Initiation of O-glycan synthesis in IgA1 hinge region is determined by a single enzyme, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2

J Biol Chem. 2003 Feb 21;278(8):5613-21. doi: 10.1074/jbc.M211097200. Epub 2002 Nov 15.

Abstract

The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • B-Lymphocytes / enzymology
  • B-Lymphocytes / immunology
  • Base Sequence
  • Cell Line
  • Humans
  • Immunoglobulin A / biosynthesis*
  • Immunoglobulin A / chemistry*
  • Kinetics
  • Lymphocytes / enzymology
  • Lymphocytes / immunology*
  • Molecular Sequence Data
  • Multiple Myeloma / immunology
  • N-Acetylgalactosaminyltransferases / metabolism*
  • Polypeptide N-acetylgalactosaminyltransferase
  • Polysaccharides / biosynthesis*
  • Reference Values
  • Reverse Transcriptase Polymerase Chain Reaction
  • Substrate Specificity
  • Transcription, Genetic
  • Tumor Cells, Cultured
  • Uridine Diphosphate N-Acetylgalactosamine / metabolism*

Substances

  • Immunoglobulin A
  • Polysaccharides
  • Uridine Diphosphate N-Acetylgalactosamine
  • N-Acetylgalactosaminyltransferases