Characterization of human C4a anaphylatoxin

Area of Science:

• Immunology
• Biochemistry
• Protein Chemistry

Background:

• The fourth component of complement (C4) plays a crucial role in the immune system.
• Anaphylatoxins are bioactive peptides derived from complement activation.
• Understanding the structure and function of anaphylatoxins is key to deciphering immune responses.

Purpose of the Study:

• To isolate and chemically characterize human C4a anaphylatoxin.
• To investigate the structural properties of C4a, including its amino acid composition and conformation.
• To compare the N-terminal sequence of C4a with other anaphylatoxins (C3a, C5a) to explore evolutionary relationships.

Main Methods:

• Isolation of C4a using ion-exchange chromatography (CM-Sephadex C-50) and gel filtration (Sephadex G-50).
• Characterization of C4a's physicochemical properties: isoelectric point (pI), residue count, molecular weight (Mr), amino acid composition, and absence of tryptophan, histidine, and carbohydrates.
• Circular dichroism spectroscopy to determine alpha-helical content.
• N-terminal amino acid sequencing and comparison with C3a and C5a sequences.

Main Results:

• Human C4a was successfully isolated and found to be a cationic polypeptide (pI 9.0-9.5) with 77 residues and Mr 8,759.
• C4a lacks tryptophan, histidine, and carbohydrates, with 54% of its backbone adopting an alpha-helical conformation.
• Sequence comparison revealed 25% homology with C3a and 33% homology with C5a in the N-terminal region.

Conclusions:

• The chemical data provide the first evidence for an evolutionary link between human anaphylatoxins C3a, C4a, and C5a.
• This suggests a conserved evolutionary relationship among their precursor proteins within the complement system.