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. 1983 Sep 1;97(3):618–626. doi: 10.1083/jcb.97.3.618

Freeze-fracture analysis of the membrane lesion of human complement

PMCID: PMC2112567  PMID: 6309866

Abstract

The structure and membrane insertion of the human C5b-9(m) complex, generated by lysis of antibody-coated sheep erythrocytes with whole human serum under conditions where high numbers of classical ring- shaped lesions form, were studied in single and complementary freeze- fracture replicas prepared by unidirectional and rotary shadowing. The intramembrane portion of the C5b-9(m) cylinder was seen on EF-faces as an elevated, circular structure. In nonetched fractures it appeared as a solid stub; in etched fractures a central pit confirmed the existence of a central, water-filled pore in the molecule. Complementary replicas showed that each EF-face ring corresponded to a hole in the lipid plateau of the PF-face. Etched fractures of proteolytically stripped membranes revealed the extramembrane annulus of the C5b-9(m) cylinder on ES-faces and putative internal openings on PS-faces. Allowing for the measured thickness of deposited Pt/C, the dimensions of EF-face rings and ES-face annuli conformed to anticipations derived from negatively stained preparations. Our results support the concept that the hollow cylindrical C5b-9(m) complex penetrates into the inner leaflet of the target erythrocyte membrane bilayer, forming a stable transmembrane protein channel.

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Selected References

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  1. BORSOS T., DOURMASHKIN R. R., HUMPHREY J. H. LESIONS IN ERYTHROCYTE MEMBRANES CAUSED BY IMMUNE HAEMOLYSIS. Nature. 1964 Apr 18;202:251–252. doi: 10.1038/202251a0. [DOI] [PubMed] [Google Scholar]
  2. Bhakdi S., Bjerrum O. J., Bhakdi-Lehnen B., Tranum-Jensen J. Complement lysis: evidence for an amphiphilic nature of the terminal membrane C5b-9 complex of human complement. J Immunol. 1978 Dec;121(6):2526–2532. [PubMed] [Google Scholar]
  3. Bhakdi S., Ey P., Bhakdi-Lehnen B. Isolation of the terminal complement complex from target sheep erythrocyte membranes. Biochim Biophys Acta. 1976 Feb 6;419(3):445–457. doi: 10.1016/0005-2736(76)90258-3. [DOI] [PubMed] [Google Scholar]
  4. Bhakdi S., Speth V., Knüfermann H., Wallach D. F., Fischer H. Complement-induced changes in the core structure of sheep erythrocyte membranes: a study by freeze-etch electron microscopy. Biochim Biophys Acta. 1974 Aug 9;356(3):300–308. doi: 10.1016/0005-2736(74)90270-3. [DOI] [PubMed] [Google Scholar]
  5. Bhakdi S., Tranum-Jensen J. Evidence for a two-domain structure of the terminal membrane C5b-9 complex of human complement. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5872–5876. doi: 10.1073/pnas.76.11.5872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhakdi S., Tranum-Jensen J., Klump O. The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel. J Immunol. 1980 May;124(5):2451–2457. [PubMed] [Google Scholar]
  7. Bhakdi S., Tranum-Jensen J. Molecular nature of the complement lesion. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5655–5659. doi: 10.1073/pnas.75.11.5655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bhakdi S., Tranum-Jensen J. Molecular weight of the membrane C5b-9 complex of human complement: characterization of the terminal complex as a C5b-9 monomer. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1818–1822. doi: 10.1073/pnas.78.3.1818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bhakdi S., Tranum-Jensen J. Re-incorporation of the terminal C5b-9 complement complex into lipid bilayers: formation and stability of reconstituted liposomes. Immunology. 1980 Nov;41(3):737–742. [PMC free article] [PubMed] [Google Scholar]
  10. Biesecker G., Podack E. R., Halverson C. A., Müller-Eberhard H. J. C5b-9 dimer: isolation from complement lysed cells and ultrastructural identification with complement-dependent membrane lesions. J Exp Med. 1979 Feb 1;149(2):448–458. doi: 10.1084/jem.149.2.448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Boyle M. D., Gee A. P., Borsos T. Studies on the terminal stages of immune hemolysis. VI. Osmotic blockers of differing Stokes' radii detect complement-induced transmembrane channels of differing size. J Immunol. 1979 Jul;123(1):77–82. [PubMed] [Google Scholar]
  12. Branton D., Bullivant S., Gilula N. B., Karnovsky M. J., Moor H., Mühlethaler K., Northcote D. H., Packer L., Satir B., Satir P. Freeze-etching nomenclature. Science. 1975 Oct 3;190(4209):54–56. doi: 10.1126/science.1166299. [DOI] [PubMed] [Google Scholar]
  13. Dalmasso A. P., Benson B. A. Lesions of different functional size produced by human and guinea pig complement in sheep red cell membranes. J Immunol. 1981 Dec;127(6):2214–2218. [PubMed] [Google Scholar]
  14. Dourmashkin R. R. The structural events associated with the attachment of complement components to cell membranes in reactive lysis. Immunology. 1978 Aug;35(2):205–212. [PMC free article] [PubMed] [Google Scholar]
  15. Esser A. F., Kolb W. P., Podack E. R., Müller-Eberhard H. J. Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1410–1414. doi: 10.1073/pnas.76.3.1410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. GREEN H., BARROW P., GOLDBERG B. Effect of antibody and complement on permeability control in ascites tumor cells and erythrocytes. J Exp Med. 1959 Nov 1;110:699–713. doi: 10.1084/jem.110.5.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gross H., Kuebler O., Bas E., Moor H. Decoration of specific sites on freeze-fractured membranes. J Cell Biol. 1978 Dec;79(3):646–656. doi: 10.1083/jcb.79.3.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hammer C. H., Nicholson A., Mayer M. M. On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte membranes. Proc Natl Acad Sci U S A. 1975 Dec;72(12):5076–5080. doi: 10.1073/pnas.72.12.5076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hammer C. H., Shin M. L., Abramovitz A. S., Mayer M. M. On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes. J Immunol. 1977 Jul;119(1):1–8. [PubMed] [Google Scholar]
  20. Hu V. W., Esser A. F., Podack E. R., Wisnieski B. J. The membrane attack mechanism of complement: photolabeling reveals insertion of terminal proteins into target membrane. J Immunol. 1981 Jul;127(1):380–386. [PubMed] [Google Scholar]
  21. Humphrey J. H., Dourmashkin R. R. The lesions in cell membranes caused by complement. Adv Immunol. 1969;11:75–115. doi: 10.1016/s0065-2776(08)60478-2. [DOI] [PubMed] [Google Scholar]
  22. Iles G. H., Seeman P., Naylor D., Cinader B. Membrane lesions in immune lysis: surface rings, globule aggregates and transient openings. J Cell Biol. 1973 Feb;56(2):528–539. doi: 10.1083/jcb.56.2.528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kolb W. P., Haxby J. A., Arroyave C. M., Müller-Eberhard H. J. Molecular analysis of the membrane attack mechanism of complement. J Exp Med. 1972 Mar 1;135(3):549–566. doi: 10.1084/jem.135.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kolb W. P., Müller-Eberhard H. J. Mode of action of human C9: adsorption of multiple C9 molecules to cell-bound C8. J Immunol. 1974 Aug;113(2):479–488. [PubMed] [Google Scholar]
  25. Lachmann P. J., Thompson R. A. Reactive lysis: the complement-mediated lysis of unsensitized cells. II. The characterization of activated reactor as C56 and the participation of C8 and C9. J Exp Med. 1970 Apr 1;131(4):643–657. doi: 10.1084/jem.131.4.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li C. K., Levine R. P. Molecular transport via the functional complement lesion. Mol Immunol. 1980 Dec;17(12):1465–1474. doi: 10.1016/0161-5890(80)90172-8. [DOI] [PubMed] [Google Scholar]
  27. Mayer M. M. Complement, past and present. Harvey Lect. 1978;72:139–193. [PubMed] [Google Scholar]
  28. Mayer M. M. Mechanism of cytolysis by complement. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2954–2958. doi: 10.1073/pnas.69.10.2954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Michaels D. W., Abramovitz A. S., Hammer C. H., Mayer M. M. Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2852–2856. doi: 10.1073/pnas.73.8.2852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Müller-Eberhard H. J. Complement. Annu Rev Biochem. 1975;44:697–724. doi: 10.1146/annurev.bi.44.070175.003405. [DOI] [PubMed] [Google Scholar]
  31. Müller M., Meister N., Moor H. Freezing in a propane jet and its application in freeze-fracturing. Mikroskopie. 1980 Sep;36(5-6):129–140. [PubMed] [Google Scholar]
  32. Podack E. R., Biesecker G., Müller-Eberhard H. J. Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins. Proc Natl Acad Sci U S A. 1979 Feb;76(2):897–901. doi: 10.1073/pnas.76.2.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Podack E. R., Müller-Eberhard H. J., Horst H., Hoppe W. Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants. J Immunol. 1982 May;128(5):2353–2357. [PubMed] [Google Scholar]
  34. Podack E. R., Stoffel W., Esser A. F., Müller-Eberhard H. J. Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4544–4548. doi: 10.1073/pnas.78.7.4544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Podack E. R., Tschoop J., Müller-Eberhard H. J. Molecular organization of C9 within the membrane attack complex of complement. Induction of circular C9 polymerization by the C5b-8 assembly. J Exp Med. 1982 Jul 1;156(1):268–282. doi: 10.1084/jem.156.1.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Podack E. R., Tschopp J. Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement. Proc Natl Acad Sci U S A. 1982 Jan;79(2):574–578. doi: 10.1073/pnas.79.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. SEARS D. A., WEED R. I., SWISHER S. N. DIFFERENCES IN THE MECHANISM OF IN VITRO IMMUNE HEMOLYSIS RELATED TO ANTIBODY SPECIFICITY. J Clin Invest. 1964 May;43:975–985. doi: 10.1172/JCI104983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shin M. L., Paznekas W. A., Abramovitz A. S., Mayer M. M. On the mechanism of membrane damage by C: exposure of hydrophobic sites on activated C proteins. J Immunol. 1977 Oct;119(4):1358–1364. [PubMed] [Google Scholar]
  39. Sims P. J., Lauf P. K. Analysis of solute diffusion across the C5b-9 membrane lesion of complement: evidence that individual C5b-9 complexes do not function as discrete, uniform pores. J Immunol. 1980 Dec;125(6):2617–2625. [PubMed] [Google Scholar]
  40. Sims P. J., Lauf P. K. Steady-state analysis of tracer exchange across the C5b-9 complement lesion in a biological membrane. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5669–5673. doi: 10.1073/pnas.75.11.5669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sleytr U. B., Robards A. W. Understanding the artefact problem in freeze-fracture replication: a review. J Microsc. 1982 Apr;126(Pt 1):101–122. doi: 10.1111/j.1365-2818.1982.tb00361.x. [DOI] [PubMed] [Google Scholar]
  42. Tranum-Jensen J., Bhakdi S., Bhakdi-Lehnen B., Bjerrum O. J., Speth V. Complement lysis: the ultrastructure and orientation of the C5b-9 complex on target sheep erythrocyte membranes. Scand J Immunol. 1978;7(1):45–46. doi: 10.1111/j.1365-3083.1978.tb00425.x. [DOI] [PubMed] [Google Scholar]
  43. Tschopp J., Müller-Eberhard H. J., Podack E. R. Formation of transmembrane tubules by spontaneous polymerization of the hydrophilic complement protein C9. Nature. 1982 Aug 5;298(5874):534–538. doi: 10.1038/298534a0. [DOI] [PubMed] [Google Scholar]
  44. Ware C. F., Wetsel R. A., Kolb W. P. Physicochemical characterization of fluid phase (SC5b-9) and membrane derived (MC5b-9) attack complexes of human complement purified by immunoadsorbent affinity chromatography or selective detergent extraction. Mol Immunol. 1981 Jun;18(6):521–531. doi: 10.1016/0161-5890(81)90130-9. [DOI] [PubMed] [Google Scholar]

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