RECOMBINANT NIDOGEN CONSISTS OF 3 GLOBULAR DOMAINS AND MEDIATES BINDING OF LAMININ TO COLLAGEN TYPE-IV
RECOMBINANT NIDOGEN CONSISTS OF 3 GLOBULAR DOMAINS AND MEDIATES BINDING OF LAMININ TO COLLAGEN TYPE-IV
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DOI：
10.1002/j.1460-2075.1991.tb04875.x
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发表时间：
1991-11-01
期刊：
影响因子：
11.4
通讯作者：
CHU, ML
中科院分区：
文献类型：
--
作者：
FOX, JW;MAYER, U;CHU, ML
重组小鼠巢蛋白和两个片段在哺乳动物细胞中产生，并从培养基中纯化，而不诉诸于变性条件。截短的产物是包含N-末端小球和杆状结构域的片段Nd-I（位置1-905）和主要对应于C-末端小球的Nd-II（位置906-1217）。重组巢蛋白与通过胍解离从肿瘤组织中获得的真实巢蛋白在大小、N-末端序列、CD光谱和免疫化学性质方面没有区别。它们在蛋白酶稳定性和形状上不同，表明更天然的重组蛋白的N-末端结构域由通过柔性片段连接的两个小球组成。这建立了巢原形状的新模型，由三个不同质量（31 - 56 kDa）的球体组成，由棒状或薄段连接。重组巢蛋白形成稳定的复合物（K（d）大于或等于1 nM）与层粘连蛋白和胶原蛋白IV在结合试验与可溶性和固定化配体，并通过电子显微镜显示。抑制试验表明，巢蛋白上的两种配体具有不同的特异性不同的结合位点。这在用片段Nd-I结合胶原IV和片段Nd-II结合层粘连蛋白片段P1的研究中得到证实。此外，重组巢蛋白，而不是钕-I能够桥接层粘连蛋白或P1和IV型胶原之间。这种三元复合物的形成暗示了巢蛋白在基底膜的超分子组织中的类似作用。
Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31 - 56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (K(d) greater-than-or-equal-to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.