Accumulation of a pentasaccharide terminating in alpha-N-acetylglucosamine in an animal cell mutant defective in heparan sulfate biosynthesis

J Biol Chem. 1995 May 26;270(21):12557-62. doi: 10.1074/jbc.270.21.12557.


Heparan sulfate biosynthesis initiates by the transfer of alpha-D-GlcNAc from UDP-GlcNAc to the D-GlcA moiety of the linkage tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-core protein. The enzyme catalyzing this reaction differs from the alpha-GlcNAc transferase involved in chain polymerization based on genetic and enzymatic studies of an animal cell mutant defective in chain polymerization (Fritz, T. A., Gabb, M. M., Wei, G., and Esko, J. D. (1994) J. Biol. Chem. 269, 28809-28814). In this report we show that this mutant also accumulates a pentasaccharide intermediate containing alpha-GlcNAc. A fusion protein was made from the IgG-binding domain of protein A and a segment of the proteoglycan, betaglycan. This segment contained one glycosaminoglycan attachment site that primes only chondroitin sulfate and another that primes both heparan sulfate and chondroitin sulfate (Zhang, L., and Esko, J. D. (1994) J. Biol. Chem. 264, 19295-19299). Expression of the chimera in the mutant resulted in the accumulation of an oligosaccharide that labeled with [6-3H]GlcN. The oligosaccharide comigrated with a pentasaccharide standard derived from chondroitin sulfate, but acid hydrolysis gave 98% [3H]GlcN. Heparin lyase III digestion yielded [3H]GlcNAc, suggesting that the GlcNAc residue was alpha-linked to the nonreducing terminus. Enzymatic treatment of [6-3H]Gal-labeled material yielded the tetrasaccharide, delta GlcA-[3H]Gal-[3H]Gal-xylitol. These findings suggest that pentasaccharide had the structure, GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl. Its accumulation in a Chinese hamster ovary cell mutant defective in the polymerizing alpha-GlcNAc transferase provides in vivo evidence that two alpha-GlcNAc transferases catalyze the formation of heparan sulfate.

MeSH terms

  • Acetylglucosamine / analysis*
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Carbohydrate Sequence
  • Cricetinae
  • Glycosaminoglycans / metabolism
  • Heparin Lyase
  • Heparitin Sulfate / biosynthesis*
  • Molecular Sequence Data
  • Mutation
  • Oligosaccharides / biosynthesis*
  • Oligosaccharides / chemistry
  • Polysaccharide-Lyases / metabolism
  • Proteoglycans / genetics
  • Proteoglycans / metabolism
  • Receptors, Transforming Growth Factor beta / genetics
  • Receptors, Transforming Growth Factor beta / metabolism
  • Recombinant Fusion Proteins / metabolism


  • Glycosaminoglycans
  • Oligosaccharides
  • Proteoglycans
  • Receptors, Transforming Growth Factor beta
  • Recombinant Fusion Proteins
  • betaglycan
  • Heparitin Sulfate
  • Polysaccharide-Lyases
  • Heparin Lyase
  • Acetylglucosamine