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Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor
K E Coyne1, S E Hall, S Thompson
1Department of Pathology, Washington University School of Medicine, St. Louis 63110.
Insights
Decay accelerating factor (DAF, CD55) protects cells from complement damage. Deleting SCR2, SCR3, or SCR4 abolished DAF function, while the S/T region acts as a crucial spacer for DAF activity.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Decay accelerating factor (DAF, CD55) is a membrane protein protecting cells from complement-mediated damage.
- DAF inhibits C3/C5 convertase formation and accelerates decay.
- Understanding DAF's structure-function relationships is crucial for its role in immune regulation.
Purpose of the Study:
- To map important structural and functional sites within the DAF molecule.
- To elucidate the role of specific domains (SCRs and S/T region) in DAF function.
- To investigate the contribution of glycosylation to DAF's protective activity.
Main Methods:
- Site-directed mutagenesis was used to create DAF deletion mutants (lacking SCRs or S/T region).
- Mutants were expressed in Chinese hamster ovary cells via stable transfection.
- Immunoprecipitation mapped antibody epitopes, and functional assays assessed protection against complement-mediated cytotoxicity.
Main Results:
- Antibody epitope mapping localized specific binding sites to SCR1 (6), SCR2 (3), SCR3 (3), SCR4 (3), and S/T region (1).
- Only antibodies targeting SCR3 completely blocked DAF function.
- Deletion of SCR2, SCR3, or SCR4 abolished DAF function, while SCR1 deletion had no effect.
- Deletion of the S/T region abrogated DAF function, but this was partially restored when fused to HLA-B44, suggesting a spacer role.
Conclusions:
- The study identified critical roles for SCR2, SCR3, and SCR4 domains in DAF's complement-inhibitory function.
- The serine/threonine-rich (S/T) region, though not essential for direct inhibition, is vital for projecting DAF domains, acting as a functional spacer.
- These findings significantly advance the understanding of DAF's structure-function dynamics and its role in immune defense.
Abstract:
Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.