Stevens P W, Raffen R, Hanson D K, Deng Y L, Berrios-Hammond M, Westholm F A, Murphy C, Eulitz M, Wetzel R, Solomon A
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Illinois 60439, USA.
Protein Sci. 1995 Mar;4(3):421-32. doi: 10.1002/pro.5560040309.
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.
目前尚不清楚致使人类单克隆轻链具有淀粉样变性的主要结构特征。为了进一步深入了解导致这些蛋白质以淀粉样纤维形式发生病理性沉积的物理和生化因素，我们挑选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。其中两种蛋白质，REC和SMA，在体内形成了淀粉样纤维。第三种蛋白质LEN，由患者以每天50微克的水平排泄，没有淀粉样沉积物的迹象。淀粉样变性蛋白REC和SMA的序列分别在14个和8个氨基酸位置与非致病性蛋白LEN的序列不同，并且已根据LEN二聚体的三维结构对这些氨基酸差异进行了分析。为了提供这些人类蛋白质的可补充来源，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了这些基因。免疫化学和生物物理比较表明，重组VkIV产物具有与患者来源的蛋白质相当的三级结构特征。这一组明确的三种具有临床特征的人类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变性轻链蛋白和同一亚组的非淀粉样变性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。
