[Show abstract][Hide abstract] ABSTRACT: The three human ficolins (H-, L- and M-ficolin) and mannan-binding lectin are pattern recognition molecules of the innate immune system mediating activation of the lectin pathway of the complement system. These four human proteins bind to some microorganisms and may be involved in the resolution of infections. We investigated the binding selectivity by examining the binding of M-ficolin to a panel of more than 100 different streptococcal strains (Streptococcus pneumoniae and Streptococcus mitis) each expressing distinct polysaccharide structures. M-ficolin binding was observed for three strains only, the pneumococcal serotypes 19B and 19C and a single mitis strain expressing a similar polysaccharide structure. The bound M-ficolin, in association with MASP-2, mediated complement factor C4 cleavage. Binding to the bacteria was inhibitable by N-acetyl glucosamine indicating that the interaction with the bacterial surface takes place via the fibrinogen-like domain. The common N-acetyl mannosamine residue present in the structures of the four capsular polysaccharides of group 19 is linked via a phosphodiester bond. This residue is apparently not a ligand for M-ficolin since the lectin binds to two of the group 19 polysaccharides only. M-ficolin bound strongly to serotype 19B and 19C polysaccharides. In contrast to the serotypes 19A and 19F these two serotypes contain an extra N-acetyl mannosamine residue linked via glycoside linkage only. Thus, this extra residue seems to be the M-ficolin ligand.In conclusion, we were able to demonstrate specific binding of M-ficolin to some capsular polysaccharides of the opportunistic pathogen S. pneumoniae and of the commensal bacterium S. mitis.
Infection and immunity 11/2012; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lectin pathway of complement is an important component of innate immunity. Its activation has been thought to occur via recognition of pathogens by mannan-binding lectin (MBL) or ficolins in complex with MBL-associated serine protease (MASP)-2, followed by MASP-2 autoactivation and cleavage of C4 and C2 generating the C3 convertase. MASP-1 and MASP-3 are related proteases found in similar complexes. MASP-1 has been shown to aid MASP-2 convertase generation by auxiliary C2 cleavage. In mice, MASP-1 and MASP-3 have been reported to be central also to alternative pathway function through activation of profactor D and factor B. In this study, we present functional studies based on a patient harboring a nonsense mutation in the common part of the MASP1 gene and hence deficient in both MASP-1 and MASP-3. Surprisingly, we find that the alternative pathway in this patient functions normally, and is unaffected by reconstitution with MASP-1 and MASP-3. Conversely, we find that the patient has a nonfunctional lectin pathway, which can be restored by MASP-1, implying that this component is crucial for complement activation. We show that, although MASP-2 is able to autoactivate under artificial conditions, MASP-1 dramatically increases lectin pathway activity at physiological conditions through direct activation of MASP-2. We further demonstrate that MASP-1 and MASP-2 can associate in the same MBL complex, and that such cocomplexes are found in serum, providing a scenario for transactivation of MASP-2. Hence, in functional terms, it appears that MASP-1 and MASP-2 act in a manner analogous to that of C1r and C1s of the classical pathway.
The Journal of Immunology 09/2012; 189(8):3957-69. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217 ng/ml, i.e., 11nM, comparable to the 7 nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent.
Journal of immunological methods 08/2011; 373(1-2):89-101. · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: M-ficolin is a PRM of the innate immune system, found in serum and associated with leukocytes. We used the soluble form to study specificity toward Gram-positive bacteria and characterized and quantified cell-associated M-ficolin. The binding of M-ficolin to capsulated and noncapsulated strains of Streptococcus agalactiae (GBS) and Staphylococcus aureus was investigated. We did not observe binding of M-ficolin to any of 13 serotypes of S. aureus. Dose-dependent binding of M-ficolin was demonstrated for all of the capsulated GBS strains. The binding was abolished by prior treatment of the bacteria with sialidase, indicating that sialic acid is the ligand for M-ficolin on these bacteria. GlcNAc could inhibit the binding, suggesting that M-ficolin binds via its FBG. M-ficolin was found associated with the complement-activating enzyme in serum, and M-ficolin bound to GBS mediated activation of the complement system. M-ficolin expression on leukocytes was evaluated by flow cytometry with anti-M-ficolin mAb. Total M-ficolin of different leukocytes was quantified in detergent extracts. Monocytes and granulocytes showed similar M-ficolin surface expression, 1.1 × 10(5) and 0.7 × 10(5) M-ficolin molecules/cell, respectively. The total M-ficolin content of the cells was 1.5 × 10(6) molecules/monocyte and approximately one-third of this for granulocytes. Lymphocytes contained <1.5% of the amount estimated for monocytes, and none was revealed on the surface of lymphocytes by flow cytometry. Immunohistochemical analysis of the distribution of M-ficolin in 25 tissues revealed staining of only granulocytes and monocytes. Reported M-ficolin expression by type II pneumocytes could not be verified. We demonstrate the specific binding of M-ficolin to sialic acids in the capsule of GBS and give quantitative aspects of the cell-associated M-ficolin.
Journal of leukocyte biology 07/2011; 90(3):425-37. · 4.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Essential effector functions of innate immunity are mediated by complement activation initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL) and the ficolins. We present a novel, phylogenetically conserved protein, MAp44, which is found in human serum at 1.4 microg/ml in Ca(2+)-dependent complexes with the soluble pattern recognition molecules. The affinity for MBL is in the nanomolar range (K(D) = 0.6 nM) as determined by surface plasmon resonance. The first eight exons of the gene for MAp44 encode four domains shared with MBL-associated serine protease (MASP)-1 and MASP-3 (CUB1-EGF-CUB2-CCP1), and a ninth exon encodes C-terminal 17 aa unique to MAp44. mRNA profiling in human tissues shows high expression in the heart. MAp44 competes with MASP-2 for binding to MBL and ficolins, resulting in inhibition of complement activation. Our results add a novel mechanism to those known to control the innate immune system.
The Journal of Immunology 11/2009; 183(11):7371-8. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: H-ficolin (Hakata antigen, ficolin-3) activates the lectin pathway of complement similar to mannose-binding lectin. However, its impact on susceptibility to infection is currently unknown. This study investigated whether the serum concentration of H-ficolin at diagnosis is associated with fever and neutropenia (FN) in paediatric cancer patients. H-ficolin was measured by time-resolved immunofluorometric assay in serum taken at cancer diagnosis from 94 children treated with chemotherapy. The association of FN episodes with H-ficolin serum concentration was analysed by multivariate Poisson regression. Median concentration of H-ficolin in serum was 26 mg/l (range 6-83). Seven (7%) children had low H-ficolin (< 14 mg/l). During a cumulative chemotherapy exposure time of 82 years, 177 FN episodes were recorded, 35 (20%) of them with bacteraemia. Children with low H-ficolin had a significantly increased risk to develop FN [relative risk (RR) 2.24; 95% confidence interval (CI) 1.38-3.65; P = 0.004], resulting in prolonged duration of hospitalization and of intravenous anti-microbial therapy. Bacteraemia occurred more frequently in children with low H-ficolin (RR 2.82; CI 1.02-7.76; P = 0.045). In conclusion, low concentration of H-ficolin was associated with an increased risk of FN, particularly FN with bacteraemia, in children treated with chemotherapy for cancer. Low H-ficolin thus represents a novel risk factor for chemotherapy-related infections.
[Show abstract][Hide abstract] ABSTRACT: Mannan-binding lectin-associated serine protease 2 (MASP-2) is an enzyme of the innate immune system. MASP-2 forms complexes with the pattern recognition molecules mannan-binding lectin (MBL), H-ficolin, L-ficolin, or M-ficolin, and is activated when one of these proteins recognizes microorganisms and subsequently cleaves complement factors C4 and C2, thus initiating the activation of the complement system. Missense polymorphisms of MASP-2 exist in different ethnic populations. To further characterize the nature of these, we have produced and characterized rMASP-2s representing the following naturally occurring polymorphisms: R99Q, D120G, P126L, H155R, 156_159dupCHNH (CHNHdup), V377A, and R439H. Only very low levels of CHNHdup were secreted from the cells, whereas quantities similar to wild-type MASP-2 were found intracellularly, indicating that this mutation results in a misfolded protein. We found that D120G and CHNHdup could not associate with MBL, whereas R99Q, P126L, H155R, V377A, R439H, and wild-type MASP-2 bound equally well to MBL. Accordingly, when D120G and CHNHdup were mixed with MBL, no activation of complement factor C4 was observed, whereas R99Q, P126L, and V377A cleaved C4 with an activity comparable to wild-type MASP-2 and H155R slightly better. In contrast, the R439H variant was deficient in this process despite its normal binding to MBL. This variant was also not able to autoactivate in the presence of MBL and mannan. We find the R439H variant is common in Sub-Saharan Africans with a gene frequency of 10%. Our results indicate that individuals with different types of MASP-2 defects may be identified through genotyping.
The Journal of Immunology 04/2009; 182(5):2939-47. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ObjectiveNecrotising enterocolitis (NEC) causes significant mortality in premature infants. The involvement of the innate immune system in the pathogenesis of NEC remains unclear. M-, L- and H-ficolins recognize microorganisms and activate the complement system, but their role in host defense is largely unknown. This study investigated whether ficolin concentrations are associated with NEC.Study designCase-control study including 30 premature infants with NEC and 60 controls. M-, L- and H-ficolins were measured in cord blood using time-resolved immunofluorometric assays. Multivariate logistic regression was performed.ResultsOf the 30 NEC cases (median gestational age, 29.5 weeks), 12 (40%) were operated and 4 (13%) died. No difference regarding ficolin concentration was found when comparing NEC cases versus controls (p > 0.05). However, infants who died of NEC had significantly lower M-ficolin cord blood concentrations than NEC survivors (for M-ficolin <300 ng/ml; multivariate OR 12.35, CI 1.03–148.59, p = 0.048). In the entire study population, M-, L- and H-ficolins were positively correlated with gestational age (p < 0.001) and birth weight (p < 0.001). Infants with low M-ficolin required significantly more often mechanical ventilation after birth multivariate (OR 10.55, CI 2.01–55.34, p = 0.005).ConclusionsM-, L- and H-ficolins are already present in cord blood and increase with gestational age. Low cord blood concentration of M-ficolin was associated with higher NEC-associated fatality and with increased need for mechanical ventilation. Future studies need to assess whether M-ficolin is involved in multiorgan failure and pulmonary disease.
[Show abstract][Hide abstract] ABSTRACT: Mannan-binding lectin (MBL) is attracting considerable interest due to its role in the immune defense. The high frequency of congenital MBL deficiency makes it feasible to evaluate clinical relevance through epidemiological investigations on fairly limited numbers of patients. MBL deficiency is determined by three mutant allotypes termed B, C and D in the coding region as well as mutations in the promoter region. It has been suggested that individuals, with deficiency-associated allotypes, may present significant amounts of low molecular weight MBL. We have compared the quantification of MBL by four commercially available assays with results obtained by our own in-house assays. Most assays are selectively sensitive for the wild type MBL (allotype A), but special combinations of antibodies also detect mutant forms of MBL. Thus a sandwich-type time-resolved immunoflourometric assay (TRIFMA), with a mouse monoclonal antibody (93C) as the catching and detecting antibody, shows B/B and D/D homozygous individuals to present signals corresponding to up to 500 ng MBL per ml (with plasma from an A/A individual as standard) as compared to less than 50 ng/ml and 200 ng/ml, respectively, when measured in other assays. In GPC at isotonic conditions the MBL in B/B and D/D individuals showed a Mr of 450 kDa. This MBL cannot bind to mannan. We further present a new method for quantifying the amount of MBL polypeptide chain. By applying plasma samples on SDS-PAGE at reducing conditions followed by Western blotting and quantification by chemiluminescense, this approach presents single polypeptide chains to the antibody independent of allotype differences in the collagen-like region. Titrations of recombinant MBL served as standard. In sera from homozygous mutants (O/O) the MBL concentrations estimated on Western blot were in the range of 100 to 500 ng/ml and correlated with that measured in the 93C-based TRIFMA.
Journal of Immunological Methods 09/2006; 315(1-2):49-60. · 2.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L-ficolin and H-ficolin are molecules of the innate immune system. Upon recognition of a suitable target they activate the complement system. The ligand recognition structure of ficolins is contained within a fibrinogen-like domain. We examined the selectivity of the ficolins through inhibiting the binding to bacteria or to beads coupled with N-acetylglucosamine. The binding of L-ficolin to Streptococcus pneumoniae 11F and the beads was inhibited by N-acetylated sugars and not by non-acetylated sugars. However, it was also inhibited by other acetylated compounds. Based on this selectivity L-ficolin is not easily defined as a lectin. The binding of H-ficolin to Aerococcus viridans was not inhibited by any of the sugars or other compounds examined. Based on the selectivity of L-ficolin we developed a new purification procedure involving affinity chromatography on N-acetylcysteine-derivatized Sepharose. The column was loaded in the presence of EDTA and high salt, and L-ficolin was eluted by decreasing the salt concentration. Further purification was achieved by ion exchange chromatography.
Journal of Biological Chemistry 12/2004; 279(46):47513-9. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mannan-binding lectin (MBL) pathway and the classical pathway of complement activation are initiated by the binding of the recognition structure of the initiator complexes, MBL and C1q, respectively, to their ligands, i.e. carbohydrate structures or immune complexes. Proenzymes associated with MBL or C1q are then activated and generate C3 convertase through the activation of C4 and C2. The cleavage product of C4, C4b, attaches covalently to nearby hydroxyl or amino groups. The current picture is that C2 must then attach to C4b before being cleaved by the same associated proteases into the enzymatically active fragment, C2b. This suggests a stringent requirement for the deposition of C4b very close to the initiator complex, or indeed onto the initiator complex. We examined the possibility of C4b being bound to the initiator complex by a solid-phase assay, allowing for the selective elution of the initiator complexes, followed by quantification of the C4b being eluted and the C4b remaining on the solid phase. Also, we estimated the generation of complexes between the released initiator complex and C4b. More than 99% of deposited C4b was bound directly to the solid phase rather than to the initiator complex. Our approach cannot answer the question of the whereabouts of the C2 when it is cleaved.
Scandinavian Journal of Immunology 07/2003; 57(6):556-61. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. The Journal of Immunology, 2000, 165: 2093-2100.
The Journal of Immunology 09/2000; 165(4). · 5.52 Impact Factor