Generation of recombinant fragments of CD11b expressing the functional beta-glucan-binding lectin site of CR3 (CD11b/CD18)

CR3 (Mac-1; alpha(M)beta(2) integrin) functions as both a receptor for the opsonic iC3b fragment of C3 triggering phagocytosis or cytotoxicity and an adhesion molecule mediating leukocyte diapedesis, Recent reports have sugge sted that a CR3 lectin site may be required for both cytotoxic responses an d adhesion, Cytotoxic responses require dual recognition of iC3b via the I domain of CD11b and specific microbial surface polysaccharides (e.g., beta- glucan) via a separate lectin site. Likewise, adhesion requires a lectin-de pendent membrane complex between CR3 and CD87, To characterize the lectin s ite further, a recombinant baculovirus (rBv) system was developed that allo wed high level expression of rCD11b on membranes and in the cytoplasm of Sf 21 insect cells. Six rBv were generated that contained truncated cDNA encod ing various CD11b domains, Immunoblotting of rBv-infected Sf21 cells showed that some native epitopes were expressed by five of six rCD11b fragments, Lectin activity of rCD11b proteins was evaluated by both how cytometry with beta-glucan-FITC and radioactive binding assays with [I-125]beta-glucan. S f21 cells expressing rCD11b that included the C-terminal region, with or wi thout the I-domain, exhibited lectin activity that was inhibited by unlabel ed beta-glucan or anti-CR3 mAbs. The smallest rCD11b fragment exhibiting le ctin activity included the C-terminus and part of the divalent cation bindi ng region. The beta-glucan binding affinities of the three C-terminal regio n-containing rCD11bs expressed on Sf21 cell membranes were not significantl y different from each other and were similar to that of neutrophil CR3, The se data suggest that the lectin site may be located entirely within CD11b, although lectin site-dependent signaling through CD18 probably occurs with the heterodimer.