A Salmonella typhi OmpC fusion protein expressing the CD154 Trp140-Ser149 amino acid strand binds CD40 and activates a lymphoma B-cell line
A Salmonella typhi OmpC fusion protein expressing the CD154 Trp140-Ser149 amino acid strand binds CD40 and activates a lymphoma B-cell line
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DOI：
10.1046/j.1365-2567.2003.01717.x
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发表时间：
2003-10-01
期刊：
影响因子：
6.4
通讯作者：
González-Bonilla, CR
中科院分区：
文献类型：
--
作者：
Vega, MI;Santos-Argumedo, L;González-Bonilla, CR
CD 154是肿瘤坏死因子（TNF）配体家族的II型糖蛋白成员，其主要表达于活化的T淋巴细胞表面。与其受体CD 40的相互作用在免疫系统的几种功能的控制中起着核心作用。基于CD 154与TNF和α-光毒素的同源性的结构模型表明，与CD 40的结合涉及氨基酸K143、R203和Q220周围的三个区域，并且链W140-S149和S198-A210对于这种相互作用是关键的。此外，已经报道了两种重组CD 154片段，包括氨基酸残基Y 45-L261或E108-L261，具有生物活性，而其他多肽，包括S149-L261，则没有生物活性。因此，我们决定构建一种融合蛋白，将W140-S149氨基酸链（WAEKGYYTMS）插入伤寒沙门氏菌外膜蛋白C（OmpC）的外环中，并评估其结合CD 40和活化B细胞的能力。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳证明嵌合OmpC-gp 39蛋白保留了其形成三聚体的能力。通过酶联免疫吸附试验的三种变体、通过用重组CD 40-Fc蛋白包被平板的直接结合试验以及通过OmpC-gp 39与重组CD 154或可溶性CD 40-Fc之间的两种竞争试验确定与CD 40的结合。流式细胞术分析表明，OmpC-gp 39增加Raji人B细胞淋巴瘤中主要组织相容性复合物II、CD 23和CD 80的表达水平，与抗CD 40抗体类似。这些结果进一步支持CD 154/CD 40相互作用类似于TNF/TNF受体。这是首次报道的细菌融合蛋白含有一个小的氨基酸链形式的配体，能够激活其同源受体。
CD154 is a type II glycoprotein member of the tumour necrosis factor (TNF) ligand family, which is expressed mainly on the surface of activated T lymphocytes. The interaction with its receptor CD40, plays a central role in the control of several functions of the immune system. Structural models based on the homology of CD154 with TNF and lymphotoxin indicate that binding to CD40 involves three regions surrounding amino acids K143, R203 and Q220, and that strands W140-S149 and S198-A210 are critical for such interactions. Also, it has been reported that two recombinant CD154 fragments, including amino acid residues Y45-L261 or E108-L261 are biologically active, whereas other polypeptides, including S149-L261, are not. Therefore, we decided to construct a fusion protein inserting the W140-S149 amino acid strand (WAEKGYYTMS) in an external loop of the outer membrane protein C (OmpC) from Salmonella enterica serovar Typhi and assess its ability to bind CD40 and activate B cells. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated that the chimeric OmpC-gp39 protein conserved its ability to form trimers. Binding to CD40 was established by three variants of enzyme-linked immunosorbent assay, a direct binding assay by coating plates with a recombinant CD40-Fc protein and through two competition assays between OmpC-gp39 and recombinant CD154 or soluble CD40-Fc. Flow cytometry analysis demonstrated that OmpC-gp39 increased the expression levels of major histocompatibility complex II, CD23, and CD80, in Raji human B-cell lymphoma similarly to an antibody against CD40. These results further support that the CD154/CD40 interaction is similar to the TNF/TNF receptor. This is the first report of a bacterial fusion protein containing a small amino acid strand form a ligand that is able to activate its cognate receptor.