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.
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November 1997
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Research Article|
November 15 1997
Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1
Stephanie WRAGG;
1Departments of Dental Research and Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Box 611, Rochester, NY 14642, U.S.A.
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K. Fred HAGEN;
K. Fred HAGEN
1
1Departments of Dental Research and Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Box 611, Rochester, NY 14642, U.S.A.
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A. Lawrence TABAK
A. Lawrence TABAK
2
1Departments of Dental Research and Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Box 611, Rochester, NY 14642, U.S.A.
2To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
May 19 1997
Revision Received:
July 21 1997
Accepted:
July 22 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 328 (1): 193–197.
Article history
Received:
May 19 1997
Revision Received:
July 21 1997
Accepted:
July 22 1997
Citation
Stephanie WRAGG, K. Fred HAGEN, A. Lawrence TABAK; Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1. Biochem J 15 November 1997; 328 (1): 193–197. doi: https://doi.org/10.1042/bj3280193
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