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Additional forms of human decay-accelerating factor (DAF)
Abstract:
Decay-accelerating factor (DAF) of human erythrocytes is a glycoprotein with a Mr of 65,000 that is anchored in the membrane via a glycolipid tail. During the purification of DAF, two lower m.w. forms were noted. DAF-A had an Mr of 63,000, and DAF-B had an Mr of 55,000. In a fluid phase assay, both forms accelerated the decay of the classical and the alternative C3 convertases with a specific activity similar to that of DAF. However, the decay-accelerating activity for the cell-bound C3 convertases was abolished, suggesting that neither could insert into E membranes and therefore that the glycolipid tail is altered. Analysis by molecular sieve high-pressure liquid chromatography demonstrated that DAF-A eluted with a Mr of approximately 450,000, similar to native DAF, and was thus in an aggregated form. In contrast, DAF-B eluted as a monomer with a Mr of approximately 60,000. DAF-A, but not DAF-B, bound to a hydrophobic column. To further characterize these two forms, surface-labeled human erythrocytes were incubated with phosphatidyl inositol-specific phospholipase C or papain. The phospholipase inefficiently released a form of DAF that was slightly larger (Mr of 64,000) than DAF-A. Papain efficiently released a 55,000 fragment that had the same Mr as DAF-B. To determine if DAF was cleaved by endogenous enzymes, surface-labeled erythrocytes were incubated with leukocytes. The kinetics of the leukocyte-induced degradation was similar to those observed with papain, and the released fragment aligned on seizing gels with the papain-derived fragment. We hypothesize that endogenous phospholipases and proteases cleave DAF to produce fragments similar to DAF-A and DAF-B, respectively.
Insights
Decay-accelerating factor (DAF) fragments, DAF-A and DAF-B, retain fluid-phase complement inhibition but lose cell-bound activity. Endogenous enzymes likely cleave DAF, altering its membrane anchor.
Area of Science:
- Biochemistry
- Immunology
- Complement System
Background:
- Decay-accelerating factor (DAF) is a 65,000 Mr glycoprotein on human erythrocytes, crucial for regulating complement activation via a glycolipid anchor.
- During DAF purification, two lower molecular weight forms, DAF-A (63,000 Mr) and DAF-B (55,000 Mr), were identified.
Purpose of the Study:
- To characterize the biochemical properties and functional activities of DAF-A and DAF-B.
- To investigate the potential cleavage of DAF by endogenous enzymes on erythrocyte membranes.
Main Methods:
- Fluid-phase and cell-bound complement convertase decay assays were used to assess DAF fragment activity.
- Molecular sieve high-pressure liquid chromatography (HPLC) analyzed the aggregation state and molecular weight of DAF forms.
- Surface-labeled erythrocytes were treated with phosphatidyl inositol-specific phospholipase C, papain, or leukocytes to identify DAF cleavage products.
Main Results:
- Both DAF-A and DAF-B inhibited fluid-phase C3 convertases but failed to inhibit cell-bound convertases, indicating altered membrane anchoring.
- DAF-A existed in an aggregated form (approx. 450,000 Mr) and bound to hydrophobic columns, while DAF-B eluted as a monomer (approx. 60,000 Mr).
- Papain efficiently released a DAF-B-sized fragment, and leukocyte incubation mimicked papain digestion, suggesting protease involvement in DAF cleavage.
Conclusions:
- DAF fragments generated by enzymatic cleavage lose their ability to associate with cell membranes, impairing their function in regulating cell-bound complement.
- Endogenous phospholipases and proteases are hypothesized to cleave DAF, producing fragments analogous to DAF-A and DAF-B observed during purification.