ABSTRACT
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.
Subject(s)
N-Acetylgalactosaminyltransferases/metabolism , Peptides/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Consensus Sequence , Conserved Sequence , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Molecular Sequence Data , N-Acetylgalactosaminyltransferases/chemistry , N-Acetylgalactosaminyltransferases/genetics , Organ Specificity , Peptides/chemistry , Rats , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Polypeptide N-acetylgalactosaminyltransferaseABSTRACT
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.
Subject(s)
DNA, Complementary/metabolism , Isoenzymes/genetics , N-Acetylgalactosaminyltransferases/genetics , Adult , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , COS Cells , Caenorhabditis elegans , Cloning, Molecular , Conserved Sequence , Humans , Mice , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNA , Polypeptide N-acetylgalactosaminyltransferaseABSTRACT
Overlapping cDNA clones which encode the protein core of a rat submandibular gland mucin-glycoprotein have been isolated and characterized. Sequence analysis revealed a translated region of 966 nucleotides encoding a protein of 322 amino acid residues. The translational start site begins with a putative signal sequence comprising the initial 22 N-terminal residues. The predicted secreted portion of the apomucin revealed three distinct domains: an N-terminal domain which is enriched in glutamine (14%), proline (13%), and tyrosine (10%); a central region which consisted of eleven, 39-base pair tandem repeats with the consensus sequence PTTDSTTPAPTTK; and a C-terminal domain which is enriched in threonine and serine residues (47%) which are not part of a repeat motif. The expression of apomucin transcript appears restricted to the rat submandibular and sublingual glands. Southern blot analysis of rat genomic DNAs suggested a low copy number (1, 2) for this apomucin gene and a limited polymorphism in the number of tandem repeats. Collectively, our sequence and expression data indicate that the cloned rat submandibular gland apomucin is distinct from any of the other salivary (bovine, porcine, or human) or rat apomucins reported thus far.
Subject(s)
Gastric Mucins , Peptide Biosynthesis , Submandibular Gland/metabolism , ABO Blood-Group System , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Cloning, Molecular , Consensus Sequence , DNA/analysis , DNA/metabolism , DNA, Complementary/isolation & purification , DNA, Complementary/metabolism , Gene Library , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptides/genetics , Peptides/isolation & purification , Polymerase Chain Reaction , Protein Conformation , Rats , Restriction MappingABSTRACT
There is a growing population who suffer from hyposalivation. These patients frequently sip liquids to alleviate their discomfort. Milk appears to have many of the physical properties of a good saliva substitute. Desalivated rats given 2% milk or lactose-reduced milk remained essentially caries-free. In contrast, animals given sucrose or lactose to drink developed caries. In addition, animals given sucrose or lactose harbored significantly higher populations of Streptococcus sobrinus than did other groups. Results showed that milk and lactose-reduced milk can be used safely by hyposalivatory patients as a saliva substitute.
