MOLECULAR RECOGNITION OF A SALMONELLA TRISACCHARIDE EPITOPE BY MONOCLONAL-ANTIBODY SE155-4
MOLECULAR RECOGNITION OF A SALMONELLA TRISACCHARIDE EPITOPE BY MONOCLONAL-ANTIBODY SE155-4
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DOI：
10.1021/bi00183a022
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发表时间：
1994-05-03
期刊：
影响因子：
2.9
通讯作者：
YOUNG, NM
中科院分区：
文献类型：
--
作者：
BUNDLE, DR;EICHLER, E;YOUNG, NM
单克隆抗体 Se155-4 的结合位点已成为晶体学和抗体工程研究成功的目标，固相免疫分析显示其结合位点与支链三糖 α-D-Galp(1-->2) [α-D-Abep(1-->3)]-α-D-Manp(1) 互补，而不是与四糖重复单元互补细菌抗原的 alpha-D-Galp(1-->2)[alpha-D-Abep(1-->3)]-alpha-D-Manp(1-->4)alpha-L-Rhap(1-)。通过筛选杂交瘤，确保副伤寒沙门氏菌 BO 抗原中存在的 3,6-双脱氧-D-木己糖（3,6-双脱氧-D-半乳糖）表位的特异性对合成寡糖衍生的糖缀合物的实验通过修饰和单脱氧寡糖衍生物进行分子识别的详细表位图谱表明，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是必需的，并且与它们参与由异构体构建的碳水化合物-蛋白质氢键的方向性一致； 3,6-二脱氧己糖、3,6-二脱氧-D-葡萄糖、帕拉糖和 3,6-二脱氧-D-甘露糖不被抗体结合，滴定微量热法测量结果与从晶体结构推断的氢键图一致，表明水分子从结合位点的位移解释了伴随天然三糖决定簇结合的有利熵。抗体 V-L 和 V-H 结构域的序列测定揭示了 V-L 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。
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(l, 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 BO-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 V-L and V-H domains reveal somatic mutation of the V-L germ line gene, implying that this antibody-binding site results from a mature antibody response.