcNAcT exon 6, encodes a region that has been proposed to be involved in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the β4-GlcNAcT gene would determine the specificity of the enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was found that deletion of only the sequence encoded by exon 8 resulted in a completely inactive enzyme. By contrast, deletion of the amino acid residues encoded by exons 7 and 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These results suggest that at least part of the donor and acceptor binding domains of the β4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity. Lymnaea stagnalisUDP-GlcNAc:GlcNAcβ-R β1→4-N-acetylglucosaminyltransferase (β4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal:GlcNAcβ-R β1→4-galactosyltransferase (β4-GalT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The β4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the β4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence similarity to part of the preceding exon (exon 6), suggesting that they have originated by exon duplication. The exon in the β4-GalT gene, corresponding to β4-GlcNAcT exon 6, encodes a region that has been proposed to be involved in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the β4-GlcNAcT gene would determine the specificity of the enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was found that deletion of only the sequence encoded by exon 8 resulted in a completely inactive enzyme. By contrast, deletion of the amino acid residues encoded by exons 7 and 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These results suggest that at least part of the donor and acceptor binding domains of the β4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity. Glycosyltransferases form a large family of functionally related, membrane-bound enzymes that are involved in the biosynthesis of the carbohydrate moieties of glycoproteins and glycolipids (1Schachter H. Curr. Opin. Struct. Biol. 1991; 1: 755-765Crossref Scopus (67) Google Scholar, 2Van den Eijnden D.H. Joziasse D.H. Curr. Opin. Struct. Biol. 1993; 3: 711-721Crossref Scopus (73) Google Scholar). Recently we have identified a novel glycosyltransferase by the isolation of a UDP-GlcNAc:GlcNAcβ-R β1→4-N-acetylglucosaminyltransferase (β4-GlcNAcT) 1The abbreviations used are: β4-GlcNAcT, UDP-GlcNAc:GlcNAcβ-R β1→4-N-acetylglucosaminyltransferase; β4-GalNAcT, UDP-GalNAc:GlcNAcβ-R β1→4-N-acetylgalactosaminyltransferase; β4-GalT, UDP-Gal:GlcNAcβ-R β1→4-galactosyltransferase; kb, kilobase pair(s); PCR, polymerase chain reaction; HPAEC, high pH anion-exchange chromatography; PAD, pulsed amperometric detection; pNP,para-nitrophenyl; WFA, W. floribundalectin. cDNA from the prostate gland of the snail Lymnaea stagnalis (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). In vitro, the recombinant β4-GlcNAcT catalyzes the transfer of GlcNAc from UDP-GlcNAc in β1→4 linkage to various β-N-acetylglucosaminides (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar, 4Bakker H. Schoenmakers P.S. Koeleman C.A.M. Joziasse D.H. Van Die I. Van den Eijnden D.H. Glycobiology. 1997; 7: 539-548Crossref PubMed Scopus (23) Google Scholar). The β4-GlcNAcT cDNA appeared to show a significant sequence similarity to the mammalian UDP-Gal:GlcNAcβ-R β1→4-galactosyltransferase (β4-GalT) cDNAs, with an overall resemblance between the predicted amino acid sequences of about 30% (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar, 5Shaper N.L. Hollis G.F. Douglas J.G. Kirsch I.R. Shaper J.H. J. Biol. Chem. 1988; 263: 10420-10428Abstract Full Text PDF PubMed Google Scholar, 6Shaper N.L. Shaper J.H. Meuth J.L. Fox J.L. Chang H. Kirsch I.R. Hollis G.F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1573-1577Crossref PubMed Scopus (161) Google Scholar, 7Masri K.A. Appert H.E. Fukuda M.N. Biochem. Biophys. Res. Commun. 1988; 157: 657-663Crossref PubMed Scopus (130) Google Scholar). Based on the genetic and enzymatic relationship of the β4-GlcNAcT and the β4-GalTs, we have suggested that these enzymes constitute a separate glycosyltransferase gene family, the members of which are capable of catalyzing the transfer of a specific sugar from their respective UDP-sugar donors in a β1→4 linkage toward a terminal β-linked GlcNAc residue in the acceptor (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar,8Van den Eijnden D.H. Neeleman A.P. Van der Knaap W.P.W. Bakker H. Agterberg M. Van Die I. Adv. Exp. Med. Biol. 1995; 376: 47-51Crossref PubMed Scopus (9) Google Scholar). Based on enzymatic properties, we have proposed that also UDP-GalNAc:GlcNAcβ-R β1→4-N-acetylgalactosaminyltransferase (β4-GalNAcT), detected in several non-vertebrate species (9Mulder H. Spronk B.A. Schachter H. Neeleman A.P. Van den Eijnden D.H. De Jong-Brink M. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1995; 227: 175-185Crossref PubMed Scopus (34) Google Scholar, 10Neeleman A.P. Van der Knaap W.P.W. Van den Eijnden D.H. Glycobiology. 1994; 4: 641-651Crossref PubMed Scopus (43) Google Scholar, 11Van Die I. Van Tetering A. Bakker H. Van den Eijnden D.H. Joziasse D.H. Glycobiology. 1996; 6: 157-164Crossref PubMed Scopus (80) Google Scholar, 12Srivatsan J. Smith D.F. Cummings R.D. J. Parasitol. 1994; 80: 884-890Crossref PubMed Scopus (33) Google Scholar, 13Neeleman A.P. Van den Eijnden D.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10111-10116Crossref PubMed Scopus (24) Google Scholar), belongs to this family (14Van den Eijnden D.H. Neeleman A.P. Van der Knaap W. Bakker H. Agterberg M. Van Die I. Biochem. Soc. Trans. 1995; 23: 175-179Crossref PubMed Scopus (57) Google Scholar). The primary structure of this enzyme, however, is still unknown. The reaction catalyzed by glycosyltransferases typically involves two substrates and often a divalent cation cofactor. This suggests that the enzymes consist of several functional domains involved in substrate and cofactor binding, respectively. Comparison of the conserved and variable regions of genetically related glycosyltransferases with different properties would open possibilities to address structure-function relationships. As snails and mammals are evolutionary distant species, comparison of the genomic organization of the genes that encode these enzymes might give insight in their way of divergence, that resulted in genes encoding enzymes with a different UDP-sugar specificity. The genomic organization of the murine and human β4-GalT genes have been described previously (15Hollis G.F. Douglas J.G. Shaper N.L. Shaper J.H. Stafford-Hollis J.M. Evans R.J. Kirsch I.R. Biochem. Biophys. Res. Commun. 1989; 162: 1069-1075Crossref PubMed Scopus (44) Google Scholar, 16Mengle-Gaw L. McCoy-Haman M.F. Tiemeier D.C. Biochem. Biophys. Res. Commun. 1991; 176: 1269-1276Crossref PubMed Scopus (41) Google Scholar). Here we report the organization of the L. stagnalis gene that codes for the β4-GlcNAcT. The intron-exon distribution was determined and compared with that of the β4-GalT gene. Mutant β4-GlcNAcT cDNAs were constructed by deletion of sequences that do not have a counterpart in the β4-GalT gene, and expressed in COS cells. Comparison of the kinetic parameters of the resulting mutant and parental enzymes showed that the insertion in the β4-GlcNAcT and its surrounding regions contribute to acceptor recognition as well as to UDP-sugar binding and specificity. Acceptor substrates were obtained from Sigma (compounds 1, 2, and3) and from Toronto Research Chemicals (compounds5 and 6). Compound 4 was a gift of Dr. O. Hindsgaul, University of Alberta, Alberta, Canada. UDP-[3H]GlcNAc, UDP-[3H]GalNAc and UDP-[3H]Gal were purchased from NEN Life Science Products, and were diluted with unlabeled UDP-sugars (Sigma) to the desired specific radioactivity. Recombinant plasmids were propagated in the Escherichia coliK12 strain XL1-Blue (Stratagene). COS-7M6 cells (ATCC 1651) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, and 1:50 penicillin/streptomycin solution (all from Life Technologies, Inc.). Synthetic oligonucleotides were obtained from Isogen Bioscience BV (Maarssen, the Netherlands). The genomic clone λ5 has been isolated previously (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar) from a genomic λEMBL3A library of L. stagnalis (17Bogerd J. Vankesteren R.E. Vanheerikhuizen H. Geraerts W.P.M. Veenstra J. Smit A.B. Joosse J. Cell Mol. Neurobiol. 1993; 13: 123-136Crossref PubMed Scopus (15) Google Scholar). Isolation of plasmid DNA was carried out by a modification of the minilysate method, as described in Ref. 18Del Sal G. Manfioletti G. Schneider C. Nucleic Acids Res. 1988; 16: 9878Crossref PubMed Scopus (230) Google Scholar. Plasmids used for transfection of COS cells were isolated by the Qiagen plasmid protocol, using a QIAGEN-tip 100 minicolumn. Restriction enzymes and other DNA-modifying enzymes were used according to the manufacturer. Dideoxynucleotide chain-terminating sequencing reactions (19Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52749) Google Scholar) were performed on double-stranded plasmid DNA, with the T7 DNA sequencing kit (Pharmacia Biotech Inc.), [α-35S]dATP (Amersham), using M13 universal primer, the KS and SK primers, and several sequence-specific synthetic oligonucleotide primers. Southern blotting was performed as described previously (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). PCR with sequence-specific primers was performed using Ultma-polymerase (Perkin-Elmer) by 25 cycles (1 min at 95 °C, 1 min at 63 °C, 1 min at 72 °C). For cloning purposes, amplified fragments were subsequently purified according to the QIAquick PCR purification protocol (Qiagen Inc.) The plasmid pMC135, containing a fusion between part of the protein A sequence and β4-GlcNAcT cDNA, was constructed as follows. ABamHI-EcoRI adapter was ligated in theBamHI site of pVTBac-P11.4, carrying 5′-truncated β4-GlcNAcT cDNA (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). The resulting 1.4-kb EcoRI fragment from this construct was ligated into anEcoRI-digested pPROTA vector (20Sanchez-Lopez R. Nicholson R. Gesnel M-C. Matrisian L.M. Breathnach R. J. Biol. Chem. 1988; 263: 11892-11899Abstract Full Text PDF PubMed Google Scholar). Plasmids carrying mutant β4-GlcNAcT genes were derived from pMC135 by exchange of the 0.52-kbXhoI-BglII fragment for a PCR fragment carrying the desired deletion. For PCR of the deletion fragments, a sense primer (bases 439–456 of the β4-GlcNAcT cDNA; Ref. 3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar) and the antisense primer ID8 (ATAGATCTTAAATCTGTCCGGGTTCACATTCCAG) or ID16 (ATAGATCTTAAATCTGTTCGGGTGCACGTTC) was used. The antisense primer ID8, used for construction of pMC142, consists of a part complementary to the 3′ end of exon 6, and a part (11 base pairs), complementary to the 5′ end of exon 9 (BglII restriction site underlined). The antisense primer ID16, used for construction of pMC166, consists of a part complementary to the 3′ end of exon 7, and the same 5′ part of exon 9 as ID8. PCR fragments obtained with these primers were digested with XhoI and BglII, and ligated into XhoI-BglII-digested pMC135. After transformation and plasmid isolation of several transformants, the desired plasmids (pMC142 encoding protA-β4-GlcNAcTΔ7–8 and pMC166 encoding protA-β4-GlcNAcTΔ8) were selected by size determination of the internal SalI fragment followed by determination of the nucleotide sequence of the complete XhoI-BglII fragment with sequence-specific primers. Recombinant pPROTA chimeric constructs were transiently transfected to COS cells (3 × 105 cells/10-cm dish), using the calcium phosphate precipitation technique as described previously (21Agterberg M. Van Die I. Yang H. Andriessen J.A. Van Tetering A. Van den Eijnden D.H. Ploegh H.L. Eur. J. Biochem. 1993; 217: 241-246Crossref PubMed Scopus (9) Google Scholar); after 24 h, the medium was replaced by fresh medium; medium was harvested at 48, 72, and 96 h after transfection and each time replaced by fresh medium. The harvested media were pooled, stored at −20 °C in portions, and used as enzyme source for glycosyltransferase assays and Western blotting. Membrane bound recombinant β4-GlcNAcT was produced with a pMT2-based construct as described previously (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). The Western blot analysis performed was aimed on detection of the protein A part of the fusion proteins. The proteins of 1 μl of pooled medium collected after transfection, were separated by SDS-polyacrylamide gel electrophoresis on 10% gels using the Mini-PROTEAN II system (Bio-Rad). Western blotting was performed essentially as described previously (21Agterberg M. Van Die I. Yang H. Andriessen J.A. Van Tetering A. Van den Eijnden D.H. Ploegh H.L. Eur. J. Biochem. 1993; 217: 241-246Crossref PubMed Scopus (9) Google Scholar). As first antibody, an arbitrary mouse IgG monoclonal antibody (ED3, Ref. 22Dijkstra C.D. Döpp E.A. Joling P. Kraal G. Immunology. 1985; 54: 589PubMed Google Scholar) was used, which reacts with the protein A part of the hybrid proteins. The second antibody used was a goat anti-mouse peroxidase conjugate (Tago, Inc. Immunodiagnostic Reagents). Standard glycosyltransferase assays were performed in a 50-μl reaction mixture containing either 25 nmol of UDP-[3H]GalNAc (2 Ci/mol), UDP-[3H]GlcNAc (1 Ci/mol), or UDP-[3H]Gal (1 Ci/mol), 5 μmol of sodium cacodylate buffer, pH 7.2, 1 μmol of MnCl2, 0.2 μmol of ATP, and 50 nmol ofp-nitrophenyl-N-acetyl-1-thio-β-d-glucosaminide (GlcNAc-S-pNP). As enzyme source, 10 μl of COS cell medium, which had been concentrated 10 times with Centriprep-10 concentrators (Amicon), was used. The product was isolated using Sep-Pak C-18 cartridges (Waters) (23Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconjugate J. 1988; 5: 49-63Crossref Scopus (279) Google Scholar). For acceptor specificity studies, acceptor substrate concentrations were kept at 1 mm, in terms of terminal GlcNAc residues. Control assays lacking the acceptor substrate were carried out to correct for incorporation into endogenous acceptors. Enzyme activity was expressed as pmol/min−1/ml−1 of the original medium. As enzyme source for the experiments studying the UDP-Gal specificity, the enzyme was isolated from the medium by binding to IgG-agarose (Sigma). 10 ml of medium was incubated with 10 μl of IgG-agarose beads for 16 h at 4 °C, carefully shaking; the beads were subsequently collected by centrifugation and resuspended in 500 μl of 0.1m sodium cacodylate buffer, pH 7, containing 1 mg/ml bovine serum albumin; 10 μl of this suspension was used in a standard assay. Kinetic parameters (K m and V) were estimated from Lineweaver-Burk plots by varying the sugar-donor concentrations from 0.05 to 0.5 mm for UDP-GlcNAc and from 0.25 to 5 mm for UDP-GalNAc while keeping the GlcNAc-S-pNP concentration at 1 mm, or by varying the acceptor substrate concentration from 0.025 to 1 mm while keeping the UDP-GlcNAc concentration at 0.5 mm. The inhibitory effect of UDP was studied at fixed concentrations of UDP-GlcNAc (0.5 mm) and GlcNAc-O-pNP (1 mm), whereas the UDP concentration was varied from 0.1 to 5 mm. Product, obtained with the enzyme protA-β4-GlcNAcTΔ7–8, GlcNAc-S-pNP, and UDP-[3H]-GalNAc, was analyzed by lectin affinity chromatography with Wisteria floribunda lectin (WFA), immobilized on azlactone/bisacrylamide polymeric beads (3m Emphaze™, Pierce) (24Do K.-Y. Do S.-I. Cummings R.D. J. Biol. Chem. 1995; 270: 18447-18451Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The product was also analyzed by HPAEC-PAD (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). As a control, both WFA lectin chromatography and HPAEC-PAD analysis were performed with reference GalNAcβ1→4GlcNAc-S-pNP produced with L. stagnalisalbumen gland β4-GalNAcT (9Mulder H. Spronk B.A. Schachter H. Neeleman A.P. Van den Eijnden D.H. De Jong-Brink M. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1995; 227: 175-185Crossref PubMed Scopus (34) Google Scholar). A genomic clone, denoted λ5, was isolated previously from a λEMBL3A library of L. stagnalis, and was shown to contain two short DNA sequences identical to β4-GlcNAcT cDNA sequences (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). A rough genomic map of λ5 was constructed by PCR and Southern blot hybridization using specific β4-GlcNAcT cDNA fragments as probes (Fig. 1). λ5 was found to encompass the complete coding sequence of the β4-GlcNAcT gene, spanning 12.5 kb of DNA, that was divided into 10 exons (Fig. 1, TableI). As probably part of the 5′-noncoding sequence is lacking from the cDNA (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar), we cannot exclude the presence in the gene of one or more exons upstream of the denoted exon 1. Exon 10 was found to encompass the complete 3′-noncoding region. All exon sequences in the genomic clone were identical to those of the cDNA (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar). Donor and acceptor splice junction sequences (Table II) are in agreement with consensus sequences reported (25Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3297) Google Scholar).Table IExons of the L. stagnalis β4-GlcNAcT geneExon no.Exon lengthCorresponding cDNA residuesAmino acids encodedbp1>52−3 –521 –17239953 –45118 –1503233452 –684151 –2284191685 –875229 –2925123876 –998293 –3336102999 –1100334 –3677781101 –1178368 –3938781179 –1256394 –41991391257 –1395420 –465102251396 –1620466 –490 Open table in a new tab Table IIIntrons of the L. stagnalis β4-GlcNAcT geneIntron no.Intron lengthSplice junction sequenceDonorAcceptorkb11.0AACAC:gtgagtaggatttctcccag:GCCAT20.7CTTAG:gtaaaaaaaatttcgaacag:CTGGA30.8AACAG:gtgagaaggagtcttggcag:ACTAC42.1TACAA:gtgagcatcctattttgcag:ACTTT52.5AACAG:gtaagacgtgtcgattgcag:GGCCG60.5CACAG:gtgagacccaatattaccag:CAAAG70.9AACAG:gtgagacccaatattaccag:CAAAT81.3GACAG:gtgagacccagtatccccag:ATTTA91.0CGAAT:gtgagtgtttctactttcag:AGCAT Open table in a new tab The cDNA encoding β4-GlcNAcT shows sequence identity with the mammalian β4-GalT cDNAs identified (3Bakker H. Agterberg M. Van Tetering A. Koeleman C.A.M. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1994; 269: 30326-30333Abstract Full Text PDF PubMed Google Scholar, 5Shaper N.L. Hollis G.F. Douglas J.G. Kirsch I.R. Shaper J.H. J. Biol. Chem. 1988; 263: 10420-10428Abstract Full Text PDF PubMed Google Scholar, 6Shaper N.L. Shaper J.H. Meuth J.L. Fox J.L. Chang H. Kirsch I.R. Hollis G.F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1573-1577Crossref PubMed Scopus (161) Google Scholar, 7Masri K.A. Appert H.E. Fukuda M.N. Biochem. Biophys. Res. Commun. 1988; 157: 657-663Crossref PubMed Scopus (130) Google Scholar). A comparison of the protein-coding exons of theL. stagnalis β4-GlcNAcT gene with those of the murine and human β4-GalT genes (15Hollis G.F. Douglas J.G. Shaper N.L. Shaper J.H. Stafford-Hollis J.M. Evans R.J. Kirsch I.R. Biochem. Biophys. Res. Commun. 1989; 162: 1069-1075Crossref PubMed Scopus (44) Google Scholar, 16Mengle-Gaw L. McCoy-Haman M.F. Tiemeier D.C. Biochem. Biophys. Res. Commun. 1991; 176: 1269-1276Crossref PubMed Scopus (41) Google Scholar) is shown in Fig. 2. The β4-GalT gene was found to be divided into six exons, whereas the β4-GlcNAcT gene appeared to contain 10 exons. Exons 3, 4, 5, 6, and 9 of the β4-GlcNAcT gene were found to show similarity to exons 2–6 of the β4-GalT gene, corresponding to the catalytic domain of the enzyme (Fig. 2). This similarity was not only confined to a high degree of sequence identity; intron/exon boundaries were also found at identical positions within the gene. The coding sequence of the β4-GlcNAcT gene is larger than that of the β4-GalT gene. These additional sequences appeared to be mainly encoded by three exons (exons 7, 8, and 10), that were not present in the β4-GalT gene. Exons 7 and 8 were found to encode a partial repeat of the sequence of exon 6 (Table III). The sequence analysis of the intron/exon borders (Table II) shows that exons 6, 7, and 8 are symmetrical exons. Additionally, it was observed that the acceptor splice junction sites of introns 6 and 7 as well as the donor splice junction sites of intron 6, 7, and 8 and the adjacent exon sequences are identical. These data strongly suggest that exons 7 and 8 of the β4-GlcNAcT gene arose by duplications (26Patthy L. Curr. Opin. Struct. Biol. 1994; 4: 383-392Crossref Scopus (76) Google Scholar), originating from the downstream part of exon 6.Table IIIExons of the β4-GlcNAcT gene encoding repeated amino acid sequencesExonAmino acid sequence encoded6AVHMKLPLLRKTLAHGLYDMVSH---VEAGWNVNPHS7KGAHSLYDMLNKALGVQAGWNVHPNS8KWPLRLFDSVNHAPAEGAGWNVNPDRIdentical amino acids are indicated in boldface, and dash indicates a gap in the amino acid sequence. Open table in a new tab Identical amino acids are indicated in boldface, and dash indicates a gap in the amino acid sequence. Deletion derivatives of the β4-GlcNAcT cDNA were constructed that lacked the sequences corresponding to exon 8 or to exons 7 and 8 (Fig. 3). Plasmids were constructed as gene fusions to part of protein A (20Sanchez-Lopez R. Nicholson R. Gesnel M-C. Matrisian L.M. Breathnach R. J. Biol. Chem. 1988; 263: 11892-11899Abstract Full Text PDF PubMed Google Scholar, 27Uhlen M. Guss B. Nilsson B. Gatenbeck S. Philipson L. Lindberg M. J. Biol. Chem. 1984; 259: 1695-1702Abstract Full Text PDF PubMed Google Scholar). pMC 135 encodes a soluble, native β4-GlcNAcT (protA-β4-GlcNAcT). pMC142 and pMC166 are plasmids encoding deletion derivatives of pMC135, designated protA-β4-GlcNAcTΔ7–8 and protA-β4-GlcNAcTΔ8, respectively. These three plasmids were transiently expressed in COS cells. Essentially equal amounts of hybrid protein per volume were secreted into the medium (Fig. 4).Figure 4Western blot analysis of protA-fusion proteins. COS-7M6 cells were transfected with the pPROTA vector and the pPROTA-fusion plasmids, carrying the β4-GlcNAcT cDNA, or deletion derivatives. Total cellular proteins, secreted into the medium, were separated by SDS-polyacrylamide gel electrophoresis (10%). The proteins were transferred to nitrocellulose. Antiserum reactions were directed against the protein A part of the proteins.Lane 1, protA-β4-GlcNAcTΔ7–8; lane 2, protA-β4-GlcNAcTΔ8; lane 3, protA-β4-GlcNAcT;lane 4, pPROTA; lane 5, molecular mass standards. Molecular masses on the right are indicated in kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The enzyme activities of the mutant hybrid proteins and the native protA-β4-GlcNAcT were assayed using similar amounts of enzyme (Fig.4). As the native membrane-bound β4-GlcNAcT shows a low GalNAcT activity (about 7% of the β4-GlcNAcT activity), the activity of the enzymes was measured with both UDP-GlcNAc and UDP-GalNAc. ProtA-β4-GlcNAcTΔ7–8 showed reproducibly an almost 2 times higher GlcNAcT activity and a 4 times higher GalNAcT activity than the parental protA-β4-GlcNAcT (Table IV). In contrast to the other chimeric proteins, protA-β4-GlcNAcTΔ8 appeared to be enzymatically inactive. To determine if the enzymes would show GalT activity, the hybrid proteins were purified using IgG-agarose beads. In this way, the recombinant enzymes were completely disposed of COS cell-derived β4-GalT. The bead-associated chimeras did not show detectable GalT activity, whereas they showed GlcNAcT and GalNAcT activities similar to those for the concentrated media (results not shown).Table IVUDP-sugar donor promiscuity of protein A-β4-GlcNAcT chimeras and deletion mutants derived thereofEnzymeGlycosyltransferase activity withUDP-GlcNAcUDP-GalNAcpmol · min −1 · ml −1protA-β4-GlcNAcT21015protA-β4-GlcNAcTΔ8<1<1protA-β4-GlcNAcTΔ7,836167Glycosyltransferase activities of the concentrated medium of transfected COS cells were assayed as described under “Experimental Procedures” using UDP-GlcNAc and UDP-GalNAc as donors, respectively. Open table in a new tab Glycosyltransferase activities of the concentrated medium of transfected COS cells were assayed as described under “Experimental Procedures” using UDP-GlcNAc and UDP-GalNAc as donors, respectively. Acceptor specificity studies, using UDP-GlcNAc as sugar donor, showed no significant differences in acceptor preference between the two active hybrid enzymes at an acceptor concentration of 1 mm(Table V; Ref. 4Bakker H. Schoenmakers P.S. Koeleman C.A.M. Joziasse D.H. Van Die I. Van den Eijnden D.H. Glycobiology. 1997; 7: 539-548Crossref PubMed Scopus (23) Google Scholar). The acceptor specificity of the mutant chimeric protein using UDP-GalNAc as a sugar-donor appeared very similar to the preference of the enzyme when using UDP-GlcNAc. By contrast, the prostate gland β4-GalNAcT is less specific for the linkage type of the terminal β-linked GlcNAc, and its acceptor substrate requirement resembles that of the albumen gland β4-GalNAcT (9Mulder H. Spronk B.A. Schachter H. Neeleman A.P. Van den Eijnden D.H. De Jong-Brink M. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1995; 227: 175-185Crossref PubMed Scopus (34) Google Scholar).Table VAcceptor specificity of recombinant protA-β4-GlcNAcT and protA-β4-GlcNAcTΔ7,8Acceptor substrateActivity withUDP-GlcNAcUDP-GalNAcprotA-β4-GlcNAcTprotA-β4-GlcNAcTΔ7,8rec-β4-GlcNAcT5-aRecombinant soluble β4-GlcNAcT from baculovirus infected insect cells (4).protA-β4-GlcNAcTΔ7,8Prostate gland membranes%1 GlcNAcβ-S-pNP1001001001001002 GlcNAcβ-O-pNP55727168283 GlcNAcβ1→4GlcNAcβ-O-pNP5711114 GlcNAcβ1→2Manα1↘6<1<1<1148 Manβ-O-R5-bR = −(CH2)8-COOCH3. GlcNAcβ1→2Manα1↗35 GlcNAcβ1→3GalNAcα-O-pNP3452476 GlcNAcβ1↘6968610413667 GalNAcα-O-pNP Galβ1↗3For the GlcNAcT assays 100% activity is 128 and 338 pmol · min−1 · ml medium−1, respectively. For the GalNAcT assays 100% activity is 110 pmol · min−1 · ml medium−1 for the mutant enzyme, and 5 nmol · min−1 · ml−1 for the prostate-derived enzyme.5-a Recombinant soluble β4-GlcNAcT from baculovirus infected insect cells (4Bakker H. Schoenmakers P.S. Koeleman C.A.M. Joziasse D.H. Van Die I. Van den Eijnden D.H. Glycobiology. 1997; 7: 539-548Crossref PubMed Scopus (23) Google Scholar).5-b R = −(CH2)8-COOCH3. Open table in a new tab For the GlcNAcT assays 100% activity is 128 and 338 pmol · min−1 · ml medium−1, respectively. For the GalNAcT assays 100% activity is 110 pmol · min−1 · ml medium−1 for the mutant enzyme, and 5 nmol · min−1 · ml−1 for the prostate-derived enzyme. To explain the differences in donor specificity in more detail, theK m and V values for UDP-GlcNAc and UDP-GalNAc were determined for both protA-β4-GlcNAcT and protA-β4-GlcNAcTΔ7–8
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