Abstract
The G domain of the laminin alpha chains consists of five homologous G modules (LG1-5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin alpha5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalian expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These results suggest that the LG4-5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4-5 modules to heparin with K(D) values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated that N-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin alpha5 chain.
Highlights
The G domain of the laminin ␣ chains consists of five homologous G modules (LG1–5) and has been implicated in various biological functions
The five laminin ␣ chains share a large domain at their C-terminal region (G domain), which consists of five homologous G modules (LG1–5), each of about 200 amino acids
Heparin and heparan sulfate were found to partly inhibit cell binding to the tandem module, whereas keratan sulfate was noninhibitory (Fig. 4B). These results suggest that human submandibular gland (HSG) cell binding to the LG4 –5 tandem module of the laminin ␣5 chain is mediated by heparan sulfate proteoglycans
Summary
Construction of Expression Plasmids—Mouse cDNA was used as a template in polymerase chain reaction to amplify sequences encoding the laminin ␣5 LG4, LG5, and LG4 –5 tandem modules. Wells were blocked at room temperature (2 h) with 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl (Tris-buffered saline), 1% BSA and washed and incubated with recombinant proteins serially diluted in the same buffer overnight at 4 °C. For competition experiments, biotinylated heparin (0.16 M) (Celsus Laboratories, Inc., Cincinnati, OH) was mixed with synthetic peptides serially diluted in Tris-buffered saline, including 0.05% Tween and 1% BSA, and incubated in wells coated with recombinant laminin ␣5 tandem module (60 ng/50 l, 19 nM). Recombinant proteins, at different concentrations, were injected on the heparin-coated surface at 30 l/min (in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.005% surfactant P20, 25 °C), and the binding and dissociation were registered (2 min each) in a BIAcoreTM 1000 instrument (BIAcore, Inc.). For experiments examining the effects of heparin and heparin-like glycosaminoglycans, substrate-coated and preblocked wells were incubated with inhibitors for 30 min at 37 °C and washed before cells were added
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