Abstract
The C-terminal G domains of laminin alpha 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, alpha4LG1-3 and alpha4LG4-5. 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 alpha4LG1-3 but not to alpha4LG4-5 clearly inhibited alpha(6)beta(1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the alpha4LG3 and alpha4LG4 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 alpha4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha4LG4-5, 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.
Highlights
The protein family of laminins consists of at least 12 different isoforms, which are mainly localized in basement membranes
A recombinant fragment corresponding to laminin ␣2LG1–5 was shown to promote the attachment of Mycobacteria leprae to Schwann cells [17, 18], indicating that LG modules are likely to be involved in pathological processes
Recombinant Production of Two Fragments Comprising the G Domain of the Laminin ␣4 Chain—The G domain of the mouse laminin ␣4 chain was prepared in the form of two recombinant fragments, ␣4LG1–3 and ␣4LG4 –5 following a previous strategy used for the laminin ␣2 chain [19]
Summary
The protein family of laminins consists of at least 12 different isoforms, which are mainly localized in basement membranes. Antibodies raised against fusion proteins of the ␣4 chain were useful in showing the extracellular deposition of the corresponding laminins by immunohistology [5, 11] This demonstrated a distinct localization in striated muscle, perineurium, capillaries, and some mesenchymal regions but only a low abundance in epithelial basement membrane zones. Based on our previous experience with the recombinant production of LG modules of laminin ␣1 and ␣2 chains in mammalian cells [13, 19], we have prepared the tandem arrays ␣4LG1–3 and ␣4LG4 –5 for the mouse laminin ␣4 chain These fragments had a strong affinity for heparin but no activity or only little activity in cell adhesion and the binding of ␣-dystroglycan.
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