Purification of the human complement control protein C3b inactivator

Area of Science:

• Biochemistry
• Immunology
• Complement System

Background:

• The complement system is crucial for innate immunity.
• Complement component C3b is a key activation product.
• C3b inactivator (C3bINA) regulates complement by hydrolyzing C3b.

Purpose of the Study:

• To develop an improved isolation method for C3bINA from human plasma.
• To characterize the role of beta 1H in C3bINA activity.
• To investigate the substrate specificity and cleavage products of C3bINA.

Main Methods:

• Chromatographic purification using QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite, and Sephacryl S-200.
• Assay development for C3bINA activity with enhanced sensitivity using beta 1H.
• Analysis of hydrolysis products using gel electrophoresis.

Main Results:

• A novel purification strategy yielded significantly more C3bINA (6 mg/500ml plasma).
• Beta 1H was found essential for C3bINA-mediated hydrolysis of C3b, in solution or cell-bound.
• C3bINA + beta 1H cleaved stored or freeze-thawed C3 into specific fragments, suggesting N-terminal and C-terminal identification.

Conclusions:

• An efficient method for C3bINA isolation has been established.
• Beta 1H is a critical cofactor for C3bINA activity.
• C3bINA exhibits specific cleavage patterns on modified C3, providing insights into its enzymatic function.