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Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55¹

Florence Teillet^*,,,, Monique Lacroix^*,,,, Steffen Thiel^¶, Dietmar Weilguny^||, Teit Agger^||, Gérard J. Arlaud^*,,, and Nicole M. Thielens^2,*,,,

^* Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France; Commissariat à l’Energie Atomique (CEA), Paris, France; Centre National de la Recherche Scientifique (CNRS), Paris, France; Université Joseph Fourier, Grenoble, France; ^¶ Department of Medical Microbiology and Immunology, University of Aarhus, Denmark; and ^|| NatImmune A/S, Copenhagen, Denmark

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys⁵⁵Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys⁵⁵Gln and Lys⁵⁵Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys⁵⁵Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg⁴⁷Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg⁴⁷Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg⁴⁷Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro⁵³Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu⁴⁹ and Leu⁵⁶ were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys⁵⁵, which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

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 the Commissariat à l’Energie Atomique, the Centre National de la Recherche Scientifique, and the Université Joseph Fourier (Grenoble, France).

² Address correspondence and reprint requests to Dr. Nicole Thielens, Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, Grenoble, France. E-mail address: nicole.thielens{at}ibs.fr

³ Abbreviations used in this paper: MBL, mannan-binding lectin; MASP, MBL-associated serine protease; MAp19, 19-kDa MBL-associated protein; CUB, module originally found in complement proteins; C1r/C1s, Uegf, and bone morphogenetic protein 1; EGF, epidermal growth factor; CCP, complement control protein; CHO, Chinese hamster ovary; WT, wild type.

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