The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.     

Characterization of an equine mannose-binding lectin and its roles in disease

The mannose-binding lectin (MBL), a pattern recognition serum protein, participates in the innate immune system of mammals as an opsonin. In humans, MBL plays a key role in first-line host defense against infection during the lag period prior to the development of a specific immune response. MBL also activates complement via the lectin pathway that requires a MBL-associated serine protease-2 (MASP-2). Homologues of human MBL (hMBL) have been identified in a variety of mammals, fish, and primitive animals such as ascidians. In this study, we report that equine MBL (eMBL) has properties that are similar to hMBL. In addition, we found low levels of MBL:MASP activity in sick horses compared to healthy horses. These results suggest that eMBL is involved in the immune response of the horse and that low MBL:MASP activity could be used to monitor immune function and clinical outcome.     

Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.     

Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio)

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.     

MBL调控MASP激活补体系统

甘露聚糖结合凝集素(MBL;或甘露聚糖结合蛋白,MBP)是由相同的多肽链组成的寡聚物,它通过结合细胞表面的碳水化合物能够有效地识别侵入体内的多种致病微生物,并激活补体来杀灭病原微生物。MBL能与血清中其相关蛋白酶(MASP)结合,MASP包含3个丝氨酸蛋白酶MASP-1、MASP-2、MASP-3和非酶蛋白MAp19。研究显示,MBL通过2种机理调控MASP-2的活性,在先天性免疫中具有重要的作用。本文简要综述MBL调控MASP激活补体的作用机理。     

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.     

Scabies Mite Inactive Serine Proteases Are Potent Inhibitors of the Human Complement Lectin Pathway

Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.     

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.     

An ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of approximately 36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.     

Disease-causing mutations in genes of the complement system

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of “conventional” complement deficiencies with these newly described developmental roles.     

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.     

Mitochondria and the Lectin Pathway of Complement

Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.     

Mannose-binding lectin-associated serine protease-1 is a significant contributor to coagulation in a murine model of occlusive thrombosis

Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl(3) treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl(3)-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl(3)-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A(-/-), or MBL-C(-/-) sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.     

Molecular cloning of the complement C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio)

The classical pathway of complement composed of C1, C4, and C2 is an antibody-dependent activation cascade that is present in jawed vertebrates. C1 is a Ca2+-dependent complex of C1q, C1r, and C1s, and analogous to an initiation complex of the lectin pathway of complement, which consists of the mannose-binding lectin (MBL) homologous to C1q and the MBL-associated serine proteases (MASPs) homologous to C1r and C1s. Thus divergence of Clq and MBL and that of C1r, C1s and the MASPs are considered to be crucial events in the establishment and evolution of the classical complement pathway. However, molecular information on the C1 subcomponents is very limited in lower vertebrates. Here we describe two distinct C1r/C1s/MASP2-like cDNA clones (C1r/s-A, C1r/s-B) isolated from the common carp (Cyprinus carpio). They share 83% identity at the amino acid level and have a domain structure similar to that of C1r/C1s/MASPs from other species. The serine protease domain of the carp homologues lacks the histidine loop and is encoded by a single exon containing an AGY codon for the active serine residue, as in mammalian C1r, C1s, and MASP2. Southern blot and PCR analyses indicated that the carp has at least three copies of the C1r/s-A gene and a single C1r/s-B gene. Although phylogenetic tree analysis does not definitively assign carp C1r/s-A and C1r/s-B, they might represent ancestral molecules which later diverged into C1r, C1s, and MASP2 of higher vertebrates.     

Cloning and characterization of mannose-binding lectin from lamprey (Agnathans)

The recognition of pathogens is mediated by a set of pattern recognition molecules that recognize conserved pathogen-associated molecular patterns shared by broad classes of microorganisms. Mannose-binding lectin (MBL) is one of the pattern recognition molecules and activates complement in association with MBL-associated serine protease (MASP) via the lectin pathway. Recently, an MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian. This ascidian lectin has a carbohydrate recognition domain, but the collagen-like domain was replaced by another sequence. To elucidate the origin of MBLs, the aim of this study is to determine the structure and function of the MBL homolog in lamprey, the most primitive vertebrate. Using an N-acetylglucosamine (GlcNAc)-agarose column, MBL-like lectin (p25) was isolated from lamprey serum and cDNA cloning was conducted. From the deduced amino acid sequence this lectin has a collagenous region and a typical carbohydrate recognition domain. This lectin also binds mannose, glucose, and GlcNAc, but not galactose, indicating that it is structurally and functionally similar to the mammalian MBLs. Furthermore, it associated with lamprey MASPs, and the MBL-MASP activated lamprey C3 in fluid-phase and on the surface of pathogens. In conjunction with the phylogenetic analysis, it seems likely that the lamprey MBL is an ortholog of the mammalian MBL. Because acquired immunity seems to have been established only from jawed vertebrates onward, the lectin complement pathway in lamprey, as one of the major contributors to innate immunity, plays a pivotal role in defending the body against microorganisms.     

Human mannose-binding lectin and L-ficolin function as specific pattern recognition proteins in the lectin activation pathway of complement

The innate immune response in vertebrates and invertebrates requires the presence of pattern recognition receptors or proteins that recognize microbial cell components including lipopolysaccharide, bacterial peptidoglycan (PGN), and fungal 1,3-beta-D-glucan. We reported previously that PGN and 1,3-beta-D-glucan recognition proteins from insect hemolymph were able to induce the activation of the prophenoloxidase-activating system, one of the major invertebrate innate immune reactions. The goal of this study was to characterize the biochemical properties and effects of the human counterparts of these molecules. Soluble pattern recognition proteins were purified from human serum and identified as human mannose-binding lectin (MBL) and L-ficolin. The use of specific microbial cell component-coupled columns demonstrated that MBL and L-ficolin bind to PGN and 1,3-beta-D-glucan, respectively. Purified MBL and L-ficolin were associated with MBL-associated serine proteases-1 and -2 (MASPs) and small MBL-associated protein as determined by Western blot analysis. Finally, the binding of purified MBL/MASP and L-ficolin/MASP complexes to PGN and 1,3-beta-D-glucan, respectively, resulted in the activation of the lectin-complement pathway. These results indicate that human PGN and 1,3-beta-D-glucan recognition proteins function as complement-activating lectins.     

The role of ficolins in the lectin pathway of innate immunity

Ficolins are a family of oligomeric proteins consisting of an N-terminal collagen-like domain and a C-terminal globular fibrinogen-like domain. They are novel lectins that employ the fibrinogen-like domain as a functional domain. Ficolins specifically recognize N-acetyl compounds such as N-acetylglucosamine, components of bacterial and fungal cell walls, and certain bacteria. Like mannose-binding lectin (MBL), ficolins circulate in complexes with MBL-associated serine proteases (MASPs). MASP complexes form with ficolins and MBL, thereby activating the complement through the lectin pathway. Upon binding of ficolins and MBL to carbohydrates on pathogens, MASPs convert to active forms, and subsequently activate the complement. The activated complements lead to pathogen phagocytosis, aggregation and lysis. In humans, three ficolins (L-, M- and H-ficolins) have been identified, which exhibit differences in tissue expression, protein location site, ligand-binding and bacteria-recognition, suggesting a specific role of each ficolin. In addition, these ficolins form complexes with three MASPs (MASP-1, MASP-2 and MASP-3) and two nonenzymatic proteins (sMAP and MAP-1), suggesting a highly sophisticated organization and regulated activation of the ficolin-dependent lectin pathway. This review provides an overview of our current knowledge of ficolins, especially human ficolins and their mouse homologues. We also discuss their possible physiological roles in innate immunity, especially their defensive role against bacterial infection.