Expression of novel 400‐kDa laminin chains by mouse and bovine endothelial cells

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Abstract
Laminin expression was studied in endothelial cells derived from different mouse tissues and primary cultures of bovine aortic endothelium (BAEC). Immunoprecipitation with polyclonal antilaminin‐1 revealed two 200‐kDa chains, a novel 400‐kDa chain and nidogen in all cells studied. Two‐dimensional electrophoresis of laminin complexes immunoprecipitated from endothelial‐cell‐conditioned medium demonstrated that the 400‐kDa chain was disulphide‐linked to the laminin β1 and γ1 chains. In addition, rotary shadowing illustrated the secretion of molecules from the endothelial cells which were morphologically similar to laminin‐1. Immunoblotting and Northern blot analysis confirmed the presence of β1 and γ1 polypeptides and the corresponding LamB1 and LamC1 mRNAs in all cells investigated. However, both polyclonal anti‐laminin‐1 and α1‐specific monoclonal antibodies failed to react with the 400‐kDa polypeptide in immunoblots and immunoprecipitations. Similarly, the expression of the gene coding for α1, LamA1, was not detected in the majority of cells by Northern blot analysis using three different cDNAs. Only in two cases, BAEC and a Simian‐virus‐40‐transformed mouse endothelial cell line (SVEC), a 10‐kb mRNA was detected by Northern analysis using a cDNA specific for the 3′ coding region (LAC) of LamA1 mRNA. However, cDNAs specific for the central and 5′ coding region of LamA1 mRNA did not show any reaction. Hybridization of LAC to BAEC and SVEC mRNA is consistant with the laminin‐like nature of the 400‐kDa chain expressed by these cells. The results demonstrate the existence of at least one and possibly two novel 400‐kDa laminin chains which complex with β1 and γ1 and occur in mouse and bovine endothelial cell basement membranes.