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Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor
W G Brodbeck1, D Liu, J Sperry
1Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Insights
Decay-accelerating factor (DAF) regulates complement pathways by dissociating C3 convertases. Specific domains, SCR-2 and SCR-3, are crucial for classical pathway regulation, while SCR-2, -3, and -4 regulate the alternative pathway.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Decay-accelerating factor (DAF) is a cell-surface protein regulating complement system activation.
- DAF protects host cells from autologous complement attack by dissociating C3 convertases.
- DAF comprises four short consensus repeats (SCRs) and is anchored by a glycoinositol phospholipid (GPI) moiety.
Purpose of the Study:
- To investigate the functional roles of individual SCR domains within DAF in regulating complement pathways.
- To determine the specific SCRs responsible for classical and alternative pathway C3 convertase regulation.
- To elucidate the mechanism of DAF's regulatory function, distinguishing domain-specific interactions from spatial arrangement.
Main Methods:
- Preparation of recombinant GPI-anchored DAF proteins lacking individual SCR domains.
- Incorporation of variant DAF proteins into sheep erythrocyte hemolytic intermediates.
- Assays to assess the intrinsic regulatory abilities of variant DAF on classical and alternative complement pathway activation.
- Fluid phase C3 activation assays to compare functional differences of variant DAF proteins.
Main Results:
- Classical pathway C3 convertase regulation is mediated by SCR-2 and SCR-3.
- Alternative pathway C3 convertase regulation involves SCR-2, SCR-3, and SCR-4.
- Functional differences arise from domain-specific interactions rather than altered spatial arrangements.
- DAF variants with SCR-1, -2, and -3 selectively inhibit classical pathway activation, excluding SCR-4.
Conclusions:
- SCR-2 and SCR-3 are essential for DAF's classical pathway regulatory function.
- SCR-2, -3, and -4 collectively contribute to DAF's alternative pathway regulatory function.
- DAF's regulatory efficacy is dependent on specific domain interactions within its SCRs.
Abstract:
Decay-accelerating factor (DAF) is a cell-associated C regulatory protein that protects host cells from autologous C attack. It functions intrinsically in host cell surface membranes to rapidly dissociate autologous classical and alternative pathway C3 convertases whenever these amplifying enzymes assemble on host cell surfaces. It is composed of four contiguous approximately 70 amino acid long regions termed short consensus repeats (SCRs) that share homology with similar units in other C3 convertase regulatory proteins. It is attached to the cell surface membrane by a glycoinositol phospholipid (GPI) anchor that is added posttranslationally. In this study, we prepared rGPI-anchored DAF proteins devoid of individual SCRs. We then incorporated the GPI-anchored products into sheep erythrocyte (Esh) hemolytic intermediates and examined their abilities to intrinsically regulate classical or alternative pathway activation. We found that classical pathway C3 convertase regulatory function resides within SCR-2 and SCR-3, while alternative pathway C3 convertase regulatory function resides within SCR-2, -3, and -4. Functional comparisons of the variant DAF proteins in fluid phase C3 activation assays established that the differences reflect domain-specific interactions rather than changes in the spatial arrangement of SCRs above the cell surface. In accordance with these findings, we found that variant DAF molecules containing SCR-1, -2, and -3, but not SCR-4, function to selectively inhibit classical pathway activation.