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 secreted O-linked glycoproteins.
Highlights
We have used degenerate primers derived from conserved sequences within a region spanning 420-aa residues to amplify putative ppGaNTases from RNA samples
We continued the analysis of the PCR products from spleen samples because in prior work we had demonstrated that the spleen expresses a low level of ppGaNTase-T1 transcript but expresses high levels of ppGaNTase enzyme activity (20)
To help define the repertoire of ppGaNTases in a tissue, we have devised a PCR-based strategy that employs a series of oligonucleotides derived from conserved sequences found within a 420-aa region of the ppGaNTase family of enzymes
Summary
We have used degenerate primers derived from conserved sequences within a region spanning 420-aa residues to amplify putative ppGaNTases from RNA samples. A 117-bp segment of the ppGaNTase-T4 cDNA region (nucleotide position [562– 679], Fig. 2) was labeled by asymmetric PCR (18) using the antisense oligonucleotide d(CCAGGAAAGTGAGGACA) and used as a probe for the ppGaNTase-T4 transcript.
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