Reinhardt D, Mann K, Nischt R, Fox J W, Chu M L, Krieg T, Timpl R
Max-Planck-Institut für Biochemie, Martinsried, Germany.
J Biol Chem. 1993 May 25;268(15):10881-7.
Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.
重组的含球状结构域G1加G2、杆状结构域以及连接球状结构G3的杆的巢蛋白片段，是从转染的人细胞克隆的培养基中制备的。此外，用胰凝乳蛋白酶切割后，结构域G1和G2彼此分离。纯化的片段通过N端序列、电泳、电子显微镜和放射免疫测定进行表征，细胞克隆则通过Northern杂交进行表征。用包含大部分结构域G3的构建体转染显示出高mRNA水平，但没有分泌蛋白，这表明存在蛋白质折叠问题。所有这些片段都用作结合试验中的可溶性和/或固定化配体。这证明了结构域G2上存在与IV型胶原和硫酸乙酰肝素蛋白聚糖的主要结合位点。在负载锌和钴的柱上进行亲和层析显示结构域G2、G3和杆状结构的结合。巢蛋白经还原和烷基化后，蛋白质结合而非金属结合被消除。这使得能够分离出几种锌结合胰蛋白酶肽，4种来自G2，2种来自杆状结构，1种来自G3结构域。这些短肽中的大多数含有几个可能介导结合的组氨酸。锌在4℃时能有效抑制G3介导的巢蛋白与层粘连蛋白的结合（IC50约为5 microM），但在较高温度下抑制作用较小。同样，锌在低温下能抑制与IV型胶原和蛋白聚糖的结合，但在高温（37℃）下则不能。这表明一些但不是所有的过渡金属对巢蛋白与其他基底膜蛋白的结合有复杂的调节作用。锌所显示的特别显著的效应是否具有生物学相关性仍有待确定。
