SC5b-7, SC5b-8 and SC5b-9 complexes of complement

Ultrastructure and localization of the S-protein (vitronectin) within the macromolecules

K. P. Preissner, E. R. Podack, H. J. Muller-Eberhard

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all anti-sera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)(n) and (C5b-8)(n), negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 < 27 S appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.

Original languageEnglish
Pages (from-to)69-75
Number of pages7
JournalEuropean Journal of Immunology
Volume19
Issue number1
StatePublished - Jan 1 1989
Externally publishedYes

Fingerprint

Vitronectin
Protein S
Complement Inactivating Agents
Electrons
Complement Membrane Attack Complex
Avidin
Ultracentrifugation
Sodium Dodecyl Sulfate
Polymerization
Gold
Sucrose
Heparin
Polyacrylamide Gel Electrophoresis
Electron Microscopy
Binding Sites
complement C5b-7 complex
SC5b-8
SC5b-9 protein complex
Membranes
Serum

ASJC Scopus subject areas

  • Immunology

Cite this

SC5b-7, SC5b-8 and SC5b-9 complexes of complement : Ultrastructure and localization of the S-protein (vitronectin) within the macromolecules. / Preissner, K. P.; Podack, E. R.; Muller-Eberhard, H. J.

In: European Journal of Immunology, Vol. 19, No. 1, 01.01.1989, p. 69-75.

Research output: Contribution to journalArticle

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abstract = "Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70{\%} yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all anti-sera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)(n) and (C5b-8)(n), negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 < 27 S appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.",
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