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.

We determined the free-amino acid content of stimulated parotid (ductal) saliva from two groups of adult subjects whose caries experiences were markedly different. The levels of free arginine and free lysine in the parotid saliva of caries-free adults were significantly higher than those found in the parotid saliva of individuals with a history of dental decay. There was no correlation, however, between the levels of these amino acids and the DMFS score within the caries-susceptible groups. Microbial catabolism of dibasic amino acids contributes to the neutralization of plaque acids and may partially account for the higher resting plaque pH observed in caries-free subjects. Alternatively, the elevations observed in free levels of arginine and lysine may reflect a systemic alteration in amino acid metabolism which is common to the caries-free group of subjects.

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...

The low-molecular-weight components of human saliva remain poorly characterized. Therefore, low-molecular-weight peptides (Mr < 3000) have been purified from human parotid saliva and characterized with respect to their amino acid sequence. From the sequences obtained, it is likely that these peptides are derived from proteolysis of the hydroxyapatite-interactive human salivary proteins, histatins, proline-rich proteins, and statherins. Since human parotid saliva is an amicrobial fluid, much of the low-molecular-weight peptide fraction of this secretion appears to be derived from the proteolytic processing of the larger proteins. Because of their small size, these peptides are likely to be in exchange with dental plaque fluid and may therefore help modulate events such as demineralization/remineralization, microbial attachment, and dental plaque metabolism at the tooth-saliva interface.

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