Simultaneous activation of complement and coagulation by MBL-associated serine protease 2

The complement system is an important immune mechanism mediating both recognition and elimination of foreign bodies. The lectin pathway is one pathway of three by which the complement system is activated. The characteristic protease of this pathway is Mannan-binding lectin (MBL)-associated serine protease 2 (MASP2), which cleaves complement proteins C2 and C4. We present a novel and alternative role of MASP2 in the innate immune system. We have shown that MASP2 is capable of promoting fibrinogen turnover by cleavage of prothrombin, generating thrombin. By using a truncated active form of MASP2 as well as full-length MASP2 in complex with MBL, we have shown that the thrombin generated is active and can cleave both factor XIII and fibrinogen, forming cross-linked fibrin. To explore the biological significance of these findings we showed that fibrin was covalently bound on a bacterial surface to which MBL/MASP2 complexes were bound. These findings suggest that, as has been proposed for invertebrates, limited clotting may contribute to the innate immune response.     

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.     

Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene

Mannan-binding lectin (MBL) forms a multimolecular complex with at least two MBL-associated serine proteases, MASP-1 and MASP-2. This complex initiates the MBL pathway of complement activation by binding to carbohydrate structures present on bacteria, yeast, and viruses. MASP-1 and MASP-2 are composed of modular structural motifs similar to those of the C1q-associated serine proteases C1r and C1s. Another protein of 19 kDa with the same N-terminal sequence as the 76-kDa MASP-2 protein is consistently detected as part of the MBL/MASP complex. In this study, we present the primary structure of this novel MBL-associated plasma protein of 19 kDa, MAp19, and demonstrate that MAp19 and MASP-2 are encoded by two different mRNA species generated by alternative splicing/polyadenylation from one structural gene.     

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.     

Responses of alternative complement expression to challenge with different combinations of Vibrio anguillarum, Escherichia coli and Staphylococcus aureus: evidence for specific immune priming in amphioxus Branchiostoma belcheri

The existence of specific immunological priming in protection upon secondary exposure in invertebrates remains controversial. By exploring the changes in the expression patterns of Bf, C3 and C6, key genes involved in the alternative complement pathway (AP), after challenge with different combinations of three bacteria Vibrio anguillarum, Escherichia coli and Staphylococcus aureus, we show that re-exposure to the same species of bacteria or the different species of the same (Gram-negative or Gram-positive) class of bacteria results in a significant increase in the expression of Bf, C3 and C6, and an earlier occurrence of gene expression peak compared with the first exposure in amphioxus Branchiostoma belcheri; in contrast, re-exposure to the different class of bacteria did not induce such responses. These characteristics appear to bear some analogy to the immunological memory in vertebrates, suggesting that amphioxus B. belcheri possesses the ability to discriminate between classes of microorganisms. Moreover, our results for the first time establish a link between the alternative complement components and the specific immune priming.     

Molecular and immunochemical demonstration of a novel member of Bf/C2 homolog in amphioxus Branchiostoma belcheri: implications for involvement of hepatic cecum in acute phase response

A complement system operating via the alternative pathway (AP) similar to that of vertebrates has been demonstrated in the primitive chordate amphioxus. However, the factor B (Bf), a key specific protease in the AP, remains elusive in amphioxus to date. We demonstrate in this study the presence of a factor B-like protein in amphioxus Branchiostoma belcheri by both immunoblotting and molecular cloning. The factor B-like protein was immunohistochemically localized in the hepatic cecum. The B. belcheri factor B-like gene, BbBf/C2, encoded a mosaic protein with three complement control protein (CCP) domains, a von Willebrand factor A (vWFA) domain and a serine protease (SP) domain. Peculiarly, BbBf/C2 had an epidermal growth factor-like domain (EGF_CA) located between CCP1 and CCP2, therefore BbBf/C2 had a modular structure of CCP-EGF_CA-CCP-CCP-vWFA-SP, making it a novel member of Bf/C2 family proteins. Real-time PCR assay revealed that lipopolysaccharide (LPS) challenge resulted in a quick and continuously significant up-regulation of BbBf/C2 expression in the hepatic cecum, while BbBf/C2 was only expressed for a short time in the hind-gut following LPS challenge though the expression level was temporarily higher than that in the hepatic cecum. Similarly, immuno-dot blotting showed that challenge with LPS triggered a significant elevation of BbBf/C2 synthesis in the hepatic cecum and hind-gut, with a higher rise in the former tissue. These results indicate that both hepatic cecum and hind-gut may be involved in the immune response induced by LPS, but the hepatic cecum, like the vertebrate liver, is the primary tissue synthesizing BbBf/C2 in response to LPS challenge, thereby playing a major role in the acute phase response.     

Molecular cloning, characterization and expression of the complement component Bf/C2 gene in grass carp

The complement system is an integral part of the host immune system and plays an immunoregulatory role at the interface between the innate and acquired immune responses. Factor B (Bf) serves as the catalytic subunit of complement C3 convertase in the alternative pathway (AP), while in the classical pathway (CP), this function is subjected to C2. In this study, we cloned and characterized the two Bf/C2 genes of grass carp, gcBf/C2A and gcBf/C2B. The gcBf/C2A and gcBf/C2B cDNA sequences are 2259 and 3004 bp in length, and the open reading frames (ORFs) of gcBf/C2A and gcBf/C2B were found to encode peptides of 752 and 837 amino acids, respectively. The genes share 30.7% amino acid identity with each other and 32.4-38.3% and 31.4-33% with the Bf and C2 genes in humans and mice. GcBf/C2A and gcBf/C2B were expressed in a wide range of grass carp tissues, with the highest level of expression of both genes detected in the liver. After a challenge with Aeromonas hydrophila, gcBf/C2A was significantly upregulated, especially at 4 h after infection, and the significantly higher expression of gcBf/C2B (27.3-fold) was found in the head kidney at 24 h post-challenge. The expression of gcBf/C2A was quickly upregulated at 1 day post-hatching and peaked at 5 days post-hatching. The maximum expression of gcBf/C2B was found at 1 day post-hatching. In conclusion, our data enables a better understanding of the physiological function of the Bf/C2 complement genes in vertebrates.     

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.     

Alternative complement pathway activation is essential for inflammation and joint destruction in the passive transfer model of collagen-induced arthritis

Activation of each complement initiation pathway (classical, alternative, and lectin) can lead to the generation of bioactive fragments with resulting inflammation in target organs. The objective of the current study was to determine the role of specific complement activation pathways in the pathogenesis of experimental anti-type II collagen mAb-passive transfer arthritis. C57BL/6 mice were used that were genetically deficient in either the alternative pathway protein factor B (Bf(-/-)) or in the classical pathway component C4 (C4(-/-)). Clinical disease activity was markedly decreased in Bf(-/-) compared with wild-type (WT) mice (0.5 +/- 0.22 (n = 6) in Bf(-/-) vs 8.83 +/- 0.41 (n = 6) in WT mice (p < 0.0001)). Disease activity scores were not different between C4(-/-) and WT mice. Analyses of joints showed that C3 deposition, inflammation, pannus, cartilage, and bone damage scores were all significantly less in Bf(-/-) as compared with WT mice. There were significant decreases in mRNA levels of C3, C4, CR2, CR3, C3aR, and C5aR in the knees of Bf(-/-) as compared with C4(-/-) and WT mice with arthritis; mRNA levels for complement regulatory proteins did not differ between the three strains. These results indicate that the alternative pathway is absolutely required for the induction of arthritis following injection of anti-collagen Abs. The mechanisms by which these target organ-specific mAbs bypass the requirements for engagement of the classical pathway remain to be defined but do not appear to involve a lack of alternative pathway regulatory proteins.     

Carp (Cyprinus carpio L.) innate immune factors are present before hatching

Expression of the innate immune factors, complement factor 3 (C3), alpha2-macroglobulin (alpha2M), serum amyloid A (SAA) and a complement factor 1 r/s--mannose binding lectin associated serine protease-like molecule (C1/MASP2), was determined with Real Time Quantitative-PCR in carp (Cyprinus carpio L.) ontogeny around hatching. Furthermore, the expression of C3 mRNA and the presence of C3 protein were studied in carp embryos and larvae using In Situ Hybridisation, Western Blotting and Immunohistochemistry. C3, alpha2M, SAA and C1/MASP2 mRNA were produced by embryos from 12 h post-fertilisation, which is relatively long before hatching (2 days post-fertilisation (dpf)), indicating either involvement of these factors in development itself or more probably a preparation of the immune system for the post-hatching period. In addition, maternal mRNA of the aforementioned innate immune factors and maternal C3- and immunoglobulin protein was present in unfertilised eggs. Furthermore, C3 mRNA production was situated in the yolk syncytial layer in embryos from 24 h post-fertilisation to 5 dpf, followed by the liver in larvae, providing a new sequence of C3 production in teleost development.     

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.     

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin 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。     

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.     

Ovarian steroid-regulated synthesis and secretion of complement C3 and factor B in mouse endometrium during the natural estrous cycle and pregnancy period.

We demonstrate the presence of complement factor B (Bf) and complement C3 in uterine luminal fluid collected from estrogen-stimulated immature and adult female mice. We examined the synthesis and secretion of these two proteins in mouse endometrium at various stages of the natural estrous cycle and during the pregnancy period. The mRNA levels of these two proteins increased markedly in proestrus and estrus and declined sharply in metestrus to an undetectable level. The Bf mRNA remained undetectable, whereas a readily detectable C3 mRNA level reappeared, in diestrus. Meanwhile, these two proteins were immunolocalized to the apical cytoplasm of glandular and luminal epithelial cells of the endometrium during the estrous cycle. Administration of an estrogenic steroid to immature or ovariectomized adult mice markedly stimulated the expression of Bf, C3, and their RNA messages in the endometrium, whereas injection of progesterone alone to ovariectomized animals did not stimulate their expression. Expression of C3 was remarkably enhanced, whereas that of Bf changed only slightly, after injection of combined estrogen and progesterone to ovariectomized animals. In pregnant mice (Day [D] 1 = day of vaginal plug), Bf mRNA was at a high level on D1 and D2, dropped to an almost undetectable level from D3 to D8, and then increased to a low level thereafter until delivery. The C3 mRNA was at a high level on D1, dropped on D2 to an almost undetectable level from D3 to D9, increased to a very high level from D10 to D18, and then declined sharply before delivery. Immunohistochemical patterns of both proteins in the endometrium during preimplantation were positively correlated with changes in their mRNA levels.     

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.