Molecular basis for protein-specific transfer of N-acetylgalactosamine to N-linked glycans by the glycosyltransferases β1,4-N-acetylgalactosaminyl transferase 3 (β4GalNAc-T3) and β4GalNAc-T4.
Two closely related β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, are thought to account for the protein-specific addition of β1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein hormone luteinizing hormone and carbonic anhydrase-6 (CA6). We have utilized soluble, secreted forms of β4GalNAc-T3 and β4GalNAc-T4 to define the basis for protein-specific GalNAc transfer in vitro to chimeric substrates consisting of Gaussia luciferase followed by a glycoprotein substrate. Transfer of GalNAc by β4GalNAc-T3 and β4GalNAc-T4 to terminal GlcNAc is divalent cation-dependent. Transfer of GalNAc to glycoprotein acceptors that contain a peptide recognition determinant is maximal between 0.5 and 1.0 mM MnCl(2); however, transfer is increasingly inhibited by concentrations of MnCl(2) above 1 mM and by anion concentrations above 15 mM. In contrast, transfer of GalNAc to the simple sugar acceptor N-acetylglucosamine-β-p-nitrophenol (GlcNAcβ-pNP) is not inhibited by concentrations of MnCl(2) or anions that would inhibit transfer to glycoprotein acceptors by >90%. This finding indicates that interaction with the peptide recognition determinant in the substrate is sensitive to the anion concentration. β4GalNAc-T3 and β4GalNAc-T4 have similar but distinct specificities, resulting in a 42-fold difference in the IC(50) for transfer of GalNAc to chimeric glycoprotein substrates by agalacto human chorionic gonadotropin, comprising 29 nM for β4GalNAc-T3 and 1.2 μM for β4GalNAc-T4. Our in vitro analysis indicates that enzymatic recognition of the peptide determinant and the oligosaccharide acceptor are independent events.
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Dorothy
D
Fiete
Mary
M
Beranek
Jacques U
JU
Baenziger
D000117:Acetylglucosamine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007986:Luteinizing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D006063:Chorionic Gonadotropin; D011993:Recombinant Fusion Proteins; D007986:Luteinizing Hormone; D017350:N-Acetylgalactosaminyltransferases; D002256:Carbonic Anhydrases; C477051:carbonic anhydrase VI; D000117:Acetylglucosamine