Accumulation of a Pentasaccharide Terminating in -N-Acetylglucosamine in an Animal Cell Mutant Defective in Heparan Sulfate Biosynthesis (*)

Abstract

Heparan sulfate biosynthesis initiates by the transfer of α-D-GlcNAc from UDP-GlcNAc to the D-GlcA moiety of the linkage tetrasaccharide, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-core protein. The enzyme catalyzing this reaction differs from the α-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 α-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 α-linked to the nonreducing terminus. Enzymatic treatment of [6-³H]Gal-labeled material yielded the tetrasaccharide, ΔGlcA-[³H]Gal-[³H]Gal-xylitol. These findings suggest that pentasaccharide had the structure, GlcNAcα1-4GlcAβ1-3Galβ1-3Galβ1-4Xyl. Its accumulation in a Chinese hamster ovary cell mutant defective in the polymerizing α-GlcNAc transferase provides in vivo evidence that two α-GlcNAc transferases catalyze the formation of heparan sulfate.