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.
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.
Girija
Umakhanth Venkatraman
UV
Department of Biochemistry, Medical Research Council Immunochemistry Unit, University of Oxford, Oxford, United Kingdom.
Dodds
Alister W
AW
Roscher
Silke
S
Reid
Kenneth B M
KB
Wallis
Russell
R
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
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