title: 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.
abstract: 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-transferase lectin domains, GalNAc-T4 and GalNAc-T2, 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 GalNAc-transferase 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.
authors.affiliation: Department of Medical Biochemistry and Genetics, Dental School, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Hans H
authors.initials: HH
authors.lastname: Wandall
authors.forename: Fernando
authors.initials: F
authors.lastname: Irazoqui
authors.forename: Mads Agervig
authors.initials: MA
authors.lastname: Tarp
authors.forename: Eric P
authors.initials: EP
authors.lastname: Bennett
authors.forename: Ulla
authors.initials: U
authors.lastname: Mandel
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Irimura
authors.forename: Ganesh
authors.initials: G
authors.lastname: Suryanarayanan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hollingsworth
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D017931:DNA Primers; D006020:Glycopeptides / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D006020:Glycopeptides; D037102:Lectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017350:N-Acetylgalactosaminyltransferases; D000116:Acetylgalactosamine