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The Role of Properdin in C5 Convertase Activity and C5b-9 Formation in the Complement Alternative Pathway

Marloes A. H. M. Michels, Rianne J. F. Maas, Thea J. A. M. van der Velden, Nicole C. A. J. van de Kar, Lambertus P. W. J. van den Heuvel, Elena B. Volokhina and on behalf of the COMBAT Consortium
J Immunol November 15, 2021, 207 (10) 2465-2472; DOI: https://doi.org/10.4049/jimmunol.2100238
Marloes A. H. M. Michels
*Radboud Institute for Molecular Life Sciences, Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, the Netherlands;
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  • ORCID record for Marloes A. H. M. Michels
Rianne J. F. Maas
†Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands;
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Thea J. A. M. van der Velden
*Radboud Institute for Molecular Life Sciences, Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, the Netherlands;
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Nicole C. A. J. van de Kar
*Radboud Institute for Molecular Life Sciences, Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, the Netherlands;
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Lambertus P. W. J. van den Heuvel
*Radboud Institute for Molecular Life Sciences, Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, the Netherlands;
†Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands;
‡Department of Pediatrics/Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium; and
§Department of Development and Regeneration, University Hospitals Leuven, Leuven, Belgium
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Elena B. Volokhina
*Radboud Institute for Molecular Life Sciences, Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, the Netherlands;
†Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands;
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Key Points

  • Properdin is required for AP-mediated C5 convertase activity and C5b-9 formation.

  • Properdin induces a switch in convertase specificity toward C5.

  • Properdin has a crucial role in AP activation and total complement activation.

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Abstract

The complement system is an important part of innate immunity. Complement activation leads to formation of convertase enzymes, switch of their specificity from C3 to C5 cleavage, and generation of lytic membrane attack complexes (C5b-9) on surfaces of pathogens. Most C5 cleavage occurs via the complement alternative pathway (AP). The regulator properdin promotes generation and stabilization of AP convertases. However, its role in C5 activation is not yet understood. In this work, we showed that serum properdin is essential for LPS- and zymosan-induced C5b-9 generation and C5b-9–mediated lysis of rabbit erythrocytes. Furthermore, we demonstrated its essential role in C5 cleavage by AP convertases. To this end, we developed a hemolytic assay in which AP convertases were generated on rabbit erythrocytes by using properdin-depleted serum in the presence of C5 inhibitor (step 1), followed by washing and addition of purified C5–C9 components to allow C5b-9 formation (step 2). In this assay, addition of purified properdin to properdin-depleted serum during convertase formation (step 1) was required to restore C5 cleavage and C5b-9–mediated hemolysis. Importantly, C5 convertase activity was also fully restored when properdin was added together with C5b-9 components (step 2), thus after convertase formation. Moreover, with C3-depleted serum, not capable of forming new convertases but containing properdin, in step 2 of the assay, again full C5b-9 formation was observed and blocked by addition of properdin inhibitor Salp20. Thus, properdin is essential for the convertase specificity switch toward C5, and this function is independent of properdin’s role in new convertase formation.

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Footnotes

  • ↵2 The members of the COMBAT Consortium are listed at the end of the article.

  • This work was supported by a grant from the Dutch Kidney Foundation (13OCA27 COMBAT Consortium).

  • M.A.H.M.M., L.P.W.J.v.d.H., and E.B.V. designed the experiments. N.C.A.J.v.d.K., L.P.W.J.v.d.H., and E.B.V. supervised the work. M.A.H.M.M., R.J.F.M., and T.J.A.M.v.d.V. performed the experiments. All authors analyzed the data. M.A.H.M.M. and E.B.V. drafted the manuscript, and all authors critically revised and approved the manuscript.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article

    AP
    alternative pathway
    ΔC3-NHS
    C3-depleted NHS
    CP
    classical pathway
    DAF/CD55
    decay-accelerating factor
    FB
    factor B
    FD
    factor D
    FH
    factor H
    FI
    factor I
    GPS
    guinea pig serum
    HI-NHS
    heat-inactivated NHS
    LP
    lectin pathway
    Mg-EGTA
    magnesium-EGTA
    NHS
    normal human serum
    PBST
    PBS supplemented with 0.05% Tween‐20
    ΔP-NHS
    properdin-depleted NHS
    TSR
    thrombospondin type I repeat

  • Received March 11, 2021.
  • Accepted September 3, 2021.
  • Copyright © 2021 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 207 (10)
The Journal of Immunology
Vol. 207, Issue 10
15 Nov 2021
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The Role of Properdin in C5 Convertase Activity and C5b-9 Formation in the Complement Alternative Pathway
Marloes A. H. M. Michels, Rianne J. F. Maas, Thea J. A. M. van der Velden, Nicole C. A. J. van de Kar, Lambertus P. W. J. van den Heuvel, Elena B. Volokhina, on behalf of the COMBAT Consortium
The Journal of Immunology November 15, 2021, 207 (10) 2465-2472; DOI: 10.4049/jimmunol.2100238

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The Role of Properdin in C5 Convertase Activity and C5b-9 Formation in the Complement Alternative Pathway
Marloes A. H. M. Michels, Rianne J. F. Maas, Thea J. A. M. van der Velden, Nicole C. A. J. van de Kar, Lambertus P. W. J. van den Heuvel, Elena B. Volokhina, on behalf of the COMBAT Consortium
The Journal of Immunology November 15, 2021, 207 (10) 2465-2472; DOI: 10.4049/jimmunol.2100238
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