Bundle D R, Eichler E, Gidney M A, Meldal M, Ragauskas A, Sigurskjold B W, Sinnott B, Watson D C, Yaguchi M, Young N M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Biochemistry. 1994 May 3;33(17):5172-82. doi: 10.1021/bi00183a022.
The binding site of monoclonal antibody Se155-4, which has been the object of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1, rather than to the tetrasaccharide repeating unit alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1-->4) alpha-L-Rhap(1- of the bacterial antigen. Specificity for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi B O-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic oligosaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives showed that complementary surfaces and three antibody-saccharide hydrogen bonds are essential for full binding activity. Both hydroxyl groups of the 3,6-dideoxy-D-galactose residue were obligatory for binding and consistent with the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-dideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mannose, tyvelose were not bound by the antibody. Titration microcalorimetry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy that accompanies binding of the native trisaccharide determinant. The protein sequences determined for the antibody VL and VH domains reveal somatic mutation of the VL germ line gene, implying that this antibody-binding site results from a mature antibody response.
单克隆抗体Se155-4的结合位点已成为成功的晶体学和抗体工程研究对象，固相免疫分析表明其与一种分支三糖α-D-半乳糖（1→2）[α-D-阿比糖（1→3）]-α-D-甘露糖（1互补，而非与细菌抗原的四糖重复单元α-D-半乳糖（1→2）[α-D-阿比糖（1→3）]-α-D-甘露糖（1→4）α-L-鼠李糖（1结合。通过用源自合成寡糖的糖缀合物进行杂交瘤筛选实验，确保了对副伤寒沙门氏菌B O抗原中存在的3,6-二脱氧-D-木糖己糖（3,6-二脱氧-D-半乳糖）表位的特异性。对修饰的和单脱氧寡糖衍生物的分子识别进行详细的表位图谱分析表明，互补表面和三个抗体-糖氢键对于完全结合活性至关重要。3,6-二脱氧-D-半乳糖残基的两个羟基对于结合都是必需的，这与它们参与碳水化合物-蛋白质氢键的方向性一致；由异构的3,6-二脱氧己糖、3,6-二脱氧-D-葡萄糖、副糖和3,6-二脱氧-D-甘露糖、泰威糖构建的相关四糖不被该抗体结合。滴定微量热法测量结果与从晶体结构推断的氢键图谱一致，并表明从结合位点置换水分子解释了天然三糖决定簇结合时伴随的有利熵变。为抗体VL和VH结构域确定的蛋白质序列揭示了VL种系基因的体细胞突变，这意味着该抗体结合位点是成熟抗体反应的结果。
