Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules

Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo, A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo, In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewis(x) (sLe(x)) tetrasaccharide (NeuNAc alpha 2-3Gal beta 1-4 (Fuc alpha 1-3)GlcNAc) during post-translational glycosylation, The resulting glycoproteins, designated sCRlsLe(x) and sCR1[desLHR-A]sLe(x), respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCRlsLe(x) and sCR1[desLHR-A]sLe(x) indicated an average incorporation of 10 and 8 mol of sLe(x)/mol of glycoprotein, respectively. sLe(x) is a carbohydrate ligand for the selectin adhesion molecules, sCR1sLe(x) was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLe(x) inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLe(x) inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG, sCR1sLe(x) and sCR1[desLHR-A]sLe(x) have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.