Hemolytically inactive C4B complement allotype caused by a proline to leucine mutation in the C5-binding site

Area of Science:

• Immunogenetics
• Complement System Biology

Background:

• The fourth component of complement (C4) comprises C4A and C4B, with C4A6 being the only known hemolytically inactive allotype.
• No hemolytically inactive C4B allotype has been previously identified, limiting understanding of C4B function.

Purpose of the Study:

• To report the identification and characterization of the first hemolytically inactive C4B allotype.
• To elucidate the molecular basis and functional consequences of this novel C4B variant.

Main Methods:

• Hemolytic overlay assays for initial identification.
• Single strand conformational polymorphism (SSCP) and allele-specific oligonucleotide (ASO) analysis for mutation detection and segregation.
• Site-directed mutagenesis and expression studies to confirm functional impact.

Main Results:

• The first hemolytically inactive C4B allotype, designated C4B*1(hi), was identified.
• A C to T transition in exon 12, resulting in a proline to leucine substitution at codon 459 (P459L), was found to cosegregate with C4B*1(hi).
• The P459L mutation was confirmed to abolish hemolytic activity through functional studies.

Conclusions:

• C4B*1(hi) represents the first identified circulating C4B protein with absent hemolytic activity.
• The P459L mutation provides critical new information regarding the C5-binding site of C4, advancing knowledge of complement system mechanisms.