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Thrombomodulin (TM) is a transmembrane protein that plays an important role in regulating the coagulation system by acting as a cofactor for thrombin in protein C activation. Additionally, TM is involved in inflammation. Previous studies have shown that soluble fragments of TM of varying sizes, which are derived from membrane-bound TM, are present in plasma and urine. Soluble fragments of TM are speculated to exhibit biological activity. Among these, a lectin-like domain fragment (TMD1) is of particular importance. Recombinant TMD1 has previously been shown to attenuate lipopolysaccharide-induced inflammation. Here, we report that thrombin cleaves recombinant soluble TM, which is used for the treatment of disseminated intravascular coagulation associated with sepsis, into TMD1 and a fragment comprising the C-terminal portion of TM (TMD23), the latter of which retains the cofactor activity for activating protein C. Our findings suggest that thrombin not only activates protein C on membrane-bound TM but may also cleave TM to generate TMD1.

Single nucleotide polymorphisms (SNPs) localised to the promoter region of the

We investigated clinical associations of ficolins and mannose-binding lectin (MBL) in 157 patients suffering from acute myeloid leukaemia (AML). Concentrations of ficolin-1, ficolin-2, ficolin-3 and MBL (before chemotherapy) in serum were determined as were selected polymorphisms of the corresponding genes (FCN1, FCN2, FCN3 and MBL2). The control group (C) consisted of 267 healthy unrelated individuals. Median level of ficolin-1 in patients was lower (p p p p = 0.0016, respectively) compared with controls. These findings were generally associated with AML itself, however the highest MBL levels predicted higher risk of severe hospital infections (accompanied with bacteremia and/or fungaemia) (p = 0.012) while the lowest ficolin-1 concentrations tended to be associated with prolonged (> 7 days) fever (p = 0.026). Genotyping indicated an association of G/G homozygosity (corresponding to FCN1 gene − 542 G > A polymorphism) with malignancy [p = 0.004, OR = 2.95, 95% CI (1.41–6.16)]. Based on ROC analysis, ficolin-1, -2 and -3 may be considered candidate supplementary biomarkers of AML. Their high potential to differentiate between patients from non-malignant controls but also from persons suffering from other haematological cancers (multiple myeloma and lymphoma) was demonstrated.

Ficolin-2 is regarded as an important innate immunity factor endowed with both lectin (carbohydrate recognition) qualities and ability to induce complement activation. The aim of this study was to investigate the association of the FCN2 3’-untranslated region (3’UTR) polymorphisms with ficolin-2 expression and perinatal complications in preterm neonates. The sequencing analysis allowed us to identify six 3’UTR polymorphisms with minor allele frequency (MAF) >1%: rs4521835, rs73664188, rs11103564, rs11103565, rs6537958 and rs6537959. Except for rs4521835, all adhered to Hardy-Weinberg expectations. Moreover, rs6537958 and rs6537959 were shown to be in perfect linkage disequilibrium (LD) with nine other genetic polymorphisms: rs7040372, rs7046516, rs747422, rs7847431, rs6537957, rs6537960, rs6537962, rs11462298 and rs7860507 together stretched on a distance of 1242 bp and very high LD with rs11103565. The 3’UTR region was shown to bind nuclear extract proteins. The polymorphisms at rs4521835 and rs73664188 were found to influence serum ficolin-2 concentration significantly. All polymorphisms identified create (together with exon 8 polymorphism, rs7851696) two haplotype blocks. Among 49 diplotypes (D1-D49) created from rs7851696 (G>T), rs4521835 (T>G), rs73664188 (T>C), rs11103564 (T>C), rs11103565 (G>A) and rs6537959 (T>A), twenty two occurred with frequency >1%. Two diplotypes: D13 (GTTTGT/GGTCGT) and D10 (GTTTGT/GGTCGA), were significantly more frequent among preterm neonates with early onset of infection and pneumonia, compared with newborns with no infectious complications (OR 2.69 and 2.81, respectively; both p

Ficolin-2 is regarded as an important innate immunity factor endowed with both lectin (carbohydrate recognition) qualities and ability to induce complement activation. The aim of this study was to investigate the association of the FCN2 3’-untranslated region (3’UTR) polymorphisms with ficolin-2 expression and perinatal complications in preterm neonates. The sequencing analysis allowed us to identify six 3’UTR polymorphisms with minor allele frequency (MAF) >1%: rs4521835, rs73664188, rs11103564, rs11103565, rs6537958 and rs6537959. Except for rs4521835, all adhered to Hardy-Weinberg expectations. Moreover, rs6537958 and rs6537959 were shown to be in perfect linkage disequilibrium (LD) with nine other genetic polymorphisms: rs7040372, rs7046516, rs747422, rs7847431, rs6537957, rs6537960, rs6537962, rs11462298 and rs7860507 together stretched on a distance of 1242 bp and very high LD with rs11103565. The 3’UTR region was shown to bind nuclear extract proteins. The polymorphisms at rs4521835 and rs73664188 were found to influence serum ficolin-2 concentration significantly. All polymorphisms identified create (together with exon 8 polymorphism, rs7851696) two haplotype blocks. Among 49 diplotypes (D1-D49) created from rs7851696 (G>T), rs4521835 (T>G), rs73664188 (T>C), rs11103564 (T>C), rs11103565 (G>A) and rs6537959 (T>A), twenty two occurred with frequency >1%. Two diplotypes: D13 (GTTTGT/GGTCGT) and D10 (GTTTGT/GGTCGA), were significantly more frequent among preterm neonates with early onset of infection and pneumonia, compared with newborns with no infectious complications (OR 2.69 and 2.81, respectively; both p

Background The M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) were identified as intrinsic antigens in primary membranous nephropathy (MN). Complement activation via the lectin pathway in intrinsic antigen-related MN is still unclear. Methods We retrospectively enrolled 60 primary Japanese MN patients and detected activated complement pathways by staining complement proteins in glomerular deposition. According to the findings of PLA2R and THSD7A staining in glomeruli, they were classified into intrinsic antigen-related or -unrelated MN. We evaluated clinicopathological characteristics and predictors of clinical outcomes in intrinsic antigen-related MN. Results Thirty-nine (65%) patients had PLA2R in glomerular deposits and two (3.3%) patients had THSD7A. One of them had both PLA2R and THSD7A (double positive). Forty patients were classified into the intrinsic antigen-related group. The other 20 patients were negative for both antigens (unrelated group). The prevalence and staining intensity of mannose-binding lectin (MBL) deposits were much higher in the intrinsic antigen-related group [55% versus 20%, P

Comprehensive studies of the complement genes in basal vertebrates have revealed that cyclostomes have apparently primitive complement systems whereas bony fish have well-developed complement systems comparable to those of mammals. Here we have performed liver transcriptome analysis of a hammerhead shark, Sphyrna zygaeana, to elucidate the early history of vertebrate complement evolution. Identified genes were; one C1qB, one C1r, one C1s, one MASP-1/-3, one MASP-2, two factor B/C2, one C3, three C4, one C5, one C6, one C7, one C8A, three C8B, one C8G, one C9, two factor I and one S protein. No MBL, ficolin, C1qA or C1qC were found. These results indicate that the lectin, classical, alternative and lytic pathways were established in the common ancestor of jawed vertebrates. In addition to the absence of MBL and ficolin, the MASP transcripts lacked the serine protease domain, suggesting that the lectin pathway was lost in the hammerhead shark lineage.

Infections are a major cause of childhood mortality. We investigated components of the lectin pathway of complement activation in the context of sepsis at both genetic and protein levels in neonates, infants and older children. Major components of the lectin pathway and two genes for Toll-like receptors were studied in 87 neonates with confirmed sepsis and compared with 40 babies with infections who did not develop sepsis (disease controls) and 273 infection-free neonatal controls. A second cohort comprised 47 older children with sepsis and 87 controls. Low MBL-conferring genotypes (LXA/O+O/O) were more frequent in sepsis patients than in healthy controls but no significant differences in the frequency of SNPs of other lectin pathway genes (FCN1, FCN2, FCN3, MASP1/3, MASP2) or TLR receptor genes (TLR2, TLR4) were found. One case of primary MASP-2 deficiency was found among healthy pre-terms and one neonate suffering from SIRS was heterozygous for the rare FCN1 gene mutation, +6658 G>A. Generally, sepsis was associated with low serum MBL and low ficolin-2 concentrations on admission. Among neonates, ficolin-1 and MASP-2 levels were elevated in sepsis relative to healthy, but not disease, controls. Unlike neonates, ficolin-3 and MASP-2 levels were lower in older patients than in healthy controls while no difference was found for ficolin-1. With the possible exception of MBL, inherited lectin pathway insufficiencies do not seem to predispose to sepsis, rather changes in protein concentrations reflect alterations in disease course.

The human ficolins are homooligomers. Each subunit is composed of an N-terminal region including two cysteine residues, a collagen-like domain, a neck region and a fibrinogen-like domain. Electron microscopy of the whole proteins of human ficolin-2 and ficolin-3 show bouquet-like images, similar to those first described for C1q. All human ficolins recognise N-acetylated carbohydrates and compounds such as N-acetylglucosamine (GlcNAc) in common, although the spectrum of ligands for each ficolin is slightly different. Human ficolin-1 binds sialic acid, and human ficolin-3 recognises d-fucose and galactose. Ficolin-2 exhibits broad binding specificities due to it having four distinct binding sites in the fibrinogen-like domain that allow it to bind various carbohydrates, including β-(1,3)-d-glucan and peptideglycan, and sulphated carbohydrates, including heparin, while ficolin-1 and ficolin-3 have only one binding site. In addition to carbohydrates, ficolin-1 binds to tectonin9, and ficolin-2 binds elastin, corticosteroid11 and DNA. Each ficolin is able to bind to a spectrum of bacteria. Ficolin-1 and ficolin-2 interact with C-reactive protein, boosting both the lectin and classical pathway. A low concentration of ficolin-1 is also present in the serum with the mean level of 1 μg/mL. Ficolin-2 and ficolin-3 are expressed in the liver and are secreted into the serum. Two cases of ficolin-3 deficiency due to homozygosity for a FCN3 frameshift mutation (c.349delC, rs28357092) lacking the entire fibrinogen-like in exon 5 leading to the abnormal ficolin-3 lacking the entire fibrinogen-like domain have been reported.

Mycobacterium tuberculosis (TB) may utilise the lectin complement pathway to facilitate entry into its niche within macrophages. Previous studies examining mannose-binding lectin (MBL) in patients with TB have been limited by failure to correlate genotype/phenotype relationships. This study investigated serum levels and genotypes of MBL, Ficolin-2, Ficolin-3 and MASP-2 in 168 patients with pulmonary tuberculosis and 168 age/sex-matched controls. Low serum levels of MBL and Ficolin-2 were defined using cut-offs previously identified in the literature. Median MBL serum levels were higher in TB patients than controls-1400 ng/ml (IQR 435-2520) vs 1030 ng/ml (350-2050), p = 0.02-but this was not mirrored by a difference in MBL haplotype frequencies (MBL deficient haplotypes were observed in 11.9 % of TB patients and 11.3 % of controls). Severe Ficolin-2 deficiency (

Both complement activation and certain infections (including those with Yersinia sp.) may contribute to the pathogenesis of juvenile idiopathic arthritis (JIA). We investigated factors specific for the lectin pathway of complement: mannose-binding lectin (MBL), ficolins and MBL-associated serine protease-2 (MASP-2), in 144 patients and 98 controls. One hundred and six patients had oligoarticular disease and 38 had polyarticular disease. In 51 patients (out of 133 tested), Yersinia-reactive antibodies were found (JIA Ye+ group). MBL deficiency was significantly more frequent in the JIA Ye+ group than in patients without Yersinia-reactive antibodies or in controls. Median serum ficolin-2 level was significantly lower (and proportion of values deemed ficolin-2 insufficient greater) in JIA patients irrespective of their Yersinia antibody status. The minority (C) allele at -64 of the FCN2 gene was less frequent among JIA patients than among control subjects. No differences were found in the frequency of FCN3 gene +1637delC or MASP2 +359 A>G mutations nor for median values of serum ficolin-1, ficolin-3 or MASP-2. However, high levels of serum ficolin-3 were under-represented in patients, in contrast to MBL. MBL, ficolin-1, ficolin-2, ficolin-3 and MASP-2 were also readily detectable in synovial fluid samples but at a considerably lower level than in serum. Our findings suggest a possible role for the lectin pathway in the pathogenesis of JIA, perhaps secondary to a role in host defence, and indicate that investigations on the specificity of lectin pathway recognition molecules towards specific infectious agents in JIA might be fruitful.

Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

We established a system to separately analyze the role of protons and bicarbonate ions in vitro in which the pH of the medium was controlled by HEPES at various concentrations of sodium bicarbonate (NaHCO3) in the absence of carbon dioxide (CO2). Using this system, we demonstrated that acidosis promoted osteoclast formation independently of extracellular NaHCO3 in a short-term culture. Protons and bicarbonate ions acted on osteoclast differentiation with opposite effects, the former positively and the latter negatively. The HEPES-based system maintained pH in the absence of extracellular NaHCO3 without CO2. Therefore, we could demonstrate that osteoblast differentiation was promoted at higher pH in a long-term culture system without NaHCO3 in which ALP activity and nodule mineralization were enhanced. This finding indicates that protons negatively control osteoblast differentiation independently of extracellular bicarbonate ions. However, the difference in the concentration of NaHCO3 did not have any influence on nodule mineralization. The opposite effects of protons, the promotion of osteoclast formation and the inhibition of osteoblast differentiation, were suppressed in the presence of 5 mM N-acetyl cysteine, a reagent activating the scavenging of reactive oxygen species (ROS), implying that ROS act on both systems, the promotion of large osteoclast formation and the deterioration of osteoblast formation under acidosis.

There are more than 70 proteins in chicken egg white, most of which have a role in host defense. We isolated a novel protein from chicken egg white which we termed EW135 by means of polyethylene glycol precipitation and ionexchange chromatography, and an amino acid sequence analysis of its tryptic peptides showed it to be a member of the group B scavenger receptor cysteine-rich domain superfamily. EW135 was speculated to be complexed with a substance (s) in egg white in a Ca 2+ -dependent manner. From a structural point of view, EW135 may play a role in host defense.

Mannose-binding lectin (MBL) and ficolin are complexed with MBL-associated serine proteases, key enzymes of complement activation via the lectin pathway, and act as soluble pattern recognition molecules in the innate immune system. Although numerous reports have revealed the importance of MBL in infectious diseases and autoimmune disorders, the role of ficolin is still unclear. To define the specific role of ficolin in vivo, we generated model mice deficient in ficolins. The ficolin A (FcnA)–deficient (Fcna−/−) and FcnA/ficolin B double-deficient (Fcna−/−b−/−) mice lacked FcnA-mediated complement activation in the sera, because of the absence of complexes comprising FcnA and MBL-associated serine proteases. When the host defense was evaluated by transnasal infection with a Streptococcus pneumoniae strain, which was recognized by ficolins, but not by MBLs, the survival rate was significantly reduced in all three ficolin-deficient (Fcna−/−, Fcnb−/−, and Fcna−/−b−/−) mice compared with wild-type mice. Reconstitution of the FcnA-mediated lectin pathway in vivo improved survival rate in Fcna−/− but not in Fcna−/−b−/− mice, suggesting that both FcnA and ficolin B are essential in defense against S. pneumoniae. These results suggest that ficolins play a crucial role in innate immunity against pneumococcal infection through the lectin complement pathway.

Thrombomodulin (TM) is a transmembrane protein expressed on vascular endothelial cells. TM has anticoagulant and anti-inflammatory properties. It has recently been reported that TM modulates complement, an immune effector system that destroys pathogens and is also involved in inflammation. TM was demonstrated to enhance the degradation of C3b into iC3b by factor I and factor H, indicating that its role is in negative regulation in the alternative pathway of the complement system. In this study, we examined the effects of recombinant human soluble TM protein composed of the extracellular domains (rTM) on the alternative pathway. The degradation of C3b into iC3b by factor I and factor H was enhanced by rTM as assessed by SDS-PAGE, confirming the previous observation. We also found that rTM enhances the cleavage of C3 into C3b as a result of activation of the alternative pathway. These results indicate that TM has both activating and inactivating functions in the alternative pathway.

Serum H-ficolin (ficolin-3) concentrations (n=613) and FCN3 genotypes (n=529) from a large group of neonates are presented. Both pre-term deliveries and low birthweight (independently of gestational age) were significantly associated with low H-ficolin concentrations but not with heterozygosity for the FCN3 1637delC frameshift mutation. The presence of the variant allele, however, apparently influenced the protein level. No association of FCN3 gene heterozygosity or relative functional H-ficolin insufficiency (determined as serum level ≤8.6 μg/ml) with perinatal infections was found. One premature newborn, with confirmed infection caused by Streptococcus agalactiae, was H-ficolin-deficient (FCN3 variant homozygote, no detectable protein). We present what is only the fourth case report of total H-ficolin deficiency in the world literature. This neonate was however previously found to be mannan-binding lectin (MBL) as well as MBL-associated serine protease-2 (MASP-2) deficient and also had low serum L-ficolin.

This guideline was provided by the Japanese Association for Complement Research targeting clinicians for making an accurate diagnosis of hereditary angioedema (HAE), and for prompt treatment of the HAE patient in Japan. This is a 2010 year version and will be updated according to any pertinent medical advancements.

Mannose-binding lectin (MBL)-associated serine proteases (MASPs) are responsible for activation of the lectin complement pathway. Three types of MASPs (MASP-1, MASP-2, and MASP-3) are complexed with MBL and ficolins in serum. Although MASP-1 and MASP-2 are known to contribute to complement activation, the function of MASP-3 remains unclear. In this study, we investigated the mechanism of MASP-3 activation and its substrate using the recombinant mouse MASP-3 (rMASP-3) and several different types of MASP-deficient mice. A proenzyme rMASP-3 was obtained that was not autoactivated during preparation. The recombinant enzyme was activated by incubation with Staphylococcus aureus in the presence of MBL-A, but not MBL-C. In vivo studies revealed the phagocytic activities of MASP-1/3–deficient mice and all MASPs (MASP-1/2/3)–deficient mice against S. aureus and bacterial clearance in these mice were lower than those in wild-type and MASP-2–deficient mice. Sera from all MASPs-deficient mice showed significantly lower C3 deposition activity on the bacteria compared with that of wild-type serum, and addition of rMASP-3 to the deficient serum restored C3 deposition. The low C3 deposition in sera from all MASPs-deficient mice was probably caused by the low level factor B activation that was ameliorated by the addition of rMASP-3. Furthermore, rMASP-3 directly activated factors B and D in vitro. These results suggested that MASP-3 complexed with MBL is converted to an active form by incubation with bacterial targets, and that activated MASP-3 triggered the initial activation step of the alternative complement pathway.

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.

Mannose-binding lectin (MBL), L-ficolin and MBL associated serine protease-2 (MASP-2) are molecules involved in initiation of the lectin pathway (LP) in the complement system. Although MBL deficiency is observed in almost 10% of healthy people, studies of associations between MBL deficiency and end-stage renal disease (ESRD) remain rare. The objective of the present study is to clarify the significance of the LP in maintenance hemodialysis (HD) patients, especially in terms of MBL levels. Two hundred and forty-four HD patients who had been followed up for 74±84months and 199 healthy controls were included in this study. Measurements of serum concentrations of MBL, L-ficolin, and MASP-2 were performed. Low serum MBL levels (

A 59-year-old man was diagnosed with IgG4-related tubulointerstitial nephritis. His symptoms as well as laboratory and imaging findings were improved after initiation of steroid therapy. Serologically, he showed hypocomplementemia (C3 23 mg/dl, C4

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

Mannose-binding lectin (MBL), L-ficolin and H-ficolin are human serum lectins, all of which form complexes with MBL-associated serine proteases (MASP). The lectin-MASP complexes bind to the surface of microbes, leading to activation of the lectin pathway of complement. Enzyme-linked immunosorbent assays (ELISA) of the lectin pathway activity reported so far determined the activity via either MBL or L-ficolin, but an assay of activity via plural host defense lectins has not been established. To measure the lectin pathway activation mediated by plural lectins simultaneously, we developed an ELISA system in which N-acetylglucosamine-pentamer conjugated to dipalmitoylphosphatidylethanolamine (GN5-DPPE) was employed as a ligand for the lectins. In our ELISA system, both purified MBL and L-ficolin isolated from serum diluted in a buffer containing high ionic NaCl bound to GN5-DPPE and activated C4. Purified H-ficolin was not capable of binding to GN5-DPPE. MBL and L-ficolin in MBL-sufficient serum also bound to GN5-DPPE and activated C4. Mannose and N-acetylgalactosamine inhibited binding of MBL and L-ficolin to GN5-DPPE, respectively. MBL-deficient serum that had been depleted of L-ficolin did not exhibit C4 activation, but addition of both or either purified MBL and/or L-ficolin to the serum restored the activation in a dose-dependent manner. Thus, C4 cleaving activity could be evaluated with the co-existence of MBL and L-ficolin in vitro. In conclusion, we propose a novel method using GN5-DPPE for investigating the MBL- and L-ficolin-dependent lectin pathway and anticipate that this method will be useful in innate immunity and clinical research.

Ficolins are a group of oligomeric lectins with subunits consisting of both collagen-like and fibrinogen-like domains. The majority of ficolins identified in vertebrates and invertebrates to date recognize N-acetylglucosamine (GlcNAc). X-ray crystallographic analysis of human ficolins has shown that the fibrinogen-like domain binds to the N-acetylated moiety. Ficolins in serum are associated with MBL-associated serine protease (MASP). The ficolin-MASP complex binds directly to carbohydrates present on the surface of a variety of Gram-positive and Gram-negative bacteria through ficolin. Binding of the complex initiates complement activation via the lectin pathway, leading to generation of opsonic fragments of complement components, such as C3b, and to lysis of the bacteria by the membrane attack complex. Thus, serum ficolins play an important role in innate immunity.

Ficolin and mannose-binding lectin (MBL) are animal lectins that are involved in innate immunity by initiating the lectin complement pathway. Here, we report that interactions between these lectins and fibrinogen/fibrin augment the lectin pathway. An ELISA revealed that recombinant mouse ficolin A (rFcnA), rMBL-A and rMBL-C bind to fibrinogen in a dose-dependent manner. Affinity Western blotting showed that these lectins bind to the Aα- and Bβ-chains of fibrinogen and the α- and β-chains of fibrin, but not to the γ-chain, and that rMBL-A and rMBL-C preferentially bind to the α- and β-chains. The C4 deposition activity on Fbg-coated plates was observed by using mouse serum, and the deposition on GlcNAc-coated plates was enhanced by fibrinogen supplementation and further enhanced by the addition of thrombin. Similar effects of fibrinogen and fibrin were observed in the bindings of these lectins to a Gram-positive pathogen, Staphylococcus aureus, and in the subsequent C3 deposition on the bacteria. In particular, the lectin pathway, through MBLs, seemed to synchronize with blood coagulation. Therefore, it is suggested that the lectin pathway collaborates with the coagulation system in the first-line host defense against pathogens under conditions such as injury and inflammation.

Udgivelsesdato: 2009-May One collectin (mannan-binding lectin, MBL) and three ficolins (M-ficolin/ficolin-1, L-ficolin/ficolin-2 and H-ficolin/ficolin-3) share the capability to activate complement via the lectin pathway. This property depends on the ability of these lectins to form complexes with MBL-associated serine proteases (MASPs), particularly MASP-2. We report the results of an investigation of cord blood MASP-2 concentrations in a large, ethnically homogeneous cohort (n=1788) of neonates. The median value of MASP-2 in cord sera was determined to be 93 ng/ml (range

Ficolins and one collectin, mannan-binding lectin (MBL), are the only factors known to activate the lectin pathway (LP) of complement. There is considerable circumstantial evidence that MBL insufficiency can increase susceptibility to various infections and influence the course of several non-infectious diseases complicated by infections. Much less information is available concerning l-ficolin. We report the results of a prospective study to investigate any association between either MBL deficiency or l-ficolin deficiency with prematurity, low birthweight or perinatal infections in a large cohort of Polish neonates, representing an ethnically homogenous population (n=1832). Cord blood samples were analysed to determine mbl-2 gene variants, MBL concentrations and MBL-MASP-2 complex activities (MBL-dependent lectin pathway activity) as well as l-ficolin levels. Median concentrations of l-ficolin and MBL were 2500 and 1124 ng/ml, respectively, while median LP activity was 272 mU/ml. After genotyping, 60.6% of babies were mbl-2 A/A, 35.4% were A/O and 4% were O/O genotypes. We found relative l-ficolin deficiency to be associated with prematurity, low birthweight and infections. l-Ficolin concentration correlated with gestational age and with birthweight, independently of gestational age. Preterm deliveries (

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K(D) of 1.5 x 10(-8) and 3.2 x 10(-9)M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 to be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.

The aim of the present study was to elucidate the correlation between complement pathways and clinicopathological findings in post-streptococcal acute glomerulonephritis (PSAGN). Immunohistological staining was performed on renal specimens obtained from 18 patients with PSAGN and 20 controls, using antibodies against IgG, IgA, IgM, C1q, C3c, C4, fibrinogen, factor B, C4-binding protein (C4-bp), C5b-9, CD59, mannose-binding lectin (MBL) and MBL-associated serine protease-1 (MASP-1). Controls showed no deposition of any antibody. In seven patients, glomerular deposits of C3c, C4, factor B, C4-bp, C5b-9, CD59, MBL and MASP-1 were found. In the remaining 11 patients, glomerular deposits of neither C4 nor MBL/MASP-1 were found, and glomerular deposits of C3c, factor B, C5b-9 and CD59 were evident. C4-bp was detected in seven of these 11 patients. Glomerular deposits of fibrinogen were detected in five of seven patients with MBL/MASP-1 deposits and in only two of 11 patients without MBL/MASP-1 deposits. Hematuria was prolonged in three of seven patients with MBL/MASP-1 deposits through follow up, whereas urinalysis was normal in all patients without MBL/MASP-1 deposits. However, the histological indicators were not different between the two groups. To the authors' knowledge this is the first report to show that complement activation through both the alternative and lectin pathways is evident in some patients with PSAGN. Complement activation is promoted in situ in the glomerulus.

The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6 -/- mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6 -/- mice and C1qtnf6 -/- embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H 2 O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases., Nature Communications, 6, 8483; 2015

In the innate immune system, a variety of recognition molecules provide the first-line host defense to prevent infection and maintain endogenous homeostasis. Ficolin is a soluble recognition molecule, which senses pathogen-associated molecular patterns on microbes and aberrant sugar structures on self-cells. It consists of a collagen-like stalk and a globular fibrinogen-like domain, the latter binding to carbohydrates such as N-acetylglucosamine. Ficolins have been widely identified in animals from higher invertebrates to mammals. In mammals, ficolins form complexes with mannose-binding lectin-associated serine proteases (MASPs), and ficolin-MASP complexes trigger complement activation via the lectin pathway. Once activated, complement mediates many immune responses including opsonization, phagocytosis, and cytokine production. Although the precise function of each ficolin is still under investigation, accumulating information suggests that ficolins have a crucial role in host defense by recognizing a variety of microorganisms and interacting with effector proteins.

In the innate immune system, a variety of recognition molecules provide the first-line host defense to prevent infection and maintain endogenous homeostasis. Ficolin is a soluble recognition molecule, which senses pathogen-associated molecular patterns on microbes and aberrant sugar structures on self-cells. It consists of a collagen-like stalk and a globular fibrinogen-like domain, the latter binding to carbohydrates such as N-acetylglucosamine. Ficolins have been widely identified in animals from higher invertebrates to mammals. In mammals, ficolins form complexes with mannose-binding lectin-associated serine proteases (MASPs), and ficolin-MASP complexes trigger complement activation via the lectin pathway. Once activated, complement mediates many immune responses including opsonization, phagocytosis, and cytokine production. Although the precise function of each ficolin is still under investigation, accumulating information suggests that ficolins have a crucial role in host defense by recognizing a variety of microorganisms and interacting with effector proteins.

Mannose-binding lectin (MBL) and ficolins are pattern recognition proteins acting in innate immunity, and they trigger the activation of the lectin complement pathway through MBL-associated serine proteases (MASPs). Upon activation of the lectin pathway, MASP-2 cleaves C4 and C2. A truncated form of MASP-2, named small MBL-associated protein (sMAP), is also associated with MBL/ficolin-MASP complexes. To clarify the role of sMAP, we have generated sMAP-deficient (sMAP−/−) mice by targeted disruption of the sMAP-specific exon. Because of the gene disruption, the expression level of MASP-2 was also decreased in sMAP−/− mice. When recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in deficient serum, the binding of these recombinant proteins to MBL was competitive, and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2, whereas the addition of rsMAP attenuated the activity. Therefore, MASP-2 is essential for the activation of C4 and sMAP plays a regulatory role in the activation of the lectin pathway.

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.

IgA nephropathy (IgAN) is characterized by glomerular co-deposition of IgA and complement components. Earlier studies showed that IgA activates the alternative pathway of complement, whereas more recent data also indicate activation of the lectin pathway. The lectin pathway can be activated by binding of mannose-binding lectin (MBL) and ficolins to carbohydrate ligands, followed by activation of MBL-associated serine proteases and C4. This study examined the potential role of the lectin pathway in IgAN. Renal biopsies of patients with IgAN (n=60) showed mesangial deposition of IgA1 but not IgA2. Glomerular deposition of MBL was observed in 15 (25%) of 60 cases with IgAN and showed a mesangial pattern. All MBL-positive case, but none of the MBL-negative cases showed glomerular co-deposition of L-ficolin, MBL-associated serine proteases, and C4d. Glomerular deposition of MBL and L-ficolin was associated with more pronounced histologic damage, as evidenced by increased mesangial proliferation, extracapillary proliferation, glomerular sclerosis, and interstitial infiltration, as well as with significantly more proteinuria. Patients who had IgAN with or without glomerular MBL deposition did not show significant differences in serum levels of MBL, L-ficolin, or IgA or in the size distribution of circulating IgA. Furthermore, in vitro experiments showed clear binding of MBL to polymeric but not monomeric patient IgA, without a significant difference between both groups. Together, these findings strongly point to a role for the lectin pathway of complement in glomerular complement activation in IgAN and suggest a contribution for both MBL and L-ficolin in the progression of the disease.

Background/Aims: Mannose-binding lectin (MBL) is characteristic of an acute-phase-reacting protein like C-reactive protein (CRP). However, the prognostic value of the serum MBL level has not been examined. The aim of this study was to evaluate whether the serum MBL level can predict all-cause mortality in hemodialysis (HD) patients. Methods: A total of 131 patients without active infection, who had been on maintenance HD for at least 2 years, were included in this study. The serum MBL, high-sensitivity CRP (hs-CRP) level, nutrition markers, and biochemical parameters were measured in June 1999. The cohort was then followed prospectively for 36 months, and clinical data were recorded. Results: The MBL level of the 131 HD patients was 9.054 ± 5.115 µg/ml (mean ± SD). During the follow-up period, 18 patients (9 males and 9 females) died and 113 (64 males and 49 females) survived. The two leading causes of death were cardiovascular events (n = 6, 33.3%) and infection (n = 4, 22.2%). The serum MBL level was significantly lower among the nonsurvivors (6.596 ± 4.990 µg/ml) than among the survivors (9.445 ± 5.046 µg/ml; p < 0.05). There was a significant, although very weak, correlation between the MBL level and albumin level (p < 0.05), but there was no correlationbetween the MBL level and the hs-CRP level. The patients were divided into two groups according to the serum MBL level (5 µg/ml). Multivariate analysis of factors predicting all-cause mortality in multivariate logistic regression analysis identified a serum MBL level Conclusions: Our findings suggest that the serum MBL level is a significant predictor of outcome in HD patients. HD patients with a low level of serum MBL should be carefully monitored.

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a coimmunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.

The ficolin 1, 2 and 3 (derived from the FCN1, 2 and 3 genes, respectively) are homologous soluble pattern recognition molecules of importance for innate immunity, comprising collagen-like and fibrinogen-like domains, binding to sugar groups on different types of microorganisms. Serum concentration of Ficolin-2 varies considerably in healthy individuals. Thus, we speculated whether this could be due to variations in the FCN2 gene. We sequenced the promoter region and the exons and intron-exon boundaries of FCN2 in Danish Caucasians. For comparison, FCN1 and FCN3 were also investigated. Ficolin-2 concentrations were measured in serum and the functional relevance of amino acid substituting polymorphisms in FCN2 was investigated by binding to and recovery from N-acetylglucosamine (GlcNAc). Both FCN1 and FCN2 contained polymorphisms in the promoters and structural parts of the genes, but only polymorphisms in FCN2 resulted in amino acid exchanges. FCN2 promoter polymorphisms were associated with marked changes in the Ficolin-2 serum concentration, whereas two polymorphisms clustered in the exon encoding the fibrinogen-like domain were associated with increased and decreased GlcNAc binding, respectively. In FCN3, only a single frame-shift deletion in exon 5 was detected. These results show that the FCN genes are polymorphic and that particularly FCN2 harbors functional polymorphic sites that regulate both the expression as well as the function of Ficolin-2, which may have pathophysiological implications for innate immunity.