Expression of an extracellular domain of soluble leukocyte integrin beta-2 subunit (sCD18) and its functional implication

Abstract

Leukocyte beta-2 integrins are heterodimeric glycoproteins, composed of a common beta chain (CD18) noncovalently associated with three distinct alpha-chain subnnits (CDlla, CDllb, CDllc). We have successfully truncated the extracellular domain of human integrin beta-2 chain by site-directed mut agenesis, and expressed the soluble CD18 subunit without the association of integrin alpha chain. Transfection of COS cells with the truncated beta 2cDNA construct produced in culture supernatant, but not on the cell surface, a secretory protein with an apparent M.W. ∼80kD as revealed by SDS-PAGE. Production of the sCDlS protein in a CHO cell expression system gave milligram quantities, and the average yield in bulk culture was approximately 5-10 mg-/L as estimated by a sandwich ELISA assay. The sCDlS protein was purified by affinity chromatography using an anti-CD18 mAb. Eight anti-CD18 mAbs were used in an ELISA to assess the structural integrity of the soluble beta chain - three mAbs bound equally well to the sCDlS and the wild-type CDllc/CDl8, whereas four other mAbs bound stronger to the native form. Interestingly, one mAb reacted strongly with the recombinant sCDlS. These binding data suggested that the isolated sCDlS subunit is structurally similar to the native heterodimeric form, but its epitope pattern was somehow modified. Furthermore, the sCDlS protein was shown to be able to partially inhibit the PMA-induced neutrophil aggregation.