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Purification of the human complement control protein C3b inactivator
Insights
A new method isolates C3b inactivator (C3bINA), a complement protein, with higher yield from human plasma. This proteinase, essential for complement regulation, requires beta 1H for C3b hydrolysis.
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.
Abstract:
An alternative method of isolation from human plasma is described for C3b inactivator, C3bINA, the proteinase that in conjunction with either beta 1H or C4b-binding protein will hydrolyse respectively C3b or C4b, the activation products of the third, C3 and fourth, C4, components of complement. The purification is by chromatography of plasma on columns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500ml of plasma) is severalfold higher than in previously described methods. The sensitivity of the assay for C3bINA has been increased by including optimal amounts of beta 1H, and it was observed that beta 1H was essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution or bound to a cell surface. Native C3 is not hydrolysed by C3bINA + beta 1H, but the haemolytically inactive form that appears on prolonged storage at 4 degrees C or on freezing and thawing is hydrolysed and gives fragments of the alpha-chain of 75000 and 43000 apparent mol.wt. As the alpha'-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67000 and 43000 apparent mol.wt. when incubated with C3bINA + beta 1H, this suggests that the larger fragment is N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clear classification of the type of proteinase to which this enzyme belongs has been obtained.