cDNA Cloning and Expression of a Novel UDP-N-acetyl-d-galactosamine:PolypeptideN-Acetylgalactosaminyltransferase*
- Fred K. Hagen,
- Kelly G. Ten Hagen,
- Thomas M. Beres,
- Marlene M. Balys,
- Brian C. VanWuyckhuyse and
- Lawrence A. Tabak‡
- From the Departments of Dental Research and Biochemistry and Biophysics, the School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642
Abstract
The cDNA for a fourth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, termed ppGaNTase-T4, has been cloned from a murine spleen cDNA library and expressed transiently in COS7 cells as a secreted functional enzyme. Degenerate primers, based upon regions that are conserved among the known mammalian members of the enzyme family (ppGaNTase-T1, -T2, and -T3) and three Caenorhabditis elegans homologues (ppGaNTase-TA, -TB, and -TC), were used in polymerase chain reactions to identify and clone this new isoform. Substrate preferences for recombinant murine ppGaNTase-T1 and ppGaNTase-T4 isozymes were readily distinguished. ppGaNTase-T1 glycosylated a broader range of synthetic peptide substrates; in contrast, the ppGaNTase-T4 preferentially glycosylated a single substrate among the panel of 11 peptides tested. Using Northern blot analysis, a ppGaNTase-T4 message of 5.5 kilobases was detectable in murine embryonic tissues, as well as the adult sublingual gland, stomach, colon, small intestine, lung, cervix, and uterus with lower levels detected in kidney, liver, heart, brain, spleen, and ovary. Thus, the pattern of expression for ppGaNTase-T4 is more restricted than for the three previously reported isoforms of the enzyme. The variation in expression patterns and substrate specificities of the ppGaNTase enzyme family suggests that differential expression of these isoenzymes may be responsible for the cell-specific repertoire of mucin-type oligosaccharides on cell-surface and secretedO-linked glycoproteins.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DE-08108 (to L. A. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U73820 (mouse ppGaNTase-T1) and U73819 (mouse ppGaNTase-T4).
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↵‡ To whom correspondence should be addressed: Depts. of Dental Research and Biochemistry and Biophysics, the School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave., Box 611, Rochester, NY 14642. Tel.: 716-275-0770; Fax: 716-473-2679; E-mail: LTAB{at}medinfo.rochester.edu.
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↵1 The abbreviations used are: ppGaNTase, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase; PCR, polymerase chain reactions; aa, amino acid; bp, base pair(s); kb, kilobase(s).
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↵2 F. K. Hagen, unpublished observations.
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↵3 K. G. Ten Hagen and L. A. Tabak, unpublished observations.
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- Received November 4, 1996.
- Revision received February 10, 1997.
