Substrate specificities of three members of the human UDP-N-acetyl-α- D-galactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc- T1, -T2, and -T3

Hans H. Wandall, Helle Hassan, Ekaterina Mirgorodskaya, Anne K. Kristensen, Peter Roepstorff, Eric P. Bennett, Peter A. Nielsen, Michael A Hollingsworth, Joy Burchell, Joyce Taylor-Papadimitriou, Henrik Clausen

Research output: Contribution to journalArticle

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Abstract

Mucin-type O-glycosylation is initiated by UDP-N- acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc- transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, - T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP- GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc- transferases.

Original languageEnglish (US)
Pages (from-to)23503-23514
Number of pages12
JournalJournal of Biological Chemistry
Volume272
Issue number38
DOIs
StatePublished - Sep 19 1997

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Acetylgalactosamine
Uridine Diphosphate
Substrate Specificity
Substrates
Glycosylation
Peptides
Tandem Repeat Sequences
Enzymes
polypeptide N-acetylgalactosaminyltransferase
Uridine Diphosphate N-Acetylgalactosamine
Capillary electrophoresis
Insect Proteins
Enzyme kinetics
Mucins
Capillary Electrophoresis
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Substrate specificities of three members of the human UDP-N-acetyl-α- D-galactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc- T1, -T2, and -T3. / Wandall, Hans H.; Hassan, Helle; Mirgorodskaya, Ekaterina; Kristensen, Anne K.; Roepstorff, Peter; Bennett, Eric P.; Nielsen, Peter A.; Hollingsworth, Michael A; Burchell, Joy; Taylor-Papadimitriou, Joyce; Clausen, Henrik.

In: Journal of Biological Chemistry, Vol. 272, No. 38, 19.09.1997, p. 23503-23514.

Research output: Contribution to journalArticle

Wandall, HH, Hassan, H, Mirgorodskaya, E, Kristensen, AK, Roepstorff, P, Bennett, EP, Nielsen, PA, Hollingsworth, MA, Burchell, J, Taylor-Papadimitriou, J & Clausen, H 1997, 'Substrate specificities of three members of the human UDP-N-acetyl-α- D-galactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc- T1, -T2, and -T3', Journal of Biological Chemistry, vol. 272, no. 38, pp. 23503-23514. https://doi.org/10.1074/jbc.272.38.23503
Wandall, Hans H. ; Hassan, Helle ; Mirgorodskaya, Ekaterina ; Kristensen, Anne K. ; Roepstorff, Peter ; Bennett, Eric P. ; Nielsen, Peter A. ; Hollingsworth, Michael A ; Burchell, Joy ; Taylor-Papadimitriou, Joyce ; Clausen, Henrik. / Substrate specificities of three members of the human UDP-N-acetyl-α- D-galactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc- T1, -T2, and -T3. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 38. pp. 23503-23514.
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abstract = "Mucin-type O-glycosylation is initiated by UDP-N- acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc- transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, - T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP- GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc- transferases.",
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AU - Wandall, Hans H.

AU - Hassan, Helle

AU - Mirgorodskaya, Ekaterina

AU - Kristensen, Anne K.

AU - Roepstorff, Peter

AU - Bennett, Eric P.

AU - Nielsen, Peter A.

AU - Hollingsworth, Michael A

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AU - Taylor-Papadimitriou, Joyce

AU - Clausen, Henrik

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