A recombinant technique for mapping functional sites of heterotrimeric collagen helices: Collagen IV CB3 fragment as a prototype for integrin binding.
A recombinant technique for mapping functional sites of heterotrimeric collagen helices: Collagen IV CB3 fragment as a prototype for integrin binding.
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DOI：
10.1016/j.jbc.2023.104901
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发表时间：
2023-07
影响因子：
4.8
通讯作者：
Lin, Fu-Yang
中科院分区：
文献类型：
--
作者：
Boudko, Sergei P.;Konopka, Elizabeth H.;Kim, Woojin;Taga, Yuki;Mizuno, Kazunori;Springer, Timothy A.;Hudson, Billy G.;Moy, Terence I.;Lin, Fu-Yang
胶原蛋白超家族是细胞外基质的主要成分。胶原蛋白缺陷是导致全球数百万人患近 40 种人类遗传疾病的原因。发病机制通常涉及三螺旋的遗传改变，三螺旋是一种标志性结构特征，赋予对拉力的特殊机械抵抗力和结合大量大分子的能力。然而，在理解三螺旋上不同位点的功能方面存在着巨大的知识差距。在这里，我们提出了一种重组技术来产生用于功能研究的三螺旋片段。该实验策略利用 IX 胶原蛋白 NC2 异三聚化结构域的独特能力来驱动三个 α 链选择并记录三螺旋交错。为了证明原理，我们生产并表征了在哺乳动物系统中表达的 IV 型胶原蛋白的长三螺旋片段。异源三聚体片段包含 IV 型胶原蛋白的 CB3 三聚体肽，其含有 α1β1 和 α2β1 整联蛋白的结合基序。片段经过表征并显示具有稳定的三螺旋、翻译后修饰以及整合素的高亲和力和特异性结合。 NC2 技术是高产生产胶原蛋白异三聚体片段的通用工具。片段适用于绘制功能位点、确定结合位点的编码序列、阐明基因突变的致病性和致病机制以及生产用于蛋白质替代疗法的片段。
Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α1β1 and α2β1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.
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影响因子：
--
作者：
Boudko SP;Danylevych N;Hudson BG;Pedchenko VK
通讯作者：
Pedchenko VK
影响因子：
5.6
作者：
Boudko, S;Frank, S;Engel, J
通讯作者：
Engel, J
影响因子：
56.9
作者：
BELLA, J;EATON, M;BERMAN, HM
通讯作者：
BERMAN, HM
影响因子：
64.5
作者：
Emsley, J;Knight, CG;Liddington, RC
通讯作者：
Liddington, RC
DOI：
10.1111/j.1432-1033.1980.tb04610.x
发表时间：
1980-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BACHINGER, HP;BRUCKNER, P;ENGEL, J
通讯作者：
ENGEL, J