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

J Biol Chem. 2012 Aug 17;287(34):29194-203. doi: 10.1074/jbc.M112.371567. Epub 2012 Jun 21.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylglucosamine / chemistry*
  • Acetylglucosamine / genetics
  • Acetylglucosamine / metabolism
  • Animals
  • Carbonic Anhydrases / chemistry*
  • Carbonic Anhydrases / genetics
  • Carbonic Anhydrases / metabolism
  • Catalysis
  • Chorionic Gonadotropin / chemistry*
  • Chorionic Gonadotropin / genetics
  • Chorionic Gonadotropin / metabolism
  • HEK293 Cells
  • Humans
  • Luteinizing Hormone / chemistry*
  • Luteinizing Hormone / genetics
  • Luteinizing Hormone / metabolism
  • Mice
  • N-Acetylgalactosaminyltransferases / chemistry*
  • N-Acetylgalactosaminyltransferases / genetics
  • N-Acetylgalactosaminyltransferases / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Chorionic Gonadotropin
  • Recombinant Fusion Proteins
  • Luteinizing Hormone
  • N-Acetylgalactosaminyltransferases
  • Carbonic Anhydrases
  • carbonic anhydrase VI
  • Acetylglucosamine