Purification and characterization of an endotoxin-binding protein with protease inhibitory activity from Limulus amebocytes.
Using a lipopolysaccharide affinity column and ion exchange chromatography, a 12-kDa protein has been purified from Limulus amebocytes. In solid phase binding assays, the radiolabeled protein binds specifically to lipopolysaccharide (LPS) with a Kd value on the order of 10(-7) M. A cDNA coding for this protein has been isolated and sequenced. The amino acid sequence deduced from the cDNA indicates that this protein shares no sequence homology with LPS-binding proteins isolated from different species of vertebrates (Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., and Ulevitch, R. J. (1990) Science 249, 1429-1431) and invertebrates (Aketagawa, J., Miyata, T., Ohtsubo, S., Nakamura, T., Morita, T., Hayashida, H., Miyata, T., Iwanaga, S., Takao, T., and Shimonishi, Y. (1986) J. Biol. Chem. 261, 7357-7365). The binding to LPS can be displaced by the unlabeled 12-kDa protein, polymyxin B, lipid A, and to a lesser extent by D-glucosamine. In whole cell binding assays, the 12-kDa protein has also been shown to bind to Escherichia coli. Using both [14C]casein and a synthetic substrate, the protein has been shown to inhibit the proteolytic activity of trypsin, with an IC50 of approximately 10(-7) M. In the presence of LPS, the antitryptic acitivity of the Limulus endotoxin-binding protein-protease inhibitor remains unaffected. The protein is a major component of the cytoplasmic proteins (1%). Immunocytochemical analysis reveals that this protein exists in the secretory granules of the amebocytes where enzymes and substrates for the clotting cascade reside. Based on the unusual dual functional properties, the newly isolated protein was named a "Limulus endotoxin-binding protein-protease inhibitor" (LEBP-PI).
Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, Maryland 20892.
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Minetti
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Lin
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Cislo
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Liu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015550:Cell Degranulation; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004220:Disulfides; D004731:Endotoxins / Q000378:metabolism*; D006737:Horseshoe Crabs / Q000166:cytology / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011112:Polymyxin B / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification*
D004220:Disulfides; D004731:Endotoxins; D008070:Lipopolysaccharides; D014361:Trypsin Inhibitors; D004247:DNA; D011112:Polymyxin B