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Cloning and Expression of a Novel, Tissue Specifically Expressed Member of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27749

1083-351X

Kelly G. Ten Hagen, Fred K. Hagen, Marlene Balys, Thomas M. Beres, Brian Van Wuyckhuyse, Lawrence A. Tabak,

Peptidase Inhibition and Analysis

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controllingO-linked glycosylation. We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controllingO-linked glycosylation. UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase polymerase chain reactions rat sublingual gland heart muscle kinase base pair(s) amino acid kilobase(s) polyacrylamide gel electrophoresis human immunodeficiency virus N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. O-Glycosidically linked oligosaccharides are responsible for the unique physical properties and extended structural conformation of molecules such as mucins and a number of membrane receptors (1Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Abstract Full Text PDF PubMed Scopus (623) Google Scholar). Additionally, O-glycans function as ligands for receptors mediating such diverse functions as sperm-egg adhesion (2Kinloch R.A. Sakai Y. Wasserman P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 263-267Crossref PubMed Scopus (95) Google Scholar) and leukocyte trafficking (3Dowbenko D. Andalibi A. Young P.E. Lusis A.J. Lasky L.A. J. Biol. Chem. 1993; 268: 4525-4529Abstract Full Text PDF PubMed Google Scholar). The initiation of O-linked glycosylation occurs through the action of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase,1 EC 2.4.1.41), which catalyzes the transfer of GalNAc from the nucleotide sugar UDP-GalNAc to the hydroxyl group of either serine or threonine.Four members of this enzyme family have been cloned and expressed thus far from mammals (ppGaNTase-T1 (4Hagen F.K. Van Wuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 5Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar), -T2 (6Sørensen T. White T. Wandall H.H. Kristense A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), -T3 (7Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 8Zara J. Hagen F.K. Ten Hagen K.G. VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar), and -T4 (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); one putative isoform has been ablated in mice with no obvious phenotype (10Hennett T. Hagen F.K. Tabak L.A. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12070-12074Crossref PubMed Scopus (225) Google Scholar). Five additional isoforms have been identified in Caenorhabditis elegans (11Hagen F.K. Nehrke K. J. Biol. Chem. 1998; 273: 8268-8277Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In vitro assays indicate that subtle differences in substrate preferences exist among the more widely expressed ppGaNTase-T1, -T2, and -T3, although there appears to be overlap (7Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 8Zara J. Hagen F.K. Ten Hagen K.G. VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar, 12Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). In contrast, ppGaNTase-T4 displays limited substrate preferences in vitro and a more restricted pattern of expression (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Thus, while it remains unclear why so many isoforms of ppGaNTase have evolved, the conservation of this family of enzymes from nematode to humans suggests that there is functional importance to the diversity of the enzymes expressed. In the present study we have cloned and expressed a new form of ppGaNTase, which we designate ppGaNTase-T5. Conceptual translation of the cDNA that encodes this isoform reveals the unusual structural feature of a long putative stem region. In addition, both the pattern of expression and in vitro substrate preferences are more restricted in nature.DISCUSSIONIn an effort to further define the repertoire of ppGaNTases, we have employed degenerate PCR to clone a novel isoform from a rat cDNA library. ppGaNTase-T5 has the attributes of a type II membrane protein, characteristic of previously identified glycosyltransferases (Fig. 6). The N-terminal, hydrophobic, and catalytic regions of ppGaNTase-T5 are similar in length to ppGaNTase-T1, -T2, -T3, and T4. However, ppGaNTase-T5 has an unusually large stem region (416 aa), which is ∼4–7 times longer than other known isoforms. Many type II membrane proteins are converted into soluble, circulating species after cleavage within the stem region by a group of enzymes referred to as "secretases" (24Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). Of interest is the large number of hydroxyamino acids and potentialN-glycosylation sites found in the ppGaNTase-T5 stem region. The presence of O- or N-glycans in this region could inhibit the action of secretases, thereby keeping this isoform resident within the Golgi complex. Alternatively, a heavilyO-glycosylated stem may function as a extended stalk (1Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Abstract Full Text PDF PubMed Scopus (623) Google Scholar), inserting the catalytic region of this enzyme further into the Golgi lumen.The aa conservation of an individual ppGaNTase isoform is greater than 90% across mammalian species, whereas different isoforms within the same species show more variability. ppGaNTase-T5 demonstrates that highest degree of similarity to ppGaNTase-T1; they share 68% aa similarity within the conserved region and 57% aa similarity across the full-length protein (Tables I and II). The reduced similarity observed when comparing full-length proteins is most likely the result of diminished conservation within the N terminus and the long stem region of ppGaNTase-T5. Previously identified isoforms (ppGaNTase-T1, -T2, -T3, and -T4) have regions of highly conserved or invariant sequences within the lumenal domain. Although ppGaNTase-T5 contains most of these conserved regions, it demonstrates the lowest aa similarity to other isoforms and has a very poorly conserved CHGXGGNQ block (which has been used in the past to identify novel isoforms) (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This result points out the limitation of using a restricted set of conserved sequences to identify new isoforms; the clones recovered by such methods may be biased against the population of transferases lacking these blocks. When taken together with the results of a site-directed mutagenesis study demonstrating that the histidine residue in this sequence is non-essential (25Wragg S. Hagen F.K. Tabak L.A. Biochem. J. 1997; 328: 193-197Crossref PubMed Scopus (22) Google Scholar), it further suggests that this conserved block per se may not play a significant functional role in the transferase activity of these enzymes.Highly tissue specifically regulated isoforms of ppGaNTases may be uniquely programmed to act only on a limited subset of substrates from a particular cell or tissue type. This may account for the relatively modest activities seen for ppGaNTase-T5 and -T4 against our limited panel of peptides. Thus, it is plausible that ppGaNTase-T5 acts on a substrate unique to the RSLG. Future studies will focus on the identification of substrates whose expression patterns mimic those of a given isoform of ppGaNTase.The highly restricted expression and unique structural features of ppGaNTase-T5 further emphasize the diversity and complexity of this family of enzymes. It is unclear why so many distinct isoforms are needed in vivo. It remains possible that some isoforms serve more general, housekeeping functions while others act on specific molecules only. However, an extensive network of transferases may be in place with enough redundancy to ensure appropriate glycosylation. Through the continued identification of novel isoforms and gene ablation studies on the diverse repertoire of members available currently, we can begin to dissect the biological significance of this family of enzymes. O-Glycosidically linked oligosaccharides are responsible for the unique physical properties and extended structural conformation of molecules such as mucins and a number of membrane receptors (1Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Abstract Full Text PDF PubMed Scopus (623) Google Scholar). Additionally, O-glycans function as ligands for receptors mediating such diverse functions as sperm-egg adhesion (2Kinloch R.A. Sakai Y. Wasserman P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 263-267Crossref PubMed Scopus (95) Google Scholar) and leukocyte trafficking (3Dowbenko D. Andalibi A. Young P.E. Lusis A.J. Lasky L.A. J. Biol. Chem. 1993; 268: 4525-4529Abstract Full Text PDF PubMed Google Scholar). The initiation of O-linked glycosylation occurs through the action of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase,1 EC 2.4.1.41), which catalyzes the transfer of GalNAc from the nucleotide sugar UDP-GalNAc to the hydroxyl group of either serine or threonine. Four members of this enzyme family have been cloned and expressed thus far from mammals (ppGaNTase-T1 (4Hagen F.K. Van Wuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 5Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar), -T2 (6Sørensen T. White T. Wandall H.H. Kristense A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), -T3 (7Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 8Zara J. Hagen F.K. Ten Hagen K.G. VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar), and -T4 (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); one putative isoform has been ablated in mice with no obvious phenotype (10Hennett T. Hagen F.K. Tabak L.A. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12070-12074Crossref PubMed Scopus (225) Google Scholar). Five additional isoforms have been identified in Caenorhabditis elegans (11Hagen F.K. Nehrke K. J. Biol. Chem. 1998; 273: 8268-8277Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In vitro assays indicate that subtle differences in substrate preferences exist among the more widely expressed ppGaNTase-T1, -T2, and -T3, although there appears to be overlap (7Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 8Zara J. Hagen F.K. Ten Hagen K.G. VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar, 12Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). In contrast, ppGaNTase-T4 displays limited substrate preferences in vitro and a more restricted pattern of expression (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Thus, while it remains unclear why so many isoforms of ppGaNTase have evolved, the conservation of this family of enzymes from nematode to humans suggests that there is functional importance to the diversity of the enzymes expressed. In the present study we have cloned and expressed a new form of ppGaNTase, which we designate ppGaNTase-T5. Conceptual translation of the cDNA that encodes this isoform reveals the unusual structural feature of a long putative stem region. In addition, both the pattern of expression and in vitro substrate preferences are more restricted in nature. DISCUSSIONIn an effort to further define the repertoire of ppGaNTases, we have employed degenerate PCR to clone a novel isoform from a rat cDNA library. ppGaNTase-T5 has the attributes of a type II membrane protein, characteristic of previously identified glycosyltransferases (Fig. 6). The N-terminal, hydrophobic, and catalytic regions of ppGaNTase-T5 are similar in length to ppGaNTase-T1, -T2, -T3, and T4. However, ppGaNTase-T5 has an unusually large stem region (416 aa), which is ∼4–7 times longer than other known isoforms. Many type II membrane proteins are converted into soluble, circulating species after cleavage within the stem region by a group of enzymes referred to as "secretases" (24Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). Of interest is the large number of hydroxyamino acids and potentialN-glycosylation sites found in the ppGaNTase-T5 stem region. The presence of O- or N-glycans in this region could inhibit the action of secretases, thereby keeping this isoform resident within the Golgi complex. Alternatively, a heavilyO-glycosylated stem may function as a extended stalk (1Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Abstract Full Text PDF PubMed Scopus (623) Google Scholar), inserting the catalytic region of this enzyme further into the Golgi lumen.The aa conservation of an individual ppGaNTase isoform is greater than 90% across mammalian species, whereas different isoforms within the same species show more variability. ppGaNTase-T5 demonstrates that highest degree of similarity to ppGaNTase-T1; they share 68% aa similarity within the conserved region and 57% aa similarity across the full-length protein (Tables I and II). The reduced similarity observed when comparing full-length proteins is most likely the result of diminished conservation within the N terminus and the long stem region of ppGaNTase-T5. Previously identified isoforms (ppGaNTase-T1, -T2, -T3, and -T4) have regions of highly conserved or invariant sequences within the lumenal domain. Although ppGaNTase-T5 contains most of these conserved regions, it demonstrates the lowest aa similarity to other isoforms and has a very poorly conserved CHGXGGNQ block (which has been used in the past to identify novel isoforms) (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This result points out the limitation of using a restricted set of conserved sequences to identify new isoforms; the clones recovered by such methods may be biased against the population of transferases lacking these blocks. When taken together with the results of a site-directed mutagenesis study demonstrating that the histidine residue in this sequence is non-essential (25Wragg S. Hagen F.K. Tabak L.A. Biochem. J. 1997; 328: 193-197Crossref PubMed Scopus (22) Google Scholar), it further suggests that this conserved block per se may not play a significant functional role in the transferase activity of these enzymes.Highly tissue specifically regulated isoforms of ppGaNTases may be uniquely programmed to act only on a limited subset of substrates from a particular cell or tissue type. This may account for the relatively modest activities seen for ppGaNTase-T5 and -T4 against our limited panel of peptides. Thus, it is plausible that ppGaNTase-T5 acts on a substrate unique to the RSLG. Future studies will focus on the identification of substrates whose expression patterns mimic those of a given isoform of ppGaNTase.The highly restricted expression and unique structural features of ppGaNTase-T5 further emphasize the diversity and complexity of this family of enzymes. It is unclear why so many distinct isoforms are needed in vivo. It remains possible that some isoforms serve more general, housekeeping functions while others act on specific molecules only. However, an extensive network of transferases may be in place with enough redundancy to ensure appropriate glycosylation. Through the continued identification of novel isoforms and gene ablation studies on the diverse repertoire of members available currently, we can begin to dissect the biological significance of this family of enzymes. In an effort to further define the repertoire of ppGaNTases, we have employed degenerate PCR to clone a novel isoform from a rat cDNA library. ppGaNTase-T5 has the attributes of a type II membrane protein, characteristic of previously identified glycosyltransferases (Fig. 6). The N-terminal, hydrophobic, and catalytic regions of ppGaNTase-T5 are similar in length to ppGaNTase-T1, -T2, -T3, and T4. However, ppGaNTase-T5 has an unusually large stem region (416 aa), which is ∼4–7 times longer than other known isoforms. Many type II membrane proteins are converted into soluble, circulating species after cleavage within the stem region by a group of enzymes referred to as "secretases" (24Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). Of interest is the large number of hydroxyamino acids and potentialN-glycosylation sites found in the ppGaNTase-T5 stem region. The presence of O- or N-glycans in this region could inhibit the action of secretases, thereby keeping this isoform resident within the Golgi complex. Alternatively, a heavilyO-glycosylated stem may function as a extended stalk (1Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Abstract Full Text PDF PubMed Scopus (623) Google Scholar), inserting the catalytic region of this enzyme further into the Golgi lumen. The aa conservation of an individual ppGaNTase isoform is greater than 90% across mammalian species, whereas different isoforms within the same species show more variability. ppGaNTase-T5 demonstrates that highest degree of similarity to ppGaNTase-T1; they share 68% aa similarity within the conserved region and 57% aa similarity across the full-length protein (Tables I and II). The reduced similarity observed when comparing full-length proteins is most likely the result of diminished conservation within the N terminus and the long stem region of ppGaNTase-T5. Previously identified isoforms (ppGaNTase-T1, -T2, -T3, and -T4) have regions of highly conserved or invariant sequences within the lumenal domain. Although ppGaNTase-T5 contains most of these conserved regions, it demonstrates the lowest aa similarity to other isoforms and has a very poorly conserved CHGXGGNQ block (which has been used in the past to identify novel isoforms) (9Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This result points out the limitation of using a restricted set of conserved sequences to identify new isoforms; the clones recovered by such methods may be biased against the population of transferases lacking these blocks. When taken together with the results of a site-directed mutagenesis study demonstrating that the histidine residue in this sequence is non-essential (25Wragg S. Hagen F.K. Tabak L.A. Biochem. J. 1997; 328: 193-197Crossref PubMed Scopus (22) Google Scholar), it further suggests that this conserved block per se may not play a significant functional role in the transferase activity of these enzymes. Highly tissue specifically regulated isoforms of ppGaNTases may be uniquely programmed to act only on a limited subset of substrates from a particular cell or tissue type. This may account for the relatively modest activities seen for ppGaNTase-T5 and -T4 against our limited panel of peptides. Thus, it is plausible that ppGaNTase-T5 acts on a substrate unique to the RSLG. Future studies will focus on the identification of substrates whose expression patterns mimic those of a given isoform of ppGaNTase. The highly restricted expression and unique structural features of ppGaNTase-T5 further emphasize the diversity and complexity of this family of enzymes. It is unclear why so many distinct isoforms are needed in vivo. It remains possible that some isoforms serve more general, housekeeping functions while others act on specific molecules only. However, an extensive network of transferases may be in place with enough redundancy to ensure appropriate glycosylation. Through the continued identification of novel isoforms and gene ablation studies on the diverse repertoire of members available currently, we can begin to dissect the biological significance of this family of enzymes. We thank Meng Qian for her help in preparing this manuscript.