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. 1995 Jul;63(7):2576–2580. doi: 10.1128/iai.63.7.2576-2580.1995

Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein.

J Schletter 1, H Brade 1, L Brade 1, C Krüger 1, H Loppnow 1, S Kusumoto 1, E T Rietschel 1, H D Flad 1, A J Ulmer 1
PMCID: PMC173345  PMID: 7540597

Abstract

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.

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Selected References

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  1. Arditi M., Zhou J., Dorio R., Rong G. W., Goyert S. M., Kim K. S. Endotoxin-mediated endothelial cell injury and activation: role of soluble CD14. Infect Immun. 1993 Aug;61(8):3149–3156. doi: 10.1128/iai.61.8.3149-3156.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bazil V., Horejsí V., Baudys M., Kristofová H., Strominger J. L., Kostka W., Hilgert I. Biochemical characterization of a soluble form of the 53-kDa monocyte surface antigen. Eur J Immunol. 1986 Dec;16(12):1583–1589. doi: 10.1002/eji.1830161218. [DOI] [PubMed] [Google Scholar]
  3. Brade L., Brade H. Characterization of two different antibody specificities recognizing distinct antigenic determinants in free lipid A of Escherichia coli. Infect Immun. 1985 Jun;48(3):776–781. doi: 10.1128/iai.48.3.776-781.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  6. Couturier C., Jahns G., Kazatchkine M. D., Haeffner-Cavaillon N. Membrane molecules which trigger the production of interleukin-1 and tumor necrosis factor-alpha by lipopolysaccharide-stimulated human monocytes. Eur J Immunol. 1992 Jun;22(6):1461–1466. doi: 10.1002/eji.1830220619. [DOI] [PubMed] [Google Scholar]
  7. Dentener M. A., Bazil V., Von Asmuth E. J., Ceska M., Buurman W. A. Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages. J Immunol. 1993 Apr 1;150(7):2885–2891. [PubMed] [Google Scholar]
  8. Di Padova F. E., Brade H., Barclay G. R., Poxton I. R., Liehl E., Schuetze E., Kocher H. P., Ramsay G., Schreier M. H., McClelland D. B. A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides. Infect Immun. 1993 Sep;61(9):3863–3872. doi: 10.1128/iai.61.9.3863-3872.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dziarski R. Cell-bound albumin is the 70-kDa peptidoglycan-, lipopolysaccharide-, and lipoteichoic acid-binding protein on lymphocytes and macrophages. J Biol Chem. 1994 Aug 12;269(32):20431–20436. [PubMed] [Google Scholar]
  10. Frey E. A., Miller D. S., Jahr T. G., Sundan A., Bazil V., Espevik T., Finlay B. B., Wright S. D. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med. 1992 Dec 1;176(6):1665–1671. doi: 10.1084/jem.176.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galanos C., Lüderitz O., Rietschel E. T., Westphal O., Brade H., Brade L., Freudenberg M., Schade U., Imoto M., Yoshimura H. Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur J Biochem. 1985 Apr 1;148(1):1–5. doi: 10.1111/j.1432-1033.1985.tb08798.x. [DOI] [PubMed] [Google Scholar]
  12. Galanos C., Lüderitz O., Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem. 1969 Jun;9(2):245–249. doi: 10.1111/j.1432-1033.1969.tb00601.x. [DOI] [PubMed] [Google Scholar]
  13. Hailman E., Lichenstein H. S., Wurfel M. M., Miller D. S., Johnson D. A., Kelley M., Busse L. A., Zukowski M. M., Wright S. D. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med. 1994 Jan 1;179(1):269–277. doi: 10.1084/jem.179.1.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Halling J. L., Hamill D. R., Lei M. G., Morrison D. C. Identification and characterization of lipopolysaccharide-binding proteins on human peripheral blood cell populations. Infect Immun. 1992 Mar;60(3):845–852. doi: 10.1128/iai.60.3.845-852.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haziot A., Rong G. W., Silver J., Goyert S. M. Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide. J Immunol. 1993 Aug 1;151(3):1500–1507. [PubMed] [Google Scholar]
  16. Jaffe E. A., Nachman R. L., Becker C. G., Minick C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–2756. doi: 10.1172/JCI107470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jesaitis A. J., Naemura J. R., Painter R. G., Sklar L. A., Cochrane C. G. Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes. Biochim Biophys Acta. 1982 Dec 17;719(3):556–568. doi: 10.1016/0304-4165(82)90246-x. [DOI] [PubMed] [Google Scholar]
  18. Kirikae T., Inada K., Hirata M., Yoshida M., Kondo S., Hisatsune K. Identification of Re lipopolysaccharide-binding protein on murine erythrocyte membrane. Microbiol Immunol. 1988;32(1):33–44. doi: 10.1111/j.1348-0421.1988.tb01363.x. [DOI] [PubMed] [Google Scholar]
  19. Kirikae T., Kirikae F., Schade F. U., Yoshida M., Kondo S., Hisatsune K., Nishikawa S., Rietschel E. T. Detection of lipopolysaccharide-binding proteins on membranes of murine lymphocyte and macrophage-like cell lines. FEMS Microbiol Immunol. 1991 Nov;3(6):327–336. doi: 10.1111/j.1574-6968.1991.tb04257.x. [DOI] [PubMed] [Google Scholar]
  20. Kirkland T. N., Virca G. D., Kuus-Reichel T., Multer F. K., Kim S. Y., Ulevitch R. J., Tobias P. S. Identification of lipopolysaccharide-binding proteins in 70Z/3 cells by photoaffinity cross-linking. J Biol Chem. 1990 Jun 5;265(16):9520–9525. [PubMed] [Google Scholar]
  21. Klempner M. S., Mikkelsen R. B., Corfman D. H., André-Schwartz J. Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation. J Cell Biol. 1980 Jul;86(1):21–28. doi: 10.1083/jcb.86.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuhn H. M., Brade L., Appelmelk B. J., Kusumoto S., Rietschel E. T., Brade H. Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A. Infect Immun. 1992 Jun;60(6):2201–2210. doi: 10.1128/iai.60.6.2201-2210.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lee J. D., Kravchenko V., Kirkland T. N., Han J., Mackman N., Moriarty A., Leturcq D., Tobias P. S., Ulevitch R. J. Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9930–9934. doi: 10.1073/pnas.90.21.9930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levy O., Weiss J., Zarember K., Ooi C. E., Elsbach P. Antibacterial 15-kDa protein isoforms (p15s) are members of a novel family of leukocyte proteins. J Biol Chem. 1993 Mar 15;268(8):6058–6063. [PubMed] [Google Scholar]
  26. Loppnow H., Libby P. Adult human vascular endothelial cells express the IL6 gene differentially in response to LPS or IL1. Cell Immunol. 1989 Sep;122(2):493–503. doi: 10.1016/0008-8749(89)90095-6. [DOI] [PubMed] [Google Scholar]
  27. Loppnow H., Libby P., Freudenberg M., Krauss J. H., Weckesser J., Mayer H. Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS. Infect Immun. 1990 Nov;58(11):3743–3750. doi: 10.1128/iai.58.11.3743-3750.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Parent J. B. Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides. J Biol Chem. 1990 Feb 25;265(6):3455–3461. [PubMed] [Google Scholar]
  30. Pugin J., Schürer-Maly C. C., Leturcq D., Moriarty A., Ulevitch R. J., Tobias P. S. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2744–2748. doi: 10.1073/pnas.90.7.2744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Read M. A., Cordle S. R., Veach R. A., Carlisle C. D., Hawiger J. Cell-free pool of CD14 mediates activation of transcription factor NF-kappa B by lipopolysaccharide in human endothelial cells. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9887–9891. doi: 10.1073/pnas.90.21.9887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rietschel E. T., Brade H. Bacterial endotoxins. Sci Am. 1992 Aug;267(2):54–61. doi: 10.1038/scientificamerican0892-54. [DOI] [PubMed] [Google Scholar]
  33. Rietschel E. T., Kirikae T., Schade F. U., Mamat U., Schmidt G., Loppnow H., Ulmer A. J., Zähringer U., Seydel U., Di Padova F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994 Feb;8(2):217–225. doi: 10.1096/fasebj.8.2.8119492. [DOI] [PubMed] [Google Scholar]
  34. Schletter J., Krüger C., Lottspeich F., Schütt C. Improved method for preparation of lipopolysaccharide-binding protein from human serum by electrophoretic and chromatographic separation techniques. J Chromatogr B Biomed Appl. 1994 Mar 18;654(1):25–34. doi: 10.1016/0378-4347(93)e0443-t. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Stefanová I., Horejsí V. Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules. J Immunol. 1991 Sep 1;147(5):1587–1592. [PubMed] [Google Scholar]
  37. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ulevitch R. J., Tobias P. S. Recognition of endotoxin by cells leading to transmembrane signaling. Curr Opin Immunol. 1994 Feb;6(1):125–130. doi: 10.1016/0952-7915(94)90043-4. [DOI] [PubMed] [Google Scholar]
  39. Weersink A. J., van Kessel K. P., van den Tol M. E., van Strijp J. A., Torensma R., Verhoef J., Elsbach P., Weiss J. Human granulocytes express a 55-kDa lipopolysaccharide-binding protein on the cell surface that is identical to the bactericidal/permeability-increasing protein. J Immunol. 1993 Jan 1;150(1):253–263. [PubMed] [Google Scholar]
  40. Weinstein S. L., June C. H., DeFranco A. L. Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14. J Immunol. 1993 Oct 1;151(7):3829–3838. [PubMed] [Google Scholar]
  41. Wright S. D., Jong M. T. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. J Exp Med. 1986 Dec 1;164(6):1876–1888. doi: 10.1084/jem.164.6.1876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wright S. D. Multiple receptors for endotoxin. Curr Opin Immunol. 1991 Feb;3(1):83–90. doi: 10.1016/0952-7915(91)90082-c. [DOI] [PubMed] [Google Scholar]
  43. Wright S. D., Ramos R. A., Patel M., Miller D. S. Septin: a factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes. J Exp Med. 1992 Sep 1;176(3):719–727. doi: 10.1084/jem.176.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wright S. D., Ramos R. A., Tobias P. S., Ulevitch R. J., Mathison J. C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990 Sep 21;249(4975):1431–1433. doi: 10.1126/science.1698311. [DOI] [PubMed] [Google Scholar]
  45. Ziegler-Heitbrock H. W., Thiel E., Fütterer A., Herzog V., Wirtz A., Riethmüller G. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int J Cancer. 1988 Mar 15;41(3):456–461. doi: 10.1002/ijc.2910410324. [DOI] [PubMed] [Google Scholar]

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