Kitagawa H, Tsutsumi K, Ujikawa M, Goto F, Tamura J, Neumann K W, Ogawa T, Sugahara K
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
Glycobiology. 1997 Jun;7(4):531-7. doi: 10.1093/glycob/7.4.531.
The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.
硫酸软骨素（CS）生物合成过程中硫酸化与聚合之间的关系一直未得到充分理解。在本研究中，我们使用结构明确的受体底物，研究了负责链延长的牛血清UDP - GalNAc：CSβ - GalNAc转移酶的特异性。它们由化学合成的四糖和六糖 - 丝氨酸以及各种在非还原末端带有GlcA残基的规则寡糖组成，这些寡糖是使用睾丸透明质酸酶从软骨素和CS制备的。通过肝素 - 琼脂糖亲和色谱法从胎牛血清中获得酶制剂。该制剂不包含最近在胎牛血清中发现的（Kitagawa等人，《生物化学杂志》，270，22190 - 22195，1995）利用常见受体底物的α - GalNAc转移酶。β - GalNAc转移酶将两个六糖 - 丝氨酸GlcAβ1 - 3GalNAcβ1 - 4GlcAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser和GlcAβ1 - 3GalNAc（4 - 硫酸酯）β1 - 4GlcAβ1 - 3Gal（4 - 硫酸酯）β1 - 3Galβ1 - 4Xylβ1 - O - Ser用作受体，但既不将单硫酸化的六糖 - 丝氨酸GlcAβ1 - 3GalNAc（4 - 硫酸酯）β1 - 4GlcAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser也不将在还原端第三个糖残基Gal - 3的C - 4处带有或不带有硫酸酯基团的四糖 - 丝氨酸用作受体。结果表明，Gal - 3 C - 4处的硫酸酯基团显著影响GalNAc向末端GlcA的转移。此外，规则四糖还原末端GalNAc的C - 4处的硫酸酯基团显著增强了GalNAc转移，表明该酶识别非还原端起至第四个糖残基。掺入到含有末端2 - O - 硫酸化GlcA残基的四糖或六糖中的水平显著，而掺入到含有末端3 - O - 硫酸化GlcA或倒数第二个4,6 - O - 二硫酸化GalNAc残基的四糖或六糖中则没有明显掺入。这些结果表明硫酸化反应在链延长和终止中起重要作用。
