Structure and Function of Laminin-5, a Cell Adhesion Protein of Basement Membrane

基底蛋白层粘连蛋白-5（LN 5）是层粘连蛋白α3、β3和γ2链的异源三聚体，有效地促进细胞粘附和运动。本研究旨在探讨LN 5的结构与功能关系及其在肿瘤侵袭中的病理作用。1)LN 5的α3链在其羧基端含有五个球状结构域（G1至G5）。为了阐明每个G结构域的功能，我们将α3链的全长和五个缺失衍生物的cDNA转染到仅表达层粘连蛋白β3和γ2链的人HT 1080细胞中，并制备缺乏每个G结构域的重组LN 5。G5或G4-5的缺失不影响LN 5的生物学活性，而G3-5缺失的LN 5几乎没有细胞粘附和迁移活性。同时发现G3含有两个不同的区域来刺激细胞粘附和迁移。此外，我们发现α3链在分泌后的G3和G4之间被切割，并确定了切割位点。2)LN 5的105-kDa γ2链在氨基末端区域被切割以产生105-kDa形式。我们发现，MT 1-MMP催化这一过程，这一过程增加了LN 5的细胞运动活性，但降低了其细胞粘附活性。这些结果表明，γ2链的氨基端区域对LN 5的生物活性具有重要影响。3)免疫组化结果显示，层粘连蛋白γ2链单体在浸润的人癌细胞中呈高表达。当γ2链氨基端区域的cDNA转染到人癌细胞系中时，该细胞系在体内的侵袭潜力大大增强。提示γ2链本身在肿瘤侵袭中起重要作用。

The basement protein laminin-5(LN5), a heterotrimer of laminin α3, β3 and γ2 chains, potently promotes cellular adhesion and motility. In this study, we investigated the structure and function relationship of LN5 and its pathological roles in tumor invasion. 1) The α3 chain of LN5 contains five globular domains (G1 to G5) in its carboxyl end. To clarify the function of each G domain, we transfected cDNAs for the full length and five deletion derivatives of the α3 chain into human HT1080 cells, which express only the laminin β3 andγ2 chains, and prepared recombinant LN5s lacking each G domain. The deletion of G5 or G4-5 did not affect the biological activities of LN5, but the LN5 without G3-5 showed little activities of cell adhesion and migration. It was also found that G3 contained two different regions to stimulate cell adhesion and migration. Furthermore, we found that the α3 chain was cleaved between G3 and G4 after secretion and determined the cleavage site. 2) The 105-kDa γ2 chain of LN5 is cleaved at an amino-terminal region to produce the 105-kDa form. We found that MT1-MMP catalyzed this processing, and that this processing increased the cell motility activity of LN5 but decreased its cell adhesion activity. These results demonstrate that the amino-terminal region of γ2 chain has an important effect on the biological activity of LN5. 3) Immunohistochemical analysis showed that the laminin γ2 chain monomer was overexpressed in invading human carcinoma cells. When a cDNA for the amino-terminal regions of the γ2 chain was transfected into a human carcinoma cell line, the invasive potential of this cell line in vivo was greatly enhanced. These results suggest that the γ2 chain itself plays some important roles in tumor invasion.