Studies on the specificity of rabbit hepatic carbohydrate-binding protein using neoglycoproteins.
Studies on the specificity of rabbit hepatic carbohydrate-binding protein using neoglycoproteins.
复制标题
利用新糖蛋白研究兔肝糖结合蛋白的特异性。
DOI：
10.1021/bi00562a031
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Lee,YC
中科院分区：
文献类型：
--
作者：
Stowell,CP;Lee,RT;Lee,YC
Christopher P. Stowell、Reiko T. Lee 和 Yuan Chuan Lee* 摘要：测量了牛血清白蛋白的脒糖蛋白与兔肝膜的结合。附着有等量的/3-D-Gal、6-0-Me-/3-o-Gal、/3-d-Fuc、l-Ara、ß-D-Glc、/3-D-Xyl 和/3-D-GalNAc 的牛血清白蛋白衍生物同样良好地与膜结合。 ao-Man、/3-l-Fuc、/3-d-G1cNAc、/3-D-阿洛糖、3-0-Me-/3-o-Glc 和 2-脱氧-/3-o-Glc 的附着不会促进强结合。通过测量新糖蛋白与固定在 Sepharose 4B 上的纯化兔肝碳水化合物结合蛋白的结合来证实与膜结合的特异性。结果表明，对于结合，（1）既不需要 6-OH（d-Fuc）也不需要 5-CH2OH（L-Ara；D-Xyl），（2）4-OH 可以是轴向（o-Gal；L-Ara）或赤道（d-G1c；D-Xyl），（3）3-OH 必须是赤道（d-G1c）而不是轴向（d-A11）也不能是赤道（d-G1c）。 (3-O-Me-D-Glc)，(4) 2-OH 必须是赤道 (d-G1c) 而不是轴向 (o-Man)，并且必须存在 (2-脱氧-o-Glc)，并且 (5) 如果 4-OH 是轴向 (D-GalNAc)，则 2-OH 可以被赤道乙酰胺基取代，但如果 4-OH 是赤道 (d-G1cNAc)，则不能被赤道乙酰胺基取代。长期以来，碳水化合物辅基一直被认为在细胞识别和结合许多糖蛋白方面发挥着作用（Roseman，1970；Ashwell & Morell，1974；Stahl 等，1978；Kaplan 等，1977；Neufeld 等，1977）。特别令人感兴趣的是观察到碳水化合物结构的结合要求非常严格。为了解决糖蛋白与细胞和受体结合的结构要求这一普遍问题，我们通过使用 2-亚氨基-2-甲氧基乙基 1-硫代糖苷将碳水化合物共价连接到蛋白质上，制备了一系列合成糖复合物（新糖蛋白）（Lee 等，1976）。特别是，我们检查了这些新糖蛋白与兔肝碳水化合物结合蛋白的结合（Krantz et al., 1976; Stowell & Lee, 1978），该蛋白已被广泛表征（Ashwell & Morell, 1974; Hudgin et al., 1974; Kawasaki & Ashwell, 1976a, b; Sarkar et al., 1979）。正如根据去糖基化血清型糖蛋白的行为所预测的那样（Ashwell & Morell，1974），/3-d-半乳糖基新糖蛋白与兔肝膜中的结合蛋白结合，而 2-乙酰氨基-2-脱氧-/3-D-葡萄糖基-和-D-甘露糖基新糖蛋白则没有（Krantz 等人，1976；Stowell） ＆李，1978）。有趣的是，还发现3-D-葡糖基新糖蛋白与结合蛋白的结合与寡糖链终止于3-D-半乳糖基残基的β-S-D-半乳糖基新糖蛋白或脱唾液酸粘蛋白一样强。这种结合蛋白的特异性有些宽松的证据促使我们前去f来自约翰·霍普金斯大学生物学系和麦科勒姆-普拉特研究所，马里兰州巴尔的摩21218。收稿日期为1980年3月31日。这项工作得到了美国公共卫生服务部、国立卫生研究院拨款AM9970的支持。来自约翰·霍普金斯大学麦科勒姆-普拉特研究所的第 1066 号贡献。使用一些新的脒糖蛋白来确定结合的结构要求（Stowell & Lee，1980）。在本研究中使用（新）糖蛋白而不是简单的寡糖或糖苷是必要的，因为低分子量碳水化合物与结合蛋白的结合比大分子的结合弱多个数量级（Stowell＆Lee，1978；Sarkar等人，1979）并且可能无法准确反映......
Christopher P. Stowell, Reiko T. Lee, and Yuan Chuan Lee* abstract: The binding of amidinoneoglycoproteins of bovine serum albumin to rabbit liver membranes was measured. Derivatives of bovine serum albumin to which equivalent amounts of/3-D-Gal, 6-0-Me-/3-o-Gal,/3-d-Fuc,«-L-Ara, ß-D-Glc,/3-D-Xyl, and/3-D-GalNAc had been attached bound to the membranes equally well. The attachment of ao-Man,/3-l-Fuc,/3-d-G1cNAc,/3-D-allose, 3-0-Me-/3-o-Glc, and 2-deoxy-/3-o-Glc did not promote strong binding. The specificity of binding to the membranes was confirmed by measuring the binding of neoglycoproteins to the purified rabbit hepatic carbohydrate-binding protein immobilized on Sepharose 4B. The results indicate that, for binding,(1) neither the 6-OH (d-Fuc) nor the 5-CH2OH (L-Ara; D-Xyl) is required,(2) the 4-OH can be axial (o-Gal; L-Ara) or equatorial (d-G1c; D-Xyl),(3) the 3-OH must be equatorial (d-G1c) not axial (d-A11) nor may it be substituted (3-O-Me-D-Glc),(4) the 2-OH must be equatorial (d-G1c) not axial (o-Man) and must be present (2-deoxy-o-Glc), and (5) the 2-OH can be replaced by an equatorial acetamido group if the 4-OH is axial (D-GalNAc) but not if it is equatorial (d-G1cNAc). e carbohydrate prosthetic group has long been implicated as having a role in the recognition and binding of many gly-coproteins by cells (Roseman, 1970; Ashwell & Morell, 1974; Stahl et al., 1978; Kaplan et al., 1977; Neufeld et al., 1977). Of particular interest is the observation that the requirements for binding with respect to carbohydrate structure are quite stringent. In order to approach the general problem of defining the structural requirements for the binding of glycoproteins to cells and receptors, we have prepared a series of synthetic glycoconjugates (neoglycoproteins) by covalently attaching carbohydrates to proteins using the 2-imino-2-methoxyethyl 1-thioglycosides (Lee et al., 1976). In particular, we have examined the binding of these neoglycoproteins to the rabbit hepatic carbohydrate-binding protein (Krantz et al., 1976; Stowell & Lee, 1978) which has been extensively characterized (Ashwell & Morell, 1974; Hudgin et al., 1974; Kawasaki & Ashwell, 1976a, b; Sarkar et al., 1979). As would have been predicted on the basis of the behavior of deglycosylated se-rum-type glycoproteins (Ashwell & Morell, 1974),/3-d-galactosylneoglycoproteins bound to the binding protein in rabbit liver membranes whereas 2-acetamido-2-deoxy-/3-D-glucosyl-and-D-mannosylneoglycoproteins did not (Krantz et al., 1976; Stowell & Lee, 1978). Interestingly,/3-d-glucosylneoglycoproteins were also found to bind to the binding protein as strongly as jS-D-galactosylneoglycoproteins or asialoorosmucoid whose oligosaccharide chains are terminated in/3-D-galactosyl residues. The evidence of the somewhat relaxed specificity of this binding protein prompted us to ex-f From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218. Received March 31, 1980. This work was supported by the US Public Health Service, National Institutes of Health Research Grant AM9970. Con-tribution No. 1066 from the McCollum-Pratt Institute, The Johns Hopkins University. amine the structural requirements for binding using some new amidinoneoglycoproteins (Stowell & Lee, 1980). The use of (neo) glycoproteins rather than simple oligosaccharides or glycosides in the present study is imperative because the binding of low molecular weight carbohydrates to the binding protein is many orders of magnitude weaker than the binding of macromolecules (Stowell & Lee, 1978; Sarkar et al., 1979) and may not accuratelyreflect the …