Crystal structure and functional characterization of the complement regulator mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1).
ABSTRACT: The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin-associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nm and 2.5 nm, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ?146 ? long. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9.
Project description:The human lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or three ficolins (ficolin-1, -2, and -3) in association with three MBL/ficolin-associated serine proteases (MASP) (MASP-1, -2, and -3) and a nonenzymatic sMAP. MASP-1 and MASP-3 (MASP1 isoforms 1 and 2, respectively) are splice variants of the MASP1 gene, whereas MASP-2 and sMAP are splice variants of the MASP2 gene. We have identified a novel serum protein of 45 kDa that is associated with MBL and the ficolins. This protein is named MBL/ficolin-associated protein 1 (MAP-1 corresponding to MASP1 isoform 3). The transcript generating MAP-1 (MASP1_v3) contains exons 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domains but contains most of the common heavy chain of MASP-1 and MASP-3. Additionally MAP-1 contains 17 unique C-terminal amino acids. By use of quantitative PCR and MAP-1-specific immunohistochemistry, we found that MAP-1 is highly expressed in myocardial and skeletal muscle tissues as well as in liver hepatocytes with a different expression profile than that observed for MASP-1 and MASP-3. MAP-1 co-precipitated from human serum with MBL, ficolin-2, and ficolin-3, and recombinant MAP-1 was able to inhibit complement C4 deposition via both the ficolin-3 and MBL pathway. In conclusion we have identified a novel 45-kDa serum protein derived from the MASP1 gene, which is highly expressed in striated muscle tissues. It is found in complex with MBL and ficolins and may function as a potent inhibitor of the complement system in vivo.
Project description:The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon in vivo pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL-MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.
Project description:Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and 3 lectin pathway-specific serine proteases. We have recently shown that the lectin pathway-specific carbohydrate recognition subcomponent mannose-binding lectin plays an essential role in the pathophysiology of thrombosis and ischemia/reperfusion injury. Thus, we hypothesized that the endogenous mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1) that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schema/reperfusion injury and thrombogenesis when used at pharmacological doses in wild-type mice.In 2 mouse models, MAP-1 preserves cardiac function, decreases infarct size, decreases C3 deposition, inhibits MBL deposition, and prevents thrombogenesis. Furthermore, we demonstrate that MAP-1 displaces MBL/ficolin-associated serine protease (MASP)-1, MASP-2, and MASP-3 from the MBL complex.Our results suggest that the natural, endogenous inhibitor MAP-1 effectively inhibits lectin pathway activation in vivo. MAP-1 at pharmacological doses represents a novel therapeutic approach for human diseases involving the lectin pathway and its associated MASPs.
Project description:Ficolins are innate immune components that bind to PAMPs and structures on apoptotic cells. Humans produce two serum forms (L- and H-ficolin) and a leukocyte-associated form (M-ficolin), whereas rodents and most other mammals produce ficolins-A and -B, orthologues of L- and M-ficolin, respectively. All three human ficolins, together with mouse and rat ficolin-A, associate with mannan-binding lectin-associated serine proteases (MASPs) and activate the lectin pathway of complement on PAMPs. By contrast, mouse ficolin-B does not bind MASPs and cannot activate complement. Because of these striking differences together with the lack of functional information for other ficolin-B orthologues, we have characterized rat ficolin-B, and compared its physical and biochemical properties with its serum counterpart. The data show that both rat ficolins have archetypal structures consisting of oligomers of a trimeric subunit. Ficolin-B recognized mainly sialyated sugars, characteristic of exogenous and endogenous ligands, whereas ficolin-A had a surprisingly narrow specificity, binding strongly to only one of 320 structures tested: an N-acetylated trisaccharide. Surprisingly, rat ficolin-B activated MASP-2 comparable to ficolin-A. Mutagenesis data reveal that lack of activity in mouse ficolin-B is probably caused by a single amino acid change in the putative MASP-binding site that blocks the ficolin-MASP interaction.
Project description:The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases.
Project description:Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3.
Project description:Components of the complement system and atypical parameters of coagulation were reported in COVID-19 patients, as well as the exacerbation of the inflammation and coagulation activity. Mannose binding lectin (MBL)- associated serine proteases (MASPs) play an important role in viral recognition and subsequent activation of the lectin pathway of the complement system and blood coagulation, connecting both processes. Genetic variants of MASP1 and MASP2 genes are further associated with different levels and functional efficiency of their encoded proteins, modulating susceptibility and severity to diseases. Our review highlights the possible role of MASPs in SARS-COV-2 binding and activation of the lectin pathway and blood coagulation cascades, as well as their associations with comorbidities of COVID-19. MASP-1 and/or MASP-2 present an increased expression in patients with COVID-19 risk factors: diabetes, arterial hypertension and cardiovascular disease, chronic kidney disease, chronic obstructive pulmonary disease, and cerebrovascular disease. Based also on the positive results of COVID-19 patients with anti-MASP-2 antibody, we propose the use of MASPs as a possible biomarker of the progression of COVID-19 and the investigation of new treatment strategies taking into consideration the dual role of MASPs, including MASP inhibitors as promising therapeutic targets against COVID-19.
Project description:Collectins and ficolins are important in the clearance of endogenous and exogenous danger materials. A new human collectin-11 was recently identified in low concentration in serum in complex with mannose-binding lectin (MBL)/ficolin-associated serine proteases. Collectin-11 binds to carbohydrate residues present on various microorganisms. Thus, we hypothesized that collectin-11 could be a novel initiation molecule in the lectin pathway of complement. We can show that collectin-11 associates with all the known MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3) as well as the lectin complement pathway regulator MAP-1. Furthermore, we found that complex formation between recombinant collectin-11 and recombinant MASP-2 on Candida albicans leads to deposition of C4b. Native collectin-11 in serum mediated complement activation and deposition of C4b and C3b, and formation of the terminal complement complex on C. albicans. Moreover, spiking collectin-11-depleted serum, which did not mediate complement activation, with recombinant collectin-11 restored the complement activation capability. These results define collectin-11 as the fifth recognition molecule in the lectin complement pathway in addition to MBL, ficolin-1, ficolin-2 and ficolin-3.
Project description:The complement system, composed of the three activation pathways, has both protective and pathogenic roles in the development of systemic lupus erythematosus (or lupus), a prototypic autoimmune disease. The classical pathway contributes to the clearance of immune complexes (ICs) and apoptotic cells, whereas the alternative pathway (AP) exacerbates renal inflammation. The role of the lectin pathway (LP) in lupus has remained largely unknown. Mannose-binding lectin (MBL)-associated serine proteases (MASPs), which are associated with humoral pattern recognition molecules (MBL or ficolins), are the enzymatic constituents of the LP and AP. MASP-1 encoded by the Masp1 gene significantly contributes to the activation of the LP. After the binding of MBL/ficolins to pathogens or self-altered cells, MASP-1 autoactivates first, then activates MASP-2, and both participate in the formation of the LP C3 convertase C4b2a, whereas, MASP-3, the splice variant of the Masp1 gene, is required for the activation of the zymogen of factor D (FD), and finally participates in the formation of the AP C3 convertase C3bBb. To investigate the roles of MASP-1 and MASP-3 in lupus, we generated Masp1 gene knockout lupus-prone MRL/lpr mice (Masp1/3-/- MRL/lpr mice), lacking both MASP-1 and MASP-3, and analyzed their renal disease. As expected, sera from Masp1/3-/- MRL/lpr mice had no or markedly reduced activation of the LP and AP with zymogen forms of complement FD. Compared to their wild-type littermates, the Masp1/3-/- MRL/lpr mice had maintained serum C3 levels, little-to-no albuminuria, as well as significantly reduced glomerular C3 deposition levels and glomerular pathological score. On the other hand, there were no significant differences in the levels of serum anti-dsDNA antibody, circulating ICs, glomerular IgG and MBL/ficolins deposition, renal interstitial pathological score, urea nitrogen, and mortality between the wild-type and Masp1/3-/- MRL/lpr mice. Our data indicate that MASP-1/3 plays essential roles in the development of lupus-like glomerulonephritis in MRL/lpr mice, most likely via activation of the LP and/or AP.
Project description:The complement system is activated cascadically via three distinct major routes: classical pathway (CP), alternative pathway (AP) or lectin pathway (LP). The unique factors associated with the latter are collectins (mannose-binding lectin, collectin-10, collectin-11), ficolins (ficolin-1, ficolin-2, ficolin-3) and proteins of the mannose-binding lectin-associated serine protease (MASP) family (MASP-1, MASP-2, MASP-3, MAp19, MAp44). Collectins and ficolins are both pattern-recognising molecules (PRM), reactive against pathogen-associated molecular patterns (PAMP) or danger-associated molecular patterns (DAMP). The MASP family proteins were first discovered as complexes with mannose-binding lectin (MBL) and therefore named MBL-associated serine proteases, but later, they were found to interact with ficolins, and later still, collectin-10 and collectin-11. As well as proteolytic enzymes (MASP-1, MASP-2, MASP-3), the group includes non-enzymatic factors (MAp19, MAp44). In this review, the association-specific factors of the lectin pathway with haematologic malignancies and related infections are discussed.