ACCUMULATION OF A PENTASACCHARIDE TERMINATING IN ALPHA-N-ACETYLGLUCOSALMINE IN AN ANIMAL-CELL MUTANT DEFECTIVE IN HEPARAN-SULFATE BIOSYNTHESIS

Heparan sulfate biosynthesis initiates by the transfer of alpha-D-GlcN Ac 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 en zyme catalyzing this reaction differs from the alpha-GlcNAc transferas e involved in chain polymerization based on genetic and enzymatic stud ies 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. 26 9, 28809-28814), In this report we show that this mutant also accumula tes a pentasaccharide intermediate containing alpha-GlcNAc, A fusion p rotein was made from the IgG-binding domain of protein A and a segment of the proteoglycan, betaglycan. This segment contained one glycosami noglycan attachment site that primes only chondroitin sulfate and anot her 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 oligo saccharide that labeled with [6-H-3]GlcN. The oligosaccharide comigrat ed with a pentasaccharide standard derived from chondroitin sulfate, b ut acid hydrolysis gave 98% [H-3]GlcN, Heparin lyase III digestion yie lded [H-3]GlcNAc, suggesting that the GlcNAc residue was alpha-linked to the nonreducing terminus, Enzymatic treatment of [6-H-3]Gal-labeled material yielded the tetrasaccharide, Delta GlcA-[H-3]Gal-[H-3]Gal-xy litol. These findings suggest that pentasaccharide had the structure, GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl. Its accumula tion in a Chinese hamster ovary cell mutant defective in the polymeriz ing alpha-GlcNAc transferase provides in vivo evidence that two alpha- GlcNAc transferases catalyze the formation of heparan sulfate.