Abstract
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.
Highlights
We predicted that a Chinese hamster ovary mutant defective in heparan sulfate polymerization (Lidholt et al, 1992) might accumulate a pentasaccharide intermediate consisting of a-GIcNAc attached to the linkage tetrasaccharide
Intermediate Purification-We have described a mutant designated pgsD-677 that lacks the a-GlcNAc transferase involved in the polymerization of heparan sulfate chains (Lidholt et al, 1992)
This finding suggested that cells use two a-GlcNAc transferases to assemble heparan sulfate, one to initiate the chain (a-GlcNAc transferase I) and another to polymerize the chain (a-GlcNAc transferase II) (Fritz et al, 1994)
Summary
Vol 270, No 21, Issue of May 26, pp. 12557-12562, 1995 Printed in U.S.A. Accumulation of a Pentasaccharide Terminating in a-N-Acetylglucosamine in an Animal Cell Mutant Defective in Heparan Sulfate Biosynthesis*. Enzymatic treatment of [6-3HlGal-labeled material yielded the tetrasaccharide, .:1GlcA-[3HlGal-[3HlGal-xylitol These findings suggest that pentasaccharide had the structure, GlcNAcal-4GlcAIJI-3GallJl-3GallJl-4Xyl. Its accumulation in a Chinese hamster ovary cell mutant defective in the polymerizing a-GlcNAc transferase provides in vivo evidence that two a-GlcNAc transferases catalyze the formation of heparan sulfate. Fritz et al (1994) reported that the enzyme transferring a-GlcNAc to the linkage tetrasaccharide differs from the one involved in forming the repeating disaccharide units of heparan sulfate (GIcAI31-4GlcNAca1-4) Based on this finding, we predicted that a Chinese hamster ovary mutant defective in heparan sulfate polymerization (Lidholt et al, 1992) might accumulate a pentasaccharide intermediate consisting of a-GIcNAc attached to the linkage tetrasaccharide. Its accumulation in the mutant provides strong evidence that different enzymes catalyze the initiation and polymerization of heparan sulfate chains
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
