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Major component of Ra-reactive factor, a complement-activating bactericidal protein, in mouse serum
S Ihara1, A Takahashi, H Hatsuse
1Department of Molecular Biology, School of Medicine, Kitasato University, Kanagawa, Japan.
Abstract:
A complement-activating bactericidal protein, Ra-reactive factor was isolated from mouse serum by an affinity method. The m.w. of the isolated RaRF estimated by glycerol density gradient sedimentation (around 300,000) was the same as that of the active material in mouse serum. As evidenced by gel filtration, the intact RaRF was decomposed into high (higher than 200,000) and low (50,000 to 200,000) m.w. components by treatment with 10% acetonitrile. SDS- and acid/urea-PAGE demonstrated that the high m.w. component was completely dissociated into equimolar quantities of two kinds of 28 kDa polypeptides, P28a and P28b, under reducing conditions, indicating that the association of these polypeptides was stabilized by disulfide bonds. The ability to bind specifically to the Ra determinant was retained in the high m.w. component, although the complement-activating potency was lost. The amino acid compositions of P28a and P28b polypeptides were compared with those of related serum proteins. The P28a and P28b polypeptides were found to have the highest homology to rat mannan-binding protein and mouse and human C1q subcomponent of complement.
Insights
Researchers isolated a mouse serum protein, Ra-reactive factor (RaRF), which activates complement. This protein comprises two polypeptides (P28a and P28b) similar to complement C1q, but loses its complement-activating function upon dissociation.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- The complement system is crucial for innate immunity, mediating pathogen clearance and inflammation.
- Bactericidal proteins in serum play a vital role in host defense against microbial infections.
- Understanding the molecular mechanisms of complement activation is essential for developing novel therapeutics.
Purpose of the Study:
- To isolate and characterize the complement-activating bactericidal protein, Ra-reactive factor (RaRF), from mouse serum.
- To elucidate the molecular structure and composition of RaRF.
- To investigate the relationship between RaRF structure and its functional properties, specifically complement activation and Ra determinant binding.
Main Methods:
- Affinity purification was used to isolate RaRF from mouse serum.
- Glycerol density gradient sedimentation and gel filtration were employed to estimate molecular weight and analyze dissociation products.
- SDS-PAGE and acid/urea-PAGE under reducing conditions were used to determine polypeptide composition and disulfide bond involvement.
- Amino acid composition analysis was performed to compare RaRF polypeptides with related serum proteins.
Main Results:
- RaRF was successfully isolated from mouse serum and confirmed to be a complement-activating protein.
- Intact RaRF (approx. 300,000 m.w.) dissociated into high and low molecular weight components upon acetonitrile treatment.
- The high molecular weight component consists of two 28 kDa polypeptides (P28a and P28b) linked by disulfide bonds.
- P28a and P28b exhibit significant homology to rat mannan-binding protein and mouse/human C1q subcomponent of complement.
- The high molecular weight component retained Ra determinant binding but lost complement-activating potency.
Conclusions:
- Ra-reactive factor is a disulfide-bonded complex of two 28 kDa polypeptides, P28a and P28b.
- These polypeptides share homology with C1q, suggesting a potential role in complement initiation.
- Dissociation of RaRF leads to loss of complement activation, highlighting the importance of its quaternary structure for function.