Recombinant glycoproteins that inhibit complement activation and also bindthe 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 classi cal and alternative complement pathways in vitro and in vivo, A truncated v ersion of sCR1 lacking the long homologous repeat-A domain (LHR-A) containi ng the C4b binding site has similarly been expressed and designated sCR1[de sLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the altern ative complement pathway in vitro and to function in vivo, In this study, s CR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alp ha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the si alyl-Lewis(x) (sLe(x)) tetrasaccharide (NeuNAc alpha 2-3Gal beta 1-4 (Fuc a lpha 1-3)GlcNAc) during post-translational glycosylation, The resulting gly coproteins, designated sCRlsLe(x) and sCR1[desLHR-A]sLe(x), respectively, r etained 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 m ol of sLe(x)/mol of glycoprotein, respectively. sLe(x) is a carbohydrate li gand for the selectin adhesion molecules, sCR1sLe(x) was shown to specifica lly 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 endo thelial 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[desL HR-A]sLe(x) have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesi on in vitro.