Structural characterization of the C4a anaphylatoxin from rat

Area of Science:

• Immunology
• Biochemistry
• Proteomics

Background:

• The complement system plays a crucial role in innate and adaptive immunity.
• Anaphylatoxins, such as C4a, are key mediators of inflammatory responses.
• Understanding anaphylatoxin structure and function is vital for immunomodulatory drug development.

Purpose of the Study:

• To purify and characterize rat C4a anaphylatoxin.
• To elucidate the complete primary structure of rat C4a.
• To assess the biological activity and glycosylation status of rat C4a.

Main Methods:

• Purification of C4a from rat sera activated by heat-aggregated IgG using a three-step procedure.
• Homogeneity assessment via electrophoresis on cellulose acetate and SDS-polyacrylamide gels.
• Contamination analysis using Ouchterlony and radioimmunoassay.
• Molecular weight determination and amino acid sequencing.

Main Results:

• Rat C4a was purified to homogeneity, confirmed by electrophoresis.
• Preparations were free of rat C5a and C3a contaminants.
• Rat C4a is a glycoprotein (11,000-12,000 mol. wt) with 76 amino acid residues and an oligosaccharide unit.
• The complete primary structure of rat C4a was elucidated.
• Rat C4a exhibited weak contractile activity on guinea pig ileum, comparable to human C4a.

Conclusions:

• Rat C4a anaphylatoxin is a glycosylated protein, distinct from non-glycosylated human and bovine C4a.
• The elucidated primary structure provides a basis for understanding rat C4a's function.
• Rat C4a's biological activity is comparable to human C4a, suggesting conserved functional domains despite glycosylation differences.