Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator

Area of Science:

• Biochemistry
• Immunology
• Proteomics

Background:

• The complement system is crucial for innate immunity.
• Understanding complement component C4b regulation is vital for immune response control.
• The C4b alpha'-chain contains a reactive thiol group involved in its function.

Purpose of the Study:

• To characterize the C4b activation product.
• To locate the reactive thiol group on the C4b alpha'-chain.
• To determine the order and properties of C4b alpha'-chain fragments.

Main Methods:

• Radioactive labeling of the C4b alpha'-chain thiol group with iodo[2-14C]acetic acid.
• Cleavage of C4b by C3bINA and isolation of fragments C4d and C4c.
• Analysis of fragment molecular weights, amino acid composition, carbohydrate content, and N-terminal/C-terminal sequences.

Main Results:

• The reactive thiol group was localized to the C4d fragment.
• The alpha'-chain fragments were identified as alpha 3 (25 kDa), C4d (44.5 kDa), and alpha 4 (12 kDa).
• The N-terminal sequence analysis confirmed alpha 3 is derived from the N-terminus, and arginine is the C-terminal residue of C4d and alpha 3.

Conclusions:

• The established order of alpha'-chain fragments is alpha 3--C4d--alpha 4.
• C3bINA specificity targets an Arg--Xaa peptide bond.
• This detailed structural analysis provides insights into C4b regulation and function.