High-affinity LPS binding domain(s) in recombinant factor C of a horseshoecrab neutralizes LPS-induced lethality

SSCrFCES is a biologically active, recombinant fragment of factor C, which is the endotoxin-sensitive serine protease of the LAL coagulation cascade. The similar to 38 kDa protein represents the LPS binding domain of factor C . A novel secretory signal directs the secretion of SSCrFCES into the cultu re supernatant of Drosophila cells, and hence it is readily purified. By di fferential ultrafiltration followed by preparative isoelectric membrane ele ctrophoresis, SSCrFCES was purified as an isoelectrically homogeneous and s table monomeric protein. The ability of SSCrFCES to bind lipid A was analyz ed using an ELISA-based assay as well as surface plasmon resonance. SSCrFCE S exhibits high positive cooperativity of binding to two or three lipid A m olecules, with a Hill's coefficient of 2.2. The 50% endotoxin-neutralizing concentration of SSCrFCES against 200 EU of endotoxin is similar to 0.069 m u M, suggesting that SSCrFCES is an effective inhibitor of LAL coagulation cascade. Although partially attenuated by human serum, as little as 1 mu M of,SSCrFCES inhibits the LPS-induced secretion of hTNF-alpha and hIL-8 by T HP-1 and human peripheral blood mononuclear cells with greater potency than polymyxin B. SSCrFCES is noncytotoxic, with a clearance rate of 4.7 ml/min . The L.D.(90) of SSCrFCES for LPS lethality is achieved at 2 mu M. These r esults demonstrate the endotoxin-neutralizing capability of SSCrFCES in vit ro and in vivo and its potential use for the treatment of endotoxin-induced septic shock.