Substrate Specificities of Three Members of the Human UDP-N-Acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase Family, GalNAc-T1, -T2, and -T3

doi:10.1074/jbc.272.38.23503

Substrate Specificities of Three Members of the Human UDP-N-Acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase Family, GalNAc-T1, -T2, and -T3*

From the ^‡School of Dentistry, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark, the ^¶Department of Molecular Biology, University of Odense, Campusvej 47, Odense, Denmark, the ^‖Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, and the ^**Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

Abstract

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptideN-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.

Footnotes

↵* This work was supported by The Danish Cancer Society, the Ingeborg Roikjer Foundation, the Velux Foundation, the Danish Medical Research Council, the Danish Natural Science Research Council, the Lundbeck Foundation, the Novo Nordisk Foundation, National Institutes of Health Grant 1 RO1 CA66234, and funds from the EU-Biotech 4th Framework.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Contributed equally to the results of this work.
↵‡ To whom the correspondence should be addressed: Henrik Clausen, School of Dentistry, Nørre Alle 20, DK-2200 Copenhagen N, Denmark. Tel.: 45-35326835; Fax: 45-35326505; E-mail:henrik.clausen{at}odont.ku.dk.
↵1 The abbreviations used are: GalNAc-transferase, UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase; MALDI-TOF, matrix-assisted laser desorption mass spectrometry; GalNAc-T1, -T2, and -T3, GalNAc-transferases cloned and expressed by Homa et al. (1), White et al. (2), and Bennett et al. (3), respectively; HPLC, high performance liquid chromatography; CE, capillary electrophoresis; PAGE, polyacrylamide gel electrophoresis; Man transferase, Dol-P-Man:polypeptide mannosyltransferases; HIV, human immunodeficiency virus; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
↵2 E. P. Bennett, D. O. Weghuis, G. Merkx, A. G. van Kessel, H. Eiberg, and H. Clausen, submitted for publication.
↵3 T. Nilsson, personal communication.
↵4 E. P. Bennett and H. Clausen, unpublished data.
↵5 N. Kaufman and M. A. Hollingsworth, unpublished data.
↵6 K. Nehrke, F. Hagen, and L. A. Tabak, personal communication.
↵7 E. P. Bennett, H. Hassan, H. Wandall, and H. Clausen, manuscript in preparation.
- Received February 22, 1997.
- Revision received June 13, 1997.