Mayer U, Kohfeldt E, Timpl R
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Ann N Y Acad Sci. 1998 Oct 23;857:130-42. doi: 10.1111/j.1749-6632.1998.tb10113.x.
High-affinity binding of nidogen to laminins involves a single binding site on the laminin gamma 1 chain and is thus a property shared by almost all laminin isoforms. This binding mediates the connection of laminins to the collagen IV network, perlecan and other proteins and is considered to be an essential step in the stabilization of basement membranes. Nidogen binding has been located to a single LE module (gamma 1III4) by recombinant analysis. Site-directed mutagenesis and X-ray crystallography demonstrated that three amino acids (Asp, Asn, Val) in loop a of gamma 1III4 are crucial for binding and are supported by some other residues. A restricted complementary binding region seems to exist on nidogen domain G3. A mutant laminin gamma 1 chain gene that lacks the region encoding gamma 1III4 was prepared in mouse embryonic stem (ES) cells by homologous recombination. ES cells homozygous for this defect were shown to assemble laminin-1 into a cruciform structure and to secrete it properly. Yet the mutant laminin failed to associate with nidogen. The mutant ES cells were still able to form embryoid bodies with a similar differentiated histology as the wild type. Immunofluorescence, however, indicated an impaired deposition of nidogen into basement membrane-like structures.
巢蛋白与层粘连蛋白的高亲和力结合涉及层粘连蛋白γ1链上的一个单一结合位点，因此几乎是所有层粘连蛋白异构体共有的特性。这种结合介导了层粘连蛋白与IV型胶原网络、基底膜聚糖及其他蛋白质的连接，被认为是基底膜稳定化的关键步骤。通过重组分析已将巢蛋白结合定位到单个LE模块（γ1III4）。定点诱变和X射线晶体学表明，γ1III4的a环中的三个氨基酸（天冬氨酸、天冬酰胺、缬氨酸）对结合至关重要，并得到其他一些残基的支持。巢蛋白结构域G3上似乎存在一个受限的互补结合区域。通过同源重组在小鼠胚胎干细胞中制备了一个缺失编码γ1III4区域的突变层粘连蛋白γ1链基因。纯合这种缺陷的胚胎干细胞显示能将层粘连蛋白-1组装成十字形结构并正常分泌。然而，突变的层粘连蛋白未能与巢蛋白结合。突变的胚胎干细胞仍能够形成具有与野生型相似分化组织学的胚状体。然而，免疫荧光表明巢蛋白沉积到基底膜样结构中的过程受损。
