High-affinity LPS binding domain(s) in recombinant factor C of a horseshoe crab 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 approximately 38 kDa protein represents the LPS binding domain of factor C. A novel secretory signal directs the secretion of SSCrFCES into the culture supernatant of Drosophila cells, and hence it is readily purified. By differential ultrafiltration followed by preparative isoelectric membrane electrophoresis, SSCrFCES was purified as an isoelectrically homogeneous and stable monomeric protein. The ability of SSCrFCES to bind lipid A was analyzed using an ELISA-based assay as well as surface plasmon resonance. SSCrFCES exhibits high positive cooperativity of binding to two or three lipid A molecules, with a Hill's coefficient of 2.2. The 50% endotoxin-neutralizing concentration of SSCrFCES against 200 EU of endotoxin is approximately 0.069 microM, suggesting that SSCrFCES is an effective inhibitor of LAL coagulation cascade. Although partially attenuated by human serum, as little as 1 microM of SSCrFCES inhibits the LPS-induced secretion of hTNF-alpha and hIL-8 by THP-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 microM. These results demonstrate the endotoxin-neutralizing capability of SSCrFCES in vitro and in vivo and its potential use for the treatment of endotoxin-induced septic shock.
Tan
N S
NS
Department of Biological Sciences and. Department of Microbiology, National University of Singapore. dbsdjl@nus.edu.sg
Ho
B
B
Ding
J L
JL
D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000295:instrumentation; D017931:DNA Primers; D004330:Drosophila; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D006737:Horseshoe Crabs; D006801:Humans; D016209:Interleukin-8 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007525:Isoelectric Focusing; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000633:toxicity; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis
D060829:Arthropod Proteins; D017931:DNA Primers; D004792:Enzyme Precursors; D016209:Interleukin-8; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C