Precise epitope determination of the anti-vimentin monoclonal antibody V9.
Precise epitope determination of the anti-vimentin monoclonal antibody V9.
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DOI：
10.3892/mmr.2017.7102
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发表时间：
2017-10
影响因子：
3.4
通讯作者：
Urano T
中科院分区：
文献类型：
--
作者：
Tomiyama L;Kamino H;Fukamachi H;Urano T
波形蛋白是典型地在间充质细胞中表达的III型中间丝蛋白。波形蛋白的过度表达经常在几种类型的癌症中观察到，并且通常与上皮向间质转化有关。近年来有研究表明，结肠和肝脏肿瘤患者血清波形蛋白水平显著升高。因此，更灵敏的波形蛋白检测系统可能有助于癌症筛查和早期检测。识别人波形蛋白的V9小鼠单克隆抗体（mAb）广泛用于常规病理学中，以使用免疫组织化学分析鉴定间充质细胞。尽管已经表明V9 mAb的表位位于波形蛋白的C-末端区域内，但其识别的精确氨基酸序列尚未确定。在本研究中，我们构建了几个波形蛋白的缺失突变体，并检查了它们与V9 mAb的反应性，以准确定位其表位。我们证实其表位位于波形蛋白的C-末端区域，氨基酸392-466之间。此外，波形蛋白的氨基酸序列比对的跨物种比较以及定点诱变揭示了一个残基，即位置417处的天冬酰胺，对于抗体结合至关重要。使用长度范围为9至13个残基的较小波形蛋白片段，每个片段含有该关键天冬酰胺，我们确定V9 mAb识别人波形蛋白所需的最小残基是位置411-423处的13个氨基酸残基（411 ISLPLPNFSSLNL 423）。
Vimentin is a type III intermediate filament protein that is typically expressed in mesenchymal cells. Overexpression of vimentin is frequently observed in several types of cancer and is often associated with epithelial-to-mesenchymal transition. It was recently reported that the serum vimentin level is significantly elevated in colon and liver tumors. Therefore, a more sensitive vimentin detection system may be useful for cancer screening and early detection. The V9 mouse monoclonal antibody (mAb), which recognizes the human vimentin protein, is widely used in routine pathology to identify mesenchymal cells using immunohistochemical analysis. Although it has been suggested that the epitope of the V9 mAb is located within the C-terminal region of vimentin, the precise amino acid sequence that it recognizes has not yet been identified. In the present study, we constructed several deletion mutants of the vimentin protein and examined their reactivity with the V9 mAb to accurately map its epitope. We confirmed that its epitope resides in the C-terminal region of vimentin, between amino acids 392–466. Additionally, cross-species comparison of amino acid sequence alignment of vimentin, as well as site-directed mutagenesis, revealed that one residue, the asparagine at position 417, is critical for antibody binding. Using smaller vimentin fragments ranging in length from 9 to 13 residues, each containing this critical asparagine, we determined that the minimal residues required for V9 mAb recognition of human vimentin are the thirteen amino acid residues at positions 411–423 (411ISLPLPNFSSLNL423).
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影响因子：
9.3
作者：
Satelli A;Brownlee Z;Mitra A;Meng QH;Li S
通讯作者：
Li S
影响因子：
4.4
作者：
Sun, Stella;Poon, Ronnie T. P.;Luk, John M.
通讯作者：
Luk, John M.
DOI：
10.1074/jbc.m113.514737
发表时间：
2013-12-13
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Matsuyama M;Tanaka H;Inoko A;Goto H;Yonemura S;Kobori K;Hayashi Y;Kondo E;Itohara S;Izawa I;Inagaki M
通讯作者：
Inagaki M
影响因子：
3.7
作者：
BOHN, W;WIEGERS, W;TRAUB, P
通讯作者：
TRAUB, P
影响因子：
5.2
作者：
Steinmetz NF;Maurer J;Sheng H;Bensussan A;Maricic I;Kumar V;Braciak TA
通讯作者：
Braciak TA