We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests that O-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.O-Linked glycans are involved in a number of biological functions including leukocyte trafficking (1) and sperm-egg adhesion (2). In addition, clusters of O-linked oligosaccharides impart a "stalk-like" conformation that is common among several membrane receptors (3). In contrast to N-linked glycosylation, O-linked glycans are synthesized stepwise. Thus, the acquisition of GalNAc represents the first step in mammalian (mucin-type) O-glycosylation. A family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase enzymes (ppGaNTase, 1 EC 2.4.1.41) is responsible for this initial enzymatic step. Five family members (ppGaNTase-T1 (4, 5), -T2 (6), -T3 (7, 8), -T4 (9), and -T5 (10)) have been identified in mammals thus far and have been shown to have unique expression patterns as well as substrate specificities. However, little is known regarding their respective activities on native substrates as well as potential inter-relationships with one another.In the present study, we have cloned a novel member of this enzyme family termed ppGaNTase-T6. When recombinant enzyme was expressed as a secreted product from COS7 cells, no ppGaNTase activity was detected in vitro against a panel of unmodified peptides, including the peptide GTTPSPVPTTSTT-SAP, which is derived from the human MUC5AC gene sequence (11). However, when this MUC5AC peptide was first glycosylated with ppGaNTase-T1 to yield mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP (but also mono-and tri-substituted species), the ppGaNTase-T6 isoform was active toward the glycopeptidic preparation. This suggests that the addition of the initial O-linked sugar may occur in a hierarchical manner wit...

We have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situ hybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development.

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