Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light‐chain amyloid proteins
Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light‐chain amyloid proteins
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DOI：
10.1002/pro.5560040309
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发表时间：
1994-09
期刊：
影响因子：
8
通讯作者：
P. W. Stevens;R. Raffen;D. Hanson;Yanxue Deng;Maria Berrios‐Hammond;F. A. Westholm;M. Schiffer;F. Stevens;C. Murphy;A. Solomon;M. Eulitz;R. Wetzel
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文献类型：
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作者：
P. W. Stevens;R. Raffen;D. Hanson;Yanxue Deng;Maria Berrios‐Hammond;F. A. Westholm;M. Schiffer;F. Stevens;C. Murphy;A. Solomon;M. Eulitz;R. Wetzel
使人单克隆轻链淀粉样变的主要结构特征目前尚不清楚。为了进一步了解导致这些蛋白质病理沉积为淀粉样纤维的物理和生化因素，我们选择了轻链可变区单基因家族VkIV的三种密切同源的蛋白质产物进行详细研究。其中两种蛋白，REC和SMA，在体内形成淀粉样纤维。第三种蛋白质LEN以50 g/天的水平由患者排出，没有淀粉样蛋白沉积的迹象。淀粉样蛋白REC和SMA的序列与非致病性蛋白LEN的序列分别在14和8个氨基酸位置不同，这些氨基酸差异已根据LEN二聚体的三维结构进行了分析。为了提供这些人类蛋白质的可降解来源，我们构建了编码REC、SMA和LEN可变结构域的合成基因，并在大肠杆菌中表达这些基因。免疫化学和生物物理学比较表明，重组VkIV产物具有与患者来源蛋白相当的三级结构特征。这组定义明确的三种临床表征的人kIV轻链，以及重组产生这些kIV蛋白的能力，为两种不同的淀粉样蛋白原轻链蛋白和同一亚组的非淀粉样蛋白原蛋白的生物物理和结构比较提供了一个系统。这项工作为未来研究轻链淀粉样变性的结构基础奠定了基础。
The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light‐chain variable‐region single‐gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three‐dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient‐derived proteins. This well‐defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light‐chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light‐chain amyloidogenicity.