Abstract
Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4- to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.
Highlights
Three-complement control protein (CCP) domain, a von Willebrand factor type A2 module, and a serine protease domain (Fig. 1)
The catalytic triad residues Asp551, His501, and Ser674 were in a typical active conformation found in serine proteases; residues that normally define the oxyanion hole produced an unusual conformation because of the inward orientation of the carbonyl group of Arg671 hydrogen-bonded to the NH group of Ser674 to form a 310 helix
When factor B was neutralized with a specific monoclonal antibody, mice were significantly protected from complement activation and fetal loss stimulated by antiphospholipid antibody [11, 26]
Summary
Wild type, factor B-deficient, or C4-deficient mice in conjunction with specific anti-factor B monoclonal antibodies have demonstrated that factor B is critical for allergen-induced development of airway hyper-responsiveness and inflammation [11, 12]. We report the development of a valuable new rapid chromogenic assay for measuring factor B activity in vitro, the discovery of some important and unexpected molecular and catalytic properties of factor B for substrate processing, and the discovery of the first substrate-based inhibitor of factor B that inhibits C3 convertase. These results provide a valuable beginning to better understanding the activity of factor B and C3 convertase, to a more robust method of screening potential inhibitors, and to the rational development of potent and selective inhibitors of C3 cleavage resulting from complement activation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
