A SINGLE EGF-LIKE MOTIF OF LAMININ IS RESPONSIBLE FOR HIGH-AFFINITY NIDOGEN BINDING
A SINGLE EGF-LIKE MOTIF OF LAMININ IS RESPONSIBLE FOR HIGH-AFFINITY NIDOGEN BINDING
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DOI：
10.1002/j.1460-2075.1993.tb05836.x
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发表时间：
1993-05-01
期刊：
影响因子：
11.4
通讯作者：
TIMPL, R
中科院分区：
文献类型：
--
作者：
MAYER, U;NISCHT, R;TIMPL, R
小鼠层粘连蛋白的主要巢蛋白结合位点先前定位于其B2链结构域III的约三个EGF样重复序列（Nos 3-5）[M.Gerl等人（1991）Eur.生物化学杂志，202，167]。通过聚合酶链反应扩增相应的cDNA，并将其插入到带有信号肽标签的真核表达载体中。稳定转染的人肾细胞克隆显示出大量加工和分泌所得片段B2 III 3 -5。在配体测定中，它具有与重组巢蛋白的高结合活性，与真实层粘连蛋白片段的亲和力相当。此外，B2 III 3 -5和巢蛋白的复合物可以通过交联剂有效地转化为共价复合物。共价复合物的蛋白水解降解证明了B2 III 3 -5与巢蛋白结构域G3的约80个残基片段的关联，其中层粘连蛋白结合先前已被归因于此。B2 III 3 -5中12个二硫键中的大多数正确形成，这是由其蛋白酶抗性和交叉反应表位的完全丧失以及还原和烷基化后巢蛋白结合活性指示的。通过相同的重组程序制备较小的片段，并显示EGF样重复序列3-4和4-5与单个重复序列4的组合而不是重复序列3或5具有完全的巢蛋白结合活性。这表明重复序列4是唯一的结合结构。重复序列4的序列在人类中是很保守的，部分在果蝇层粘连蛋白B2链中是保守的。进一步显示，抗B2 III 3 -5的抗体抑制层粘连蛋白与巢蛋白的结合，表明重复序列4代表层粘连蛋白的唯一高亲和力结合位点。
A major nidogen binding site of mouse laminin was previously localized to about three EGF-like repeats (Nos 3-5) of its B2 chain domain III [M.Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3-5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3-5 and nidogen could be efficiently converted into a covalent complex by cross-linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3-5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3-5 was indicated from its protease resistance and the complete loss of cross-reacting epitopes as well as of nidogen-binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF-like repeats 3-4 and 4-5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen-binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain. It was further shown that antibodies against B2III3-5 inhibit laminin binding to nidogen, indicating that repeat 4 represents the only high affinity binding site of laminin.