Structural and functional analysis of the recombinant G domain of the laminin alpha 4 chain and its proteolytic processing in tissues

The C-terminal G domains of laminin a chains have been implicated in variou s cellular and other interactions. The G domain of the alpha4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules , alpha 4LG1-3 and alpha 4LG4-5. The recombinant fragments Were shown to fo ld into globular structures and could be distinguished by specific antibodi es. Both fragments were able to bind to heparin, sulfatides, and the microf ibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for c ell adhesion and had only a low affinity;for the alpha -dystroglycan recept or when compared with: the G domains of the laminin alpha1 and alpha2 chain s. Yet antibodies to alpha 4LG1-3 but not to alpha 4LG4-5 clearly inhibited alpha (6)beta (1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha 4LG1-3 in a cell-adhesive structure of higher c omplexity. Proteolytic: processing within a link region between the alpha 4 LG3 and alpha 4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the a lpha 4LG4-5 segment. Immunohistology demonstrated abundant staining of alph a 4LG1-3 in vessel walls, adipose, and perineural tissue. No significant st aining was found for alpha 4LG4-5 indicating their loss from tissues. Immun ogold staining demonstrated an association of the alpha4 chain primarily wi th microfibrillar regions rather than with basement membranes, while lamini n alpha2 chains appear primarily associated with various basement membranes .