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. 1991 Sep 1;174(3):649–655. doi: 10.1084/jem.174.3.649

Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils

PMCID: PMC2118937  PMID: 1875165

Abstract

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes (PMN) is a potent cytotoxin, specific for Gram-negative bacteria, that also inhibits endotoxin activity by neutralizing isolated bacterial lipopolysaccharides (LPS). We have previously shown that an isolated 25 kD N-terminal fragment of human BPI carries all the antibacterial activities of the parent 55-60 kD molecule. In this study we have compared the LPS-neutralizing activities of human holo-BPI, the N-terminal fragment and a 30 kD C- terminal fragment that we have now isolated. We show that the N- terminal fragment also has LPS-neutralizing activity as detected by inhibition (up to 95%) of (a) activation by LPS of procoagulant proteases in Limulus amebocyte lysates, (b) LPS "priming" of PMN, and (c) LPS-mediated production of tumor necrosis factor in whole human blood. Holo-BPI and the 25 kD fragment have similar neutralizing potency (in nanomolar range) in all assays toward "smooth" LPS from Escherichia coli O111:B4 and O55:B5 (possessing long chain polysaccharide or O-antigen), and "deep rough" LPS from Salmonella minnesota Re595 mutant (possessing no O-antigen). The C-terminal fragment of BPI is devoid of antibacterial activity when tested against BPI-sensitive E. coli J5, but does have endotoxin-neutralizing activity. This activity is weak relative to holo-BPI and the 25 kD N- terminal fragment in the Limulus and PMN-priming assay, but is comparable for inhibition of TNF production in whole blood. We conclude that the principal determinants for LPS recognition and neutralization, like those for antibacterial action, reside in the N-terminal half of the BPI molecule, but that sites within the C-terminal half can also contribute to BPI-LPS interaction once LPS is detached from the bacterial envelope.

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Selected References

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  1. Beutler B., Cerami A. Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature. 1986 Apr 17;320(6063):584–588. doi: 10.1038/320584a0. [DOI] [PubMed] [Google Scholar]
  2. Desch C. E., Kovach N. L., Present W., Broyles C., Harlan J. M. Production of human tumor necrosis factor from whole blood ex vivo. Lymphokine Res. 1989 Summer;8(2):141–146. [PubMed] [Google Scholar]
  3. Doerfler M. E., Danner R. L., Shelhamer J. H., Parrillo J. E. Bacterial lipopolysaccharides prime human neutrophils for enhanced production of leukotriene B4. J Clin Invest. 1989 Mar;83(3):970–977. doi: 10.1172/JCI113983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elsbach P., Weiss J., Franson R. C., Beckerdite-Quagliata S., Schneider A., Harris L. Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship. J Biol Chem. 1979 Nov 10;254(21):11000–11009. [PubMed] [Google Scholar]
  5. Gray P. W., Flaggs G., Leong S. R., Gumina R. J., Weiss J., Ooi C. E., Elsbach P. Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations. J Biol Chem. 1989 Jun 5;264(16):9505–9509. [PubMed] [Google Scholar]
  6. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  7. Mannion B. A., Kalatzis E. S., Weiss J., Elsbach P. Preferential binding of the neutrophil cytoplasmic granule-derived bactericidal/permeability increasing protein to target bacteria. Implications and use as a means of purification. J Immunol. 1989 Apr 15;142(8):2807–2812. [PubMed] [Google Scholar]
  8. Mannion B. A., Weiss J., Elsbach P. Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest. 1990 Mar;85(3):853–860. doi: 10.1172/JCI114512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Marom Z., Shelhamer J. H., Kaliner M. Human pulmonary macrophage-derived mucus secretagogue. J Exp Med. 1984 Mar 1;159(3):844–860. doi: 10.1084/jem.159.3.844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Marra M. N., Wilde C. G., Griffith J. E., Snable J. L., Scott R. W. Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity. J Immunol. 1990 Jan 15;144(2):662–666. [PubMed] [Google Scholar]
  11. Morrison D. C., Ryan J. L. Endotoxins and disease mechanisms. Annu Rev Med. 1987;38:417–432. doi: 10.1146/annurev.me.38.020187.002221. [DOI] [PubMed] [Google Scholar]
  12. Munford R. S., Andersen J. M., Dietschy J. M. Sites of tissue binding and uptake in vivo of bacterial lipopolysaccharide-high density lipoprotein complexes: studies in the rat and squirrel monkey. J Clin Invest. 1981 Dec;68(6):1503–1513. doi: 10.1172/JCI110404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Munford R. S., Hall C. L. Detoxification of bacterial lipopolysaccharides (endotoxins) by a human neutrophil enzyme. Science. 1986 Oct 10;234(4773):203–205. doi: 10.1126/science.3529396. [DOI] [PubMed] [Google Scholar]
  14. Munford R. S., Hall C. L., Lipton J. M., Dietschy J. M. Biological activity, lipoprotein-binding behavior, and in vivo disposition of extracted and native forms of Salmonella typhimurium lipopolysaccharides. J Clin Invest. 1982 Oct;70(4):877–888. doi: 10.1172/JCI110684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ooi C. E., Weiss J., Elsbach P., Frangione B., Mannion B. A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein. J Biol Chem. 1987 Nov 5;262(31):14891–14894. [PubMed] [Google Scholar]
  16. Raetz C. R., Dowhan W. Biosynthesis and function of phospholipids in Escherichia coli. J Biol Chem. 1990 Jan 25;265(3):1235–1238. [PubMed] [Google Scholar]
  17. Roeder D. J., Lei M. G., Morrison D. C. Endotoxic-lipopolysaccharide-specific binding proteins on lymphoid cells of various animal species: association with endotoxin susceptibility. Infect Immun. 1989 Apr;57(4):1054–1058. doi: 10.1128/iai.57.4.1054-1058.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schumann R. R., Leong S. R., Flaggs G. W., Gray P. W., Wright S. D., Mathison J. C., Tobias P. S., Ulevitch R. J. Structure and function of lipopolysaccharide binding protein. Science. 1990 Sep 21;249(4975):1429–1431. doi: 10.1126/science.2402637. [DOI] [PubMed] [Google Scholar]
  19. Shak S. Leukotriene B4 catabolism: quantitation of leukotriene B4 and its omega-oxidation products by reversed-phase high-performance liquid chromatography. Methods Enzymol. 1987;141:355–371. doi: 10.1016/0076-6879(87)41083-5. [DOI] [PubMed] [Google Scholar]
  20. Tobias P. S., Mathison J. C., Ulevitch R. J. A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis. J Biol Chem. 1988 Sep 25;263(27):13479–13481. [PubMed] [Google Scholar]
  21. Tobias P. S., Soldau K., Ulevitch R. J. Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein. J Biol Chem. 1989 Jun 25;264(18):10867–10871. [PubMed] [Google Scholar]
  22. Tobias P. S., Soldau K., Ulevitch R. J. Isolation of a lipopolysaccharide-binding acute phase reactant from rabbit serum. J Exp Med. 1986 Sep 1;164(3):777–793. doi: 10.1084/jem.164.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ulevitch R. J., Johnston A. R., Weinstein D. B. New function for high density lipoproteins. Isolation and characterization of a bacterial lipopolysaccharide-high density lipoprotein complex formed in rabbit plasma. J Clin Invest. 1981 Mar;67(3):827–837. doi: 10.1172/JCI110100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vosbeck K., Tobias P., Mueller H., Allen R. A., Arfors K. E., Ulevitch R. J., Sklar L. A. Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein. J Leukoc Biol. 1990 Feb;47(2):97–104. doi: 10.1002/jlb.47.2.97. [DOI] [PubMed] [Google Scholar]
  26. Weiss J., Beckerdite-Quagliata S., Elsbach P. Resistance of gram-negative bacteria to purified bactericidal leukocyte proteins: relation to binding and bacterial lipopolysaccharide structure. J Clin Invest. 1980 Mar;65(3):619–628. doi: 10.1172/JCI109707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weiss J., Elsbach P., Olsson I., Odeberg H. Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J Biol Chem. 1978 Apr 25;253(8):2664–2672. [PubMed] [Google Scholar]
  28. Weiss J., Muello K., Victor M., Elsbach P. The role of lipopolysaccharides in the action of the bactericidal/permeability-increasing neutrophil protein on the bacterial envelope. J Immunol. 1984 Jun;132(6):3109–3115. [PubMed] [Google Scholar]
  29. Weiss J., Olsson I. Cellular and subcellular localization of the bactericidal/permeability-increasing protein of neutrophils. Blood. 1987 Feb;69(2):652–659. [PubMed] [Google Scholar]
  30. Weiss J., Victor M., Elsbach P. Role of charge and hydrophobic interactions in the action of the bactericidal/permeability-increasing protein of neutrophils on gram-negative bacteria. J Clin Invest. 1983 Mar;71(3):540–549. doi: 10.1172/JCI110798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wright S. D., Tobias P. S., Ulevitch R. J., Ramos R. A. Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages. J Exp Med. 1989 Oct 1;170(4):1231–1241. doi: 10.1084/jem.170.4.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ziegler E. J., Fisher C. J., Jr, Sprung C. L., Straube R. C., Sadoff J. C., Foulke G. E., Wortel C. H., Fink M. P., Dellinger R. P., Teng N. N. Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1A Sepsis Study Group. N Engl J Med. 1991 Feb 14;324(7):429–436. doi: 10.1056/NEJM199102143240701. [DOI] [PubMed] [Google Scholar]

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