WARP interacts with collagen VI-containing microfibrils in the pericellular matrix of human chondrocytes.
WARP interacts with collagen VI-containing microfibrils in the pericellular matrix of human chondrocytes.
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DOI：
10.1371/journal.pone.0052793
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Fitzgerald J
中科院分区：
文献类型：
--
作者：
Hansen U;Allen JM;White R;Moscibrocki C;Bruckner P;Bateman JF;Fitzgerald J
VI 型胶原蛋白和 WARP 是存在于软骨中的细胞外结构大分子，与非骨骼组织中的 BM 上部结构相关。我们之前已经证明，在 WARP 缺陷的小鼠中，VI 型胶原蛋白在表达 WARP 的周围神经 ECM 区域中特异性减少，表明这两种大分子是同一上层结构的一部分。本研究的目的是在软骨（一种富含 WARP 和 VI 型胶原蛋白的组织）中对 WARP 与 VI 型胶原蛋白的体外相互作用进行详细分析。小鼠和人关节软骨的免疫组织化学分析表明，WARP 和 VI 型胶原蛋白共定位于浅层关节软骨细胞的细胞周基质中。对人类关节软骨提取物的电镜分析表明，WARP 与含有 VI 型胶原的上层结构密切相关。免疫金电镜和免疫印迹分析提供了相互作用的额外证据，表明 WARP 存在于从软骨中分离的含胶原蛋白 VI 的网络中。通过固相结合研究和使用纯化的重组 WARP 和分离的胶原 VI 的重构实验进行了进一步的表征。 VI 胶原蛋白以大约 22 nM 的表观 Kd 与 WARP 结合，并且 WARP 的结合位点位于三螺旋结构域内，因为 WARP 与完整的 VI 胶原蛋白四聚体和胃蛋白酶化的 VI 胶原蛋白结合。这些数据共同证实并扩展了我们之前的发现，证明了 WARP 和 VI 型胶原蛋白在体内软骨中形成高亲和力缔合。我们得出的结论是，WARP 非常适合作为软骨细胞周基质中的衔接蛋白。
Collagen VI and WARP are extracellular structural macromolecules present in cartilage and associated with BM suprastructures in non-skeletal tissues. We have previously shown that in WARP-deficient mice, collagen VI is specifically reduced in regions of the peripheral nerve ECM where WARP is expressed, suggesting that both macromolecules are part of the same suprastructure. The object of this study was to conduct a detailed analysis of WARP-collagen VI interactions in vitro in cartilage, a tissue rich in WARP and collagen VI. Immunohistochemical analysis of mouse and human articular cartilage showed that WARP and collagen VI co-localize in the pericellular matrix of superficial zone articular chondrocytes. EM analysis on extracts of human articular cartilage showed that WARP associates closely with collagen VI-containing suprastructures. Additional evidence of an interaction is provided by immunogold EM and immunoblot analysis showing that WARP was present in collagen VI-containing networks isolated from cartilage. Further characterization were done by solid phase binding studies and reconstitution experiments using purified recombinant WARP and isolated collagen VI. Collagen VI binds to WARP with an apparent Kd of approximately 22 nM and the binding site(s) for WARP resides within the triple helical domain since WARP binds to both intact collagen VI tetramers and pepsinized collagen VI. Together, these data confirm and extend our previous findings by demonstrating that WARP and collagen VI form high affinity associations in vivo in cartilage. We conclude that WARP is ideally placed to function as an adapter protein in the cartilage pericellular matrix.
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影响因子：
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