Mapping the functional domains of human transcobalamin using monoclonal antibodies
Mapping the functional domains of human transcobalamin using monoclonal antibodies
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DOI：
10.1111/j.1742-4658.2005.04805.x
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发表时间：
2005-08-01
期刊：
影响因子：
5.4
通讯作者：
Petersen, TE
中科院分区：
文献类型：
--
作者：
Fedosov, SN;Örning, L;Petersen, TE
重组人转钴胺素（TC）探测与17个单克隆抗体（mAb），使用表面等离子体共振测量。这些实验鉴定了holo-TC表面上的五个不同的表位簇。对TC的CNBr切割片段的Western印迹分析使我们能够将表位分布在两个区域之间，这两个区域跨越TC序列GQLA的第二个四分之一。TAAM（103-198）或C-末端肽LEPA... LVSW（316-427）。利用TC蛋白水解片段和合成肽进一步确定TC的表位图和功能结构域。只有一个抗体对钴胺素（Cbl）与TC的结合有一定的干扰作用，其相应的表位位于TQAS的C端。QLLR（372-399）。我们探讨了几种单克隆抗体和肝素的受体阻断作用，以确定TC域之间的相互作用所必需的holo-TC和受体。受体相关表位位于TC序列GQLA内。HHSV（103-159）。假定的肝素结合位点对应于带正电荷的片段KRSN…RTVR（207-227），这似乎也是受体结合所必需的。我们的结论是，构象变化TC CBL结合时伴随着多个结构域的收敛，只有组装构象的蛋白质（即holo-TC）具有高亲和力的受体。
Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.