Glycobiology Advance Access published online on January 10, 2007
Glycobiology, doi:10.1093/glycob/cwl082
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The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate binding specificity for GalNAc: Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.
1 Department of Medical Biochemistry and Genetics and the Department of Oral Diagnostics, Dental School, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
3 CIQUIBIC-CONICET / Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Cordoba, 5000 Cordoba, Argentina
4 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
5 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198
Address correspondence to: Henrik Clausen, Department of Medical Biochemistry and Genetics, the Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Tel.: +45 35327797, Fax: +45 35367980, E-mail: hc{at}imbg.ku.dk
Received on August 9, 2006; revised on November 21, 2006; accepted on December 19, 2006
Initiation of mucin-type O-glycosylation is controlled by a large family of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here we report the direct carbohydrate binding of two GalNAc-transferases, GalNAc-T4 and GalNAc-T2, and their isolated lectins domains, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNActransferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.
Key words: GalNAc / transferases / lectins / glycans / mucins
2 Present address: Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
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