Characterization of tryptic fragments of human complement factor C3

Area of Science:

• Biochemistry
• Immunology
• Proteomics

Background:

• Human complement component 3 (C3) is a central protein in the complement system.
• Understanding C3 fragment structure is crucial for deciphering complement-mediated immune responses.
• Previous studies have identified various C3 fragments, but detailed characterization of tryptic fragments is ongoing.

Purpose of the Study:

• To prepare and purify C3c and C3d fragments from trypsin-digested human C3.
• To isolate and characterize the individual polypeptide chains of tryptic C3c.
• To determine the molecular weights, amino acid composition, and N-terminal sequences of these fragments.

Main Methods:

• Purification of C3c and C3d fragments using gel filtration chromatography.
• Isolation of C3c chains in 6M guanidinium hydrochloride.
• Characterization of polypeptide chains by molecular weight (Mr), amino acid composition, and N-terminal sequencing.
• Analysis of C3d heterogeneity using SDS-PAGE.
• Immunoblotting with antisera against C3 and C3c subunits.

Main Results:

• Tryptic C3c consisted of a beta-chain fragment (Mr 64,000) and two alpha'-chain fragments (Mr 40,000 and 23,000).
• The beta-chain fragment originated from the C-terminal end.
• The 23,000-Mr alpha'-chain fragment was N-terminal, and the 40,000-Mr fragment was C-terminal.
• Tryptic C3d showed microheterogeneity but had a homogeneous N-terminal sequence.
• A degradation scheme for C3 by trypsin was proposed, indicating fragment positions.

Conclusions:

• The study successfully isolated and characterized major fragments of trypsin-digested human C3.
• Detailed structural information on C3c and C3d fragments was obtained, including their origins within the intact C3 molecule.
• The proposed degradation scheme provides insights into the mechanism of C3 cleavage by trypsin.