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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.
Journal of Immunology (Baltimore, Md. : 1950)
|March 15, 1991
Summary
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.