Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity

Glycobiology. 2006 Jul;16(7):584-93. doi: 10.1093/glycob/cwj090. Epub 2006 Feb 16.

Abstract

Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antigens, Neoplasm / analysis
  • Antigens, Neoplasm / biosynthesis*
  • Antigens, Neoplasm / immunology
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Fucosyltransferases / genetics
  • Fucosyltransferases / metabolism
  • Galactoside 2-alpha-L-fucosyltransferase
  • Galactosyltransferases / genetics
  • Galactosyltransferases / metabolism
  • Glycoproteins / chemistry
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism*
  • Humans
  • Lewis Blood Group Antigens
  • Mice
  • Oligosaccharides / analysis
  • Oligosaccharides / biosynthesis*
  • Oligosaccharides / immunology
  • Polysaccharides / metabolism
  • Serum Albumin / chemistry
  • Substrate Specificity

Substances

  • 3-aminopropyl fucopyranosyl-1-2-galactopyranosyl-1-3-(fucopyranosyl-1-4)-2-acetamido-2-deoxyglucopyranosyl-1-3-galactopyranosyl-1-4-glucopyranoside
  • Antibodies, Monoclonal
  • Antigens, Neoplasm
  • DU-PAN-2 antigen, human
  • Glycoproteins
  • Lewis Blood Group Antigens
  • Lewis a oligosaccharide
  • Oligosaccharides
  • Polysaccharides
  • Serum Albumin
  • Glycosyltransferases
  • B3GALT5 protein, human
  • Fucosyltransferases
  • Galactosyltransferases