Zhang L, Esko J D
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham 35294, USA.
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.
硫酸乙酰肝素的生物合成起始于将α-D-葡萄糖胺从UDP-葡萄糖胺转移至连接四糖的D-葡萄糖醛酸部分，即葡萄糖醛酸β1-3半乳糖β1-3半乳糖β1-4木糖β1-核心蛋白。基于对链聚合缺陷的动物细胞突变体的遗传和酶学研究，催化此反应的酶不同于参与链聚合的α-葡萄糖胺转移酶（弗里茨，T.A.，加布，M.M.，魏，G.，和埃斯科，J.D.（1994年）《生物化学杂志》269卷，28809 - 28814页）。在本报告中，我们表明该突变体还积累了一种含α-葡萄糖胺的五糖中间体。一种融合蛋白由蛋白A的IgG结合结构域和蛋白聚糖β-聚糖的一个片段制成。该片段包含一个仅引发硫酸软骨素的糖胺聚糖连接位点和另一个引发硫酸乙酰肝素及硫酸软骨素的连接位点（张，L.，和埃斯科，J.D.（1994年）《生物化学杂志》264卷，19295 - 19299页）。该嵌合体在突变体中的表达导致积累了一种用[6-³H]葡糖胺标记的寡糖。该寡糖与源自硫酸软骨素的五糖标准品共迁移，但酸水解产生98%的[³H]葡糖胺。肝素酶III消化产生[³H]葡萄糖胺，表明葡萄糖胺残基以α-连接至非还原末端。对用[6-³H]半乳糖标记的物质进行酶处理产生四糖，Δ葡萄糖醛酸-[³H]半乳糖-[³H]半乳糖-木糖醇。这些发现表明五糖具有结构，葡萄糖胺α1-4葡萄糖醛酸β1-3半乳糖β1-3半乳糖β1-4木糖。它在聚合α-葡萄糖胺转移酶缺陷的中国仓鼠卵巢细胞突变体中的积累提供了体内证据，证明两种α-葡萄糖胺转移酶催化硫酸乙酰肝素的形成。
