The activation of human complement component C5 by a fluid phase C5 convertase.

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Complement component C5 (C5) is a crucial protein in the complement system.
• C5 activation leads to the formation of C5a and C5b fragments, initiating downstream inflammatory and cytolytic cascades.
• Understanding C5 activation mechanisms is vital for comprehending immune responses and developing therapeutic strategies.

Purpose of the Study:

• To investigate the conformational changes and binding interactions during C5 activation.
• To elucidate the role of the C5 convertase (CVF,Bb) in C5 cleavage and subsequent complex formation.
• To characterize the structural and functional properties of C5b and its interaction with C6.

Main Methods:

• Proteolytic cleavage assays using cobra venom factor-dependent C3/C5 convertase (CVF,Bb).
• Circular dichroism spectroscopy to analyze conformational changes in C5.
• Transmission electron microscopy (TEM) to visualize C5 and C5b structures.
• Biochemical assays to study the formation of C5b,6 complexes.

Main Results:

• CVF,Bb selectively cleaves C5 at the arginyl-leucine peptide bond (positions 74-75) in the alpha chain.
• Circular dichroism confirmed conformational changes in C5 upon activation.
• C5 activation generates a transient binding site for complement component C6, essential for C5b,6 complex formation.
• C5b aggregation occurs in the absence of C6, suggesting exposure of hydrophobic sites.
• TEM revealed a multilobal ultrastructure for C5 and globular particles for aggregated C5b.

Conclusions:

• C5 activation by CVF,Bb induces a conformational change and exposes a transient binding site for C6.
• Unlike C3, C5 lacks a labile thiol ester linkage, and its interactions with C6 and membranes are noncovalent.
• These findings provide critical insights into the molecular mechanisms governing complement system activation and effector functions.