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

Abstract

The C-terminal G domains of laminin α chains have been implicated in various cellular and other interactions. The G domain of the α4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, α4LG1-3 and α4LG4-5. 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 α-dystroglycan receptor when compared with the G domains of the laminln α1 and α2 chains. Yet antibodies to α4LG1-3 but not to α4LG4-5 clearly inhibited α6β1 integrin-mediated cell adhesion to laminin-8, indicating the participation of α4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the α4LG3 and α4LG4 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 α4LG4-5 segment. Immunohistology demonstrated abundant staining of α4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for α4LG4-5, indicating their loss from tissues. Immunogold staining demonstrated an association of the α4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin α2 chains appear primarily associated with various basement membranes.