Association of the FCN2 Gene Promoter Region Polymorphisms with Very Low Birthweight in Preterm Neonates.

Single nucleotide polymorphisms (SNPs) localised to the promoter region of the FCN2 gene are known to influence the concentration of ficolin-2 in human serum and therefore potentially have clinical associations. We investigated the relationships between SNPs at positions −986 (A > G), −602 (G > A), −64 (A > C) and −4 (A > G) and clinical complications in 501 preterms. Major alleles at positions −986 and −64 and A/A homozygosity for both polymorphisms were less frequent among babies with very low birthweight (VLBW, ≤1500 g) compared with the reference group (OR = 0.24, p = 0.0029; and OR = 0.49, p = 0.024, respectively for A/A genotypes). A lower frequency of G/G homozygosity at position −4 was associated with gestational age <33 weeks and VLBW (OR = 0.38, p = 0.047; and OR = 0.07, p = 0.0034, respectively). The AGAG haplotype was protective for VLBW (OR = 0.6, p = 0.0369), whilst the GGCA haplotype had the opposite effect (OR = 2.95, p = 0.0249). The latter association was independent of gestational age. The AGAG/GGAA diplotype favoured both shorter gestational age and VLBW (OR = 1.82, p = 0.0234 and OR = 1.95, p = 0.0434, respectively). In contrast, AGAG homozygosity was protective for lower body mass (OR = 0.09, p = 0.0155). Our data demonstrate that some FCN2 variants associated with relatively low ficolin-2 increase the risk of VLBW and suggest that ficolin-2 is an important factor for fetal development/intrauterine growth.

Glomerular mannose-binding lectin deposition in intrinsic antigen-related membranous nephropathy.

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 < 0.010, 1.0 (interquartile range 1.0-2.0) versus 1.0 (0.0-1.0), P = 0.01, respectively]. The staining intensity of MBL in glomeruli also correlated with the IgG4 staining intensity. In intrinsic antigen-related MN, MBL staining intensity was an unfavorable predictor for remission of proteinuria [hazard ratio (HR) 0.40, P < 0.01] and renal dysfunction (HR 3.81, P = 0.01) in Cox proportional hazards analysis. Moreover, the glomerular MBL-positive group showed more severe interstitial fibrosis and worse clinical outcomes. Conclusions: Intrinsic antigen-related MN was more strongly associated with complement activation by the lectin pathway, which may contribute to a less favorable clinical outcome.

Lectin pathway factors in patients suffering from juvenile idiopathic arthritis.

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.

Glycoepitopes of staphylococcal wall teichoic acid govern complement-mediated opsonophagocytosis via human serum antibody and mannose-binding lectin.

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or ß-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, ß-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ß-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact ß-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-ß-GlcNAc-specific WTA IgGs, anti-ß-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.

Proton concentrations can be a major contributor to the modification of osteoclast and osteoblast differentiation, working independently of extracellular bicarbonate ions.

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.

Mice deficient in ficolin, a lectin complement pathway recognition molecule, are susceptible to Streptococcus pneumoniae infection.

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.

Specific serum Ig recognizing staphylococcal wall teichoic acid induces complement-mediated opsonophagocytosis against Staphylococcus aureus.

Wall teichoic acid (WTA) of Staphylococcus aureus is a major cell envelope-associated glycopolymer that is a key molecule in promoting colonization during S. aureus infection. The complement system plays a key role in the opsonization and clearance of pathogens. We recently reported that S. aureus WTA functions as a ligand of human serum mannose-binding lectin (MBL), a recognition molecule of the lectin complement pathway. Intriguingly, serum MBL in adults does not bind to WTA because of an inhibitory effect of serum anti-WTA-IgG. In this study, serum anti-WTA-IgG was purified to homogeneity using a purified S. aureus WTA-coupled affinity column to examine the biological function of human anti-WTA-IgG. The purified anti-WTA-IgG contained the IgG2 subclass as a major component and specifically induced C4 and C3 deposition on the S. aureus surface in the anti-WTA-IgG-depleted serum, but not in C1q-deficient serum. Furthermore, the anti-WTA-IgG-dependent C3 deposition induced phagocytosis of S. aureus cells by human polymorphonuclear leukocytes. These results demonstrate that serum anti-WTA-IgG is a real trigger for the induction of classical complement-dependent opsonophagocytosis against S. aureus. Our results also support the fact that a lack of the lectin complement pathway in MBL-deficient adults is compensated by Ag-specific, Ab-mediated adaptive immunity.

Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.

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.

Ficolin-2 and ficolin-3 in women with malignant and benign ovarian tumours.

Ficolins are serum pattern recognition molecules. They have opsonic properties and are able to activate complement via the lectin pathway. This paper reports investigations concerning ficolin-2 and ficolin-3 in ovarian cancer (OC). Their serum levels, single nucleotide polymorphisms of the corresponding FCN2 and FCN3 genes and specific mRNA expression in ovarian sections were investigated in 128 patients suffering from primary OC and 197 controls operated on for reasons other than malignancies. The latter consisted of two reference groups: those with benign tumours (n = 123) and those with normal ovaries (NO) (n = 74). Serum ficolin-2 and ficolin-3 concentrations were higher among patients with malignant disease when compared with either of the reference groups. A significant correlation between ficolin-2 and ficolin-3 concentrations was found, while no correlations with CA125 antigen or CRP were observed. No differences in the frequency of single nucleotide polymorphisms at sites -64, -4 (promoter), +6359, or +6424 (exon 8) (FCN2 gene) nor in the frame-shift mutation 1637delC (FCN3 gene) were found between investigated groups. In contrast to serum concentrations, the expression of FCN2 gene (reported for the first time in ovarian sections) was significantly lower in women with OC in comparison with patients with NO but not with benign ovarian tumours. In case of FCN3 gene, its expression levels in OC group inversely correlated with serum ficolin-3 and were lower in comparison with controls.

The role of mannose-binding lectin-associated serine protease-3 in activation of the alternative complement pathway.

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.

Association of low ficolin-2 concentration in cord serum with respiratory distress syndrome in preterm newborns.

Introduction: Ficolin-2 is a serum pattern recognition molecule, involved in complement activation via the lectin pathway. This study aimed to investigate the association of ficolin-2 concentration in cord blood serum with complications related to premature birth. Methods: 546 premature neonates were included. The concentration of ficolin-2 in cord blood serum was determined by a sandwich TRIFMA method. FCN2 genetic variants were analysed with RFLP-PCR, allele-specific PCR, Sanger sequencing or allelic discrimination using TaqMan probes method. Findings: Cord blood serum ficolin-2 concentration correlated positively with Apgar score and inversely with the length of hospitalisation and stay at Neonatal Intensive Care Unit (NICU). Multivariate logistic regression analysis indicated that low ficolin-2 increased the possibility of respiratory distress syndrome (RDS) diagnosis [OR=2.05, 95% CI (1.24-3.37), p=0.005]. Median ficolin-2 concentration was significantly lower in neonates with RDS than in premature babies without this complication, irrespective of FCN2 gene polymorphisms localised to promoter and 3'untranslated regions: for patients born <33 GA: 1471 ng/ml vs. 2115 ng/ml (p=0.0003), and for patients born ≥33 GA 1610 ng/ml vs. 2081 ng/ml (p=0.012). Ficolin-2 level was also significantly lower in neonates requiring intubation in the delivery room (1461 ng/ml vs. 1938 ng/ml, p=0.023) and inversely correlated weakly with the duration of respiratory support (R=-0.154, p<0.001). Interestingly, in the neonates born at GA <33, ficolin-2 concentration permitted differentiation of those with/without RDS [AUC=0.712, 95% CI (0.612-0.817), p<0.001] and effective separation of babies with mild RDS from those with moderate/severe form of the disease [AUC=0.807, 95% CI (0.644-0.97), p=0.0002]. Conclusion: Low cord serum ficolin-2 concentration (especially in neonates born at GA <33 weeks) is associated with a higher risk of developing moderate/severe RDS, requiring respiratory support and intensive care.

L-ficolin and capsular polysaccharide-specific IgG in cord serum contribute synergistically to opsonophagocytic killing of serotype III and V group B streptococci.

Group B streptococci (GBS; Streptococcus agalactiae) are the most common cause of neonatal sepsis and meningitis. Serotype-specific IgG antibody is known to protect neonates against GBS infections by promoting opsonophagocytosis. The L-ficolin-mediated lectin pathway of the complement is also a potential mechanism for opsonization of GBS, because L-ficolin activates the complement after binding to serotype Ib, III, V, VI, and VIII GBS. In the present study, we investigated how L-ficolin and serotype-specific IgG in cord sera contribute to opsonophagocytic killing of GBS. Neither L-ficolin nor serotype-specific IgG concentrations correlated with C3b deposition on serotype Ib and VI GBS, suggesting L-ficolin- and serotype-specific IgG-independent mechanisms of complement activation. The percentage of serotype VIII GBS killed was high regardless of the concentration of L-ficolin and IgG. In contrast, L-ficolin and serotype-specific IgG can each initiate C3b deposition on serotype III and V GBS and promote phagocytosis by polymorphonuclear leukocytes, but L-ficolin and serotype-specific IgG together promote opsonophagocytic killing to a greater extent than does either alone in vitro. This synergy was observed when serotype III-specific IgG concentrations were between 1 and 6 µg/ml and when serotype V-specific IgG concentrations were between 2 and 5 µg/ml. Concentrations of serotype III-specific IgG in cord blood above 7 µg/ml are considered protective for neonates colonized with GBS, but most neonates with IgG levels of less than 7 µg/ml do not develop GBS infections. The data presented here suggest that L-ficolin enhances opsonophagocytosis of serotype III and V GBS when serotype-specific IgG alone is suboptimal for protection.

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei.

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.

Mouse ficolin B has an ability to form complexes with mannose-binding lectin-associated serine proteases and activate complement through the lectin pathway.

Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

Guideline for hereditary angioedema (HAE) 2010 by the Japanese Association for Complement Research - secondary publication.

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.

Thrombin cleaves recombinant soluble thrombomodulin into a lectin-like domain fragment and a fragment with protein C-activating cofactor activity.

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.

Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus, which is restricted in infancy.

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.

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway.

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.

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.

The N2N3 domains of ClfA, FnbpA and FnbpB in Staphylococcus aureus bind to human complement factor H, and their antibodies enhance the bactericidal capability of human blood.

In the complement system, the opsonin C3b binds to the bacterial cell surface and mediates the opsonophagocytosis. However, the cell-wall protein SdrE of Staphylococcus aureus inhibits the C3b activity by recruiting the complement regulatory protein factor H (fH). SdrE binds to fH via its N-terminal N2N3 domain, which are also found in six other staphylococcal cell-wall proteins. In this study, we report that not only the N2N3 domain of SdrE but also those of ClfA, FnbpA and FnbpB can bind to fH. When immobilized on a microplate, the N2N3 domains recruited fH and enhanced the factor I (fI)-mediated cleavage of C3b. When mixed with fH and S. aureus cells, the N2N3 domains inhibited the fH binding to S. aureus cells and reduced the fI-mediated C3b cleavage on the bacterial cell surface. The F(ab)'2 fragments of the rabbit N2N3 antibodies also inhibited the fH binding to the S. aureus cell surface. When added to human blood, the N2N3 antibodies or the N2N3 domain proteins significantly increased the bactericidal activity. Based on these results, we conclude that, in S. aureus, not only SdrE but also ClfA, FnbpA and FnbpB can contribute to the inhibition of C3b-mediated opsonophagocytosis.