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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 Ficolins¹

Umakhanth Venkatraman Girija^*, Alister W. Dodds^*, Silke Roscher, Kenneth B. M. Reid^* and Russell Wallis^2,*,,

^* Department of Biochemistry, Medical Research Council Immunochemistry Unit, University of Oxford, Oxford, United Kingdom; and Department of Infection, Immunity and Inflammation and Department of Biochemistry, University of Leicester, Leicester, United Kingdom

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, Lys⁵⁶ 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant 077400 from The Wellcome Trust and Grant G0501425 from the Medical Research Council. The analytical ultracentrifugation facility at Oxford was set up using grants from the Biotechnology and Biological Sciences Research Council and The Wellcome Trust. R.W. is a Research Council U.K. Academic Fellow.

² Address correspondence and reprint requests to Dr. Russell Wallis, Department of Infection, Immunity and Inflammation, Medical Science Building, University of Leicester, University Road, Leicester, U.K. E-mail address: rw73{at}le.ac.uk

³ Abbreviations used in this paper: MBL, mannose-binding lectin; MASP, MBL-associated serine protease.

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