STRUCTURAL REQUIREMENT FOR CELL-ADHESION TO KALININ (LAMININ-5)
STRUCTURAL REQUIREMENT FOR CELL-ADHESION TO KALININ (LAMININ-5)
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DOI：
10.1074/jbc.270.23.13766
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发表时间：
1995-06-09
影响因子：
4.8
通讯作者：
AUMAILLEY, M
中科院分区：
文献类型：
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作者：
ROUSSELLE, P;GOLBIK, R;AUMAILLEY, M
通过单克隆抗体 BM165 上的亲和层析从用过的细胞培养基（SCC25 细胞）中纯化 Laminin-5（kalinin）。根据还原条件下的 SDS 聚丙烯酰胺凝胶电泳分析判断，蛋白质被回收为 165-155、140 和 105 kDa 的典型多肽的混合物。纯化的层粘连蛋白-5的氨基酸组成与最近公布的α(3)-、β(3)-和γ(2)-层粘连蛋白链的cDNA序列汇编的氨基酸组成一致。此外，laminin-5中半胱氨酸残基的含量约为laminin-1中的三分之二，这证实了在三条链的氨基末端部分中存在较少数量的表皮生长因子样重复的预测。 CD 光谱测定的卷曲螺旋 α 螺旋含量 (27%) 与报道的 laminin-1 相当，这表明 laminin-5 的长臂部分与其他层粘连蛋白亚型的长臂部分相当。通过 CD 监测热变性和复性过程中卷曲螺旋结构的展开和重折叠，记录熔化温度为 72 摄氏度。因此，层粘连蛋白-5的热稳定性显着高于层粘连蛋白-1或含有α(2)链的层粘连蛋白，这表明层粘连蛋白-5的三个多肽链之间存在更高的离子相互作用。研究发现，层粘连蛋白 5 的细胞粘附促进活性严格且完全依赖于卷曲螺旋结构的存在，在 65 摄氏度以上的蛋白质热变性后，其细胞粘附促进活性逐渐降低，并在 75 摄氏度时完全消失。这与层粘连蛋白 1 不同，层粘连蛋白 1 分别在长臂结构域和短臂结构域上包含构象依赖性和非依赖性细胞结合位点。
Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged by SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha(3)-, beta(3)-, and gamma(2)-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha(2)-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin 5 was found to be strictly and entirely dependent on the presence of coiled-coil structures, It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.