Structural and functional analysis of the recombinant G domain of the laminin α4 chain and its proteolytic processing in tissues

The C-terminal G domains of laminin a chains have been implicated in various 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 fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity;for the alpha -dystroglycan receptor when compared with: the G domains of the laminin alpha1 and alpha2 chains. 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 complexity. Proteolytic: processing within a link region between the alpha 4LG3 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 alpha 4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha 4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha 4LG4-5 indicating their loss from tissues. Immunogold staining demonstrated an association of the alpha4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin alpha2 chains appear primarily associated with various basement membranes.