Nadanaka S, Kitagawa H, Sugahara K
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
J Biol Chem. 1998 Dec 11;273(50):33728-34. doi: 10.1074/jbc.273.50.33728.
Thrombomodulin (TM), a cell surface glycoprotein, is a critical mediator of endothelial anticoagulant defenses occurring both as a chondroitin sulfate proteoglycan (beta-TM) and a protein (alpha-TM) unsubstituted by chondroitin sulfate (CS), hence its description as a "part-time" proteoglycan (PG) (Fransson, L. A. (1987) Trends Biochem. Sci. 12, 406-411). Sugar analysis was performed on alpha-TM to investigate a possible biosynthetic mechanism for part-time PGs. Recombinant human alpha-TM, which was expressed in CHO-K1 cells, separated by anion-exchange chromatography from beta-TM, and purified by immunoaffinity chromatography (Nawa, K., Sakano, K., Fujiwara, H., Sato, Y., Sugiyama, N., Teruuchi, T., Iwamoto, M., and Marumoto, Y. (1990) Biochem. Biophys. Res. Commun. 171, 729-737), was used for analysis. Preliminary sugar composition analysis after acid hydrolysis showed Xyl in addition to Gal, GalNAc, GlcNAc, Man, Fuc, and Glc. O-Glycosidically-linked oligosaccharides were liberated by mild alkaline treatment and purified. The isolated oligosaccharide fraction was derivatized with a fluorophore 2-aminobenzamide (2AB), resulting in two fluorescent components, a 2AB-oligosaccharide and a putative 2AB-Glc. Based on structural analysis by a combination of sequential exoglycosidase digestion and 500-MHz 1H NMR spectroscopy of the 2AB-oligosaccharide, the structure of the oligosaccharide was elucidated as GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl, which turned out to represent a glycosaminoglycan (GAG)-protein linkage region tetrasaccharide common to various PGs and was considered to be a biosynthetic intermediate of an immature GAG chain. The results may indicate that at least one class of the so-called part-time PGs bear the linkage tetrasaccharide at the GAG attachment sites and that the critical determining step or the rate-limiting step for PG biosynthesis is the transfer of the fifth sugar residue, the first hexosamine, rather than xylose.
血栓调节蛋白（TM）是一种细胞表面糖蛋白，是内皮抗凝防御的关键介质，它以硫酸软骨素蛋白聚糖（β-TM）和未被硫酸软骨素（CS）取代的蛋白质（α-TM）两种形式存在，因此被描述为“兼职”蛋白聚糖（PG）（弗兰松，L.A.（1987年）《生物化学趋势》12卷，406 - 411页）。对α-TM进行了糖分析，以研究兼职PG可能的生物合成机制。在CHO - K1细胞中表达的重组人α-TM，通过阴离子交换色谱与β-TM分离，并通过免疫亲和色谱纯化（名和，K.，坂野，K.，藤原，H.，佐藤，Y.，杉山，N.，照内，T.，岩本，M.，丸本，Y.（1990年）《生物化学与生物物理研究通讯》171卷，729 - 737页），用于分析。酸水解后的初步糖组成分析显示，除了半乳糖（Gal）、N - 乙酰半乳糖胺（GalNAc）、N - 乙酰葡糖胺（GlcNAc）、甘露糖（Man）、岩藻糖（Fuc）和葡萄糖（Glc）外，还有木糖（Xyl）。通过温和的碱性处理释放并纯化了O - 糖苷键连接的寡糖。分离出的寡糖部分用荧光团2 - 氨基苯甲酰胺（2AB）衍生化，产生了两个荧光成分，一个2AB - 寡糖和一个假定的2AB - Glc。基于对2AB - 寡糖进行顺序外切糖苷酶消化和500兆赫兹1H核磁共振光谱相结合的结构分析，寡糖的结构被阐明为GlcAbeta₁ - 3Galbeta₁ - 3Galbeta₁ - 4Xyl，结果表明它代表了各种PG共有的糖胺聚糖（GAG） - 蛋白质连接区四糖，并且被认为是未成熟GAG链的生物合成中间体。结果可能表明，至少一类所谓的兼职PG在GAG连接位点带有连接四糖，并且PG生物合成的关键决定步骤或限速步骤是第五个糖残基（第一个己糖胺）的转移，而不是木糖的转移。
