title: Localization and characterization of the mannose-binding lectin (MBL)-associated-serine protease-2 binding site in rat ficolin-A: equivalent binding sites within the collagenous domains of MBLs and ficolins.
abstract: Ficolins and mannose-binding lectins (MBLs) are the first components of the lectin branch of the complement system. They comprise N-terminal collagen-like domains and C-terminal pathogen-recognition domains (fibrinogen-like domains in ficolins and C-type carbohydrate-recognition domains in MBLs), which target surface-exposed N-acetyl groups or mannose-like sugars on microbial cell walls. Binding leads to activation of MBL-associated serine protease-2 (MASP-2) to initiate complement activation and pathogen neutralization. Recent studies have shown that MASP-2 binds to a short segment of the collagen-like domain of MBL. However, the interaction between ficolins and MASP-2 is relatively poorly understood. In this study, we show that the MASP-2 binding site on rat ficolin-A is also located within the collagen-like domain and encompasses a conserved motif that is present in both MBLs and ficolins. Characterization of this motif using site-directed mutagenesis reveals that a lysine residue in the X position of the Gly-X-Y collagen repeat, Lys(56) in ficolin-A, which is present in all ficolins and MBLs known to activate complement, is essential for MASP-2 binding. Adjacent residues also make important contributions to binding as well as to MASP activation probably by stabilizing the local collagen helix. Equivalent binding sites and comparable activation kinetics of MASP-2 suggest that complement activation by ficolins and MBLs proceeds by analogous mechanisms.
authors.affiliation: Department of Biochemistry, Medical Research Council Immunochemistry Unit, University of Oxford, Oxford, United Kingdom.
authors.forename: Umakhanth Venkatraman
authors.initials: UV
authors.lastname: Girija
authors.forename: Alister W
authors.initials: AW
authors.lastname: Dodds
authors.forename: Silke
authors.initials: S
authors.lastname: Roscher
authors.forename: Kenneth B M
authors.initials: KB
authors.lastname: Reid
authors.forename: Russell
authors.initials: R
authors.lastname: Wallis
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D003167:Complement Activation; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D007700:Kinetics; D037102:Lectins / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000276:immunology; D000097604:Ficolins
chemical_list: D003094:Collagen; D037102:Lectins; D008239:Lysine; D037241:Mannose-Binding Lectins; D050606:Mannose-Binding Protein-Associated Serine Proteases; D011994:Recombinant Proteins; D000097604:Ficolins; C491338:Masp2 protein, rat