Alister W. Dodds

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition

ABSTRACT Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. DiverseCandida species, Aspergillus fumigatus,Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci,Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

Mannose-Binding Lectin Recognizes Peptidoglycan via the N-Acetyl Glucosamine Moiety, and Inhibits Ligand-Induced Proinflammatory Effect and Promotes Chemokine Production by Macrophages

Peptidoglycan (PGN) is the major cell wall component (90%, w/w) of Gram-positive bacteria and consists of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide repeating arrays that are cross-linked by short peptides. We hypothesized that PGN is a ligand for pathogen-associated pattern-recognition proteins. Mannose-binding lectin (MBL) and serum amyloid component P are two carbohydrate-binding innate immune proteins present in the blood. In this study we show that human MBL, but not serum amyloid component P, binds significantly to PGN via its C-type lectin domains, and that the interaction can be more effectively competed by GlcNAc than by MurNAc. Surface plasmon resonance analyses show that native MBL binds immobilized PGN with high avidity. Competition experiments also show that both native MBL and MBL(n/CRD), a 48-kDa recombinant trimeric fragment of MBL containing neck and carbohydrate recognition domains, have higher affinity for GlcNAc than for MurNAc. Protein arrays and ELISA show that PGN increases the secretion of TNF-α, IL-8, IL-10, MCP-2, and RANTES from PMA-stimulated human monocytic U937 cells. Interestingly, the presence of MBL together with PGN increases the production of IL-8 and RANTES, but reduces that of TNF-α. Our results indicate that Gram-positive bacterial is a biologically relevant ligand for MBL, and that the collectin preferentially binds to the GlcNAc moiety of the PGN via its C-type lectin domains. MBL inhibits PGN-induced production of proinflammatory cytokines while enhancing the production of chemokines by macrophages, which suggests that MBL may down-regulate macrophage-mediated inflammation while enhancing phagocyte recruitment.

Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement

The classical and lectin pathways of complement activation neutralize pathogens and stimulate key immunological processes. Both pathways are initiated by collagen-containing, soluble pattern recognition molecules associated with specific serine proteases. In the classical pathway, C1q binds to Ab-Ag complexes or bacterial surfaces to activate C1r and C1s. In the lectin pathway, mannan-binding lectin and ficolins bind to carbohydrates on pathogens to activate mannan-binding lectin-associated serine protease 2. To characterize the interactions leading to classical pathway activation, we have analyzed binding between human C1q, C1r, and C1s, which associate to form C1, using full-length and truncated protease components. We show that C1r and C1s bind to C1q independently. The CUB1-epidermal growth factor fragments contribute most toward binding, but CUB2 of C1r, but not of C1s, is also important. Each C1rs tetramer presents a total of six binding sites, one for each of the collagenous domains of C1q. We also demonstrate that subcomponents of the lectin and classical pathways cross-interact. Thus, although the stoichiometries of complexes differ, interactions are analogous, with equivalent contacts between recognition and protease subcomponents. Importantly, these new data are contrary to existing models of C1 and enable us to propose a new model using mannan-binding lectin-mannan-binding lectin-associated serine protease interactions as a template.

Molecular Interactions between MASP-2, C4, and C2 and Their Activation Fragments Leading to Complement Activation via the Lectin Pathway

Activation of component C3 is central to the pathways of complement and leads directly to neutralization of pathogens and stimulation of adaptive immune responses. The convertases that catalyze this reaction assemble from fragments of complement components via multistep reactions. In the lectin pathway, mannose-binding lectin (MBL) and ficolins bind to pathogens and activate MBL-associated serine protease-2 (MASP-2). MASP-2 cleaves C4 releasing C4a and generating C4b, which attaches covalently to the pathogen surface upon exposure of its reactive thioester. C2 binds to C4b and is also cleaved by MASP-2 to form the C3 convertase (C4b2a). To understand how this complex process is coordinated, we have analyzed the interactions between MASP-2, C4, C2, and their activation fragments and have compared MASP-2-catalyzed cleavage of C4b2 and C2. The data show that C2 binds tightly to C4b but not to C4, implying that C4 and C2 do not circulate as preformed complexes but that C2 is recruited only after prior activation of C4. Following cleavage of C4, C4b still binds to MASP-2 (KD ∼ 0.6 μm) and dissociates relatively slowly (koff ∼ 0.06 s–1) compared with the half-life of the thioester (≤0.7 s) (Sepp, A., Dodds, A. W., Anderson, M. J., Campbell, R. D., Willis, A. C., and Law, S. K. (1993) Protein Sci. 2, 706–716). We propose that the C4b·MASP-2 interaction favors attachment of C4b near to the activating MBL·MASP complex on the bacterial surface so that, following recruitment of C2, the proximity of enzyme and substrate (C4b2) combined with more favorable reaction kinetics drive the formation of the C3 convertase, promoting complement activation.

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, 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.

Enzyme-independent, orientation-selective conjugation of whole human complement C3 to protein surfaces

Complement C3 is a central component of the humoral immune system. Upon triggering of the complement cascade, proteolytic fragments of C3 mediate important processes such as opsonization and lymphocyte activation. C3 possesses an internal thioester that mediates covalent attachment of proteolytically activated C3 to target surfaces. Treatment of native C3 with methylamine cleaves the thioester bond and exposes a free sulfhydryl group at the target-binding face of the protein. Through the use of sulfhydryl-reactive heterobifunctional cross-linking and biotinylation reagents, we demonstrate the capacity to form stable, multimeric whole human C3-protein conjugates in a fashion reflecting the orientation of physiologically-activated C3. We speculate that this C3 conjugation strategy presents a route for targeting dendritic cells and macrophages. In addition, manipulation of the thioester bond could enhance the study of biological roles of C3 and related proteins such as C4, and also of transmissible agents that exploit complement function such as prions.

A monoclonal antibody to C1q which appears to interact with C1r2C1s2-binding site

A monoclonal antibody (SB‐4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 1010 M−1. It has been shown that the antibody binds to the A‐B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of preformed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2‐C1s2 complex.

Molecular Biology of the Human Complement Class III Products of the MHC: Factor I and Its Cofactors

Deletion analysis of the 5′ flanking region of the factor B gene indicates the presence of a region with enhancer-like activity in addition to the promoter element and indicates that both elements are important in the tissue-specific expression of factor B. DNA sequencing of a number of C4 allotypes has allowed definition of the structural basis of the polymorphism found in C4A and C4B types in terms of gene size, reactivity, and antigenicity. It has been established that the regulatory proteins C4bp, CR1, and H as well as the enzymes C2, B, and C1r are structurally related since they contain two or more internal repeating units of 60 amino acids. Thus these complement proteins appear to be members of a new superfamily since this structural feature is widespread, being found in a number of noncomplement proteins.