Expression and supramolecular assembly of recombinant α1(VIII) and α2(VIII) collagen homotrimers
Expression and supramolecular assembly of recombinant α1(VIII) and α2(VIII) collagen homotrimers
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DOI：
10.1074/jbc.m305805200
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发表时间：
2004-05-14
影响因子：
4.8
通讯作者：
Kielty, CM
中科院分区：
文献类型：
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作者：
Stephan, S;Sherratt, MJ;Kielty, CM
胶原VIII是一种细胞外基质大分子，由两条多肽链alpha1（VIII）和alpha2（VIII）组成，在体内和体外均可形成同型三聚体。在这里，表达重组胶原VIII以研究其分泌后的超分子组装。转染了alpha1（VIII）或alpha2（VIII）的细胞组装并分泌在变性条件下稳定的同源三聚体，SDS-PAGE凝胶上的分子质量与180 kDa相似。与脯氨酰4-羟化酶共转染产生具有稳定耐胃蛋白酶三螺旋结构域的同源三聚体。对有或没有脯氨酸4-羟化酶表达的天然重组胶原VIII分子进行大小分离，鉴定出在Superose 6HR 10/30柱空隙体积中洗脱的尿素敏感的高分子质量组合和类似于700 kDa的尿素抗性组合，它们都是由同源三聚体组成的。免疫荧光分析强调了重组alpha1(VIII)(3)、alpha2(VIII)(3)和共表达alpha1(VIII)(3)/alpha2(VIII)(3)的细胞外沉积。重组胶原VIII的显微镜分析发现，长134 nm的棒状分子组装成角阵列，分支角度接近114度，并形成广泛的网络。基于这些数据，我们提出了一个胶原VIII组装模型，其中四个同源三聚体形成一个四面体，由中心相互作用的c端NC1三聚体稳定。四面体可以作为三维六边形晶格的构建块，这些晶格是由涉及末端序列和螺旋序列的次级相互作用产生的。
Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, alpha1(VIII) and alpha2(VIII), that can form homotrimers in vitro and in vivo. Here, recombinant collagen VIII was expressed to study its supramolecular assembly following secretion. Cells transfected with alpha1(VIII) or alpha2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of similar to180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated homotrimers with stable pepsin-resistant triple-helical domains. Size fractionation of native recombinant collagen VIII molecules expressed with or without prolyl 4-hydroxylase identified urea-sensitive high molecular mass assemblies eluting in the void volume of a Superose 6HR 10/30 column and urea-resistant assemblies of similar to700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant alpha1(VIII)(3), alpha2(VIII)(3), and co-expressed alpha1(VIII)(3)/alpha2(VIII)(3). Microscopy analysis of recombinant collagen VIII identified rod-like molecules of 134 nm in length that assembled into angular arrays with branching angles of similar to114degrees and extensive networks. Based on these data, we propose a model of collagen VIII assembly in which four homotrimers form a tetrahedron stabilized by central interacting C-terminal NC1 trimers. Tetrahedrons may then act as building blocks of three-dimensional hexagonal lattices generated by secondary interactions involving terminal and helical sequences.