title: Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4.
abstract: The complement system is fundamental to both innate and adaptive immunity and can be initiated via the classical, lectin or alternative pathways. Cleavage of C4 by MASP-2, the initiating protease of the lectin pathway, is a crucial event in the activation of this pathway, preceding the eventual formation of the C3 convertase (C4bC2a) complex on the pathogen surface. Interactions required for the cleavage of C4 by MASP-2 are likely to be facilitated by the initial binding of C4 to an exosite on the protease. We have shown that both proteolytically active and catalytically inactive CCP1-CCP2-serine protease (CCP1-CCP2-SP) forms bind C4 with similar affinity. Interestingly, proteins containing the CCP1-CCP2 domains or the SP domain alone bound C4 with much lower affinity than the CCP1-CCP2-SP protein, suggesting that the CCP domains cooperate positively with the active site to mediate efficient binding and cleavage of C4. In addition, mutation of residue K342 to alanine in the CCP1 domain abolished binding to both C4 and C4b in its CCP1-CCP2 form, suggesting a key electrostatic role for this amino acid. The presented data indicates that all of the domains are required in order to mediate high affinity interaction with C4.
authors.affiliation: Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Renee C
authors.initials: RC
authors.lastname: Duncan
authors.forename: Frida
authors.initials: F
authors.lastname: Bergström
authors.forename: Theresa H
authors.initials: TH
authors.lastname: Coetzer
authors.forename: Anna M
authors.initials: AM
authors.lastname: Blom
authors.forename: Lakshmi C
authors.initials: LC
authors.lastname: Wijeyewickrema
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
chemical_list: D003181:Complement C4; D037102:Lectins; D011994:Recombinant Proteins; D050577:Complement C3-C5 Convertases; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases