PMID:9359852

Citation	Wragg, S, Hagen, FK and Tabak, LA (1997) Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1. Biochem. J. 328 ( Pt 1):193-7
Abstract	UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) catalyse the initial step of mucin-type O-glycosylation. The activity of bovine ppGaNTase-T1 isoenzyme was inhibited by diethyl pyrocarbonate (DEPC) modification. Activity was partially restored by hydroxylamine treatment, indicating that one of the reactive residues was a histidine. The transferase was protected against DEPC inactivation when UDP-GalNAc and EPO-G, a peptide pseudo-substrate PPDAAGAAPLR, were simultaneously present, while presence of EPO-G alone did not alter DEPC inactivation. However, inclusion of UDP-GalNAc alone potentiated DEPC-inhibition of the enzyme, suggesting that UDP-GalNAc binding changes the accessibility or reactivity of an essential histidine residue. Deletion of the first 56 amino acids (including one hisitidine residue) yielded a fully active secreted form of the bovine ppGaNTase-T1 enzyme. Each of the 14 remaining histidines in the enzyme were mutated to alanine, and the recombinant mutants were recovered from COS7 cells. The mutation of histidine residues His211-->Ala and His344-->Ala resulted in recombinant proteins with no detectable enzymic activity. A significant decrease in the initial rate of GalNAc transfer to the substrate was observed with mutants His125-->Ala and His341-->Ala (1% and 6% of wild-type activity respectively). Mutation of the remaining ten histidine residues yielded mutants that were indistinguishable from the wild-type enzyme. Mutagenesis and SDS/PAGE analysis of all N-glycosylation sequons revealed that positions N-95 and N-552 are occupied by N-linked sugars in COS7 cells. Ablation of either site did not perturb enzyme biosynthesis or enzyme activity.
Links	PubMed PMC1218905
Keywords	Animals; Binding Sites/genetics; Cattle; Diethyl Pyrocarbonate/pharmacology; Enzyme Activation/drug effects; Glycosylation; Histidine/metabolism; Isoenzymes/drug effects; Isoenzymes/genetics; Isoenzymes/metabolism; Kinetics; N-Acetylgalactosaminyltransferases/drug effects; N-Acetylgalactosaminyltransferases/genetics; N-Acetylgalactosaminyltransferases/metabolism; Recombinant Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BOVIN:GALT1		GO:0004653: polypeptide N-acetylgalactosaminyltransferase activity	ECO:0000314:		F		Table 2 is a list of Km values determined from an kinetics experiment, the purified wild-type is the first entry in table, and all mutant proteins are normalized to wild-type activity. The glycoproteins formed were separated later with anion-exchanged chromatography.	complete CACAO 3615
BOVIN:GALT1	enables	GO:0004653: polypeptide N-acetylgalactosaminyltransferase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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