Binding Of Mannan-binding Lectin Research Articles

Mannan-Binding Lectin Is Associated with Inflammation and Kidney Damage in a Mouse Model of Type 2 Diabetes.

Autoreactivity of the complement system may escalate the development of diabetic nephropathy. We used the BTBR OB mouse model of type 2 diabetes to investigate the role of the complement factor mannan-binding lectin (MBL) in diabetic nephropathy. Female BTBR OB mice (n = 30) and BTBR non-diabetic WT mice (n = 30) were included. Plasma samples (weeks 12 and 21) and urine samples (week 19) were analyzed for MBL, C3, C3-fragments, SAA3, and markers for renal function. Renal tissue sections were analyzed for fibrosis, inflammation, and complement deposition. The renal cortex was analyzed for gene expression (complement, inflammation, and fibrosis), and isolated glomerular cells were investigated for MBL protein. Human vascular endothelial cells cultured under normo- and hyperglycemic conditions were analyzed by flow cytometry. We found that the OB mice had elevated plasma and urine concentrations of MBL-C (p < 0.0001 and p < 0.001, respectively) and higher plasma C3 levels (p < 0.001) compared to WT mice. Renal cryosections from OB mice showed increased MBL-C and C4 deposition in the glomeruli and increased macrophage infiltration (p = 0.002). Isolated glomeruli revealed significantly higher MBL protein levels (p < 0.001) compared to the OB and WT mice, and no renal MBL expression was detected. We report that chronic inflammation plays an important role in the development of DN through the binding of MBL to hyperglycemia-exposed renal cells.

Complement lectin pathway components MBL and MASP-1 promote haemostasis upon vessel injury in a microvascular bleeding model.

BackgroundComplement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model.MethodsWe studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry.ResultsUpon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time.ConclusionWe show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system.

Oligomerization of Mannan-binding Lectin Dictates Binding Properties and Complement Activation.

The complement system is a part of the innate immune system and is involved in recognition and clearance of pathogens and altered-self structures. The lectin pathway of the complement system is initiated when soluble pattern recognition molecules (PRMs) with collagen-like regions bind to foreign or altered self-surfaces. Associated with the collagen-like stems of these PRMs are three mannan-binding lectin (MBL)-associated serine proteases (MASPs) and two MBL-associated proteins (MAps). The most studied of the PRMs, MBL, is present in serum mainly as trimeric and tetrameric oligomers of the structural subunit. We hypothesized that oligomerization of MBL may influence both the potential to bind to micro organisms and the interaction with the MASPs and MAps, thus influencing the ability to initiate complement activation. When testing binding at 37°C, we found higher binding of tetrameric MBL to Staphylococcus aureus (S.aureus) than trimeric and dimeric MBL. In serum, we found that tetrameric MBL was the main oligomeric form present in complexes with the MASPs and MAp44. Such preference was confirmed using purified forms of recombinant MBL (rMBL) oligomers, where tetrameric rMBL interacted stronger with all of the MASPs and MAp44, compared to trimeric MBL. As a direct consequence of the weaker interaction with the MASPs, we found that trimeric rMBL was inferior to tetrameric rMBL in activating the complement system. Our data suggest that the oligomeric state of MBL is crucial both for the binding properties and the effector function of MBL.

MBL-associated serine proteases (MASPs) and infectious diseases.

The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases.

Differential ability to resist to complement lysis and invade host cells mediated by MBL in R4 and 860 strains of Trypanosoma cruzi

To produce an infection Trypanosoma cruzi must evade lysis by the complement system. During early stages of infection, the lectin pathway plays an important role in host defense and can be activated by binding of mannan-binding lectin (MBL) to carbohydrates on the surface of pathogens. We hypothesized that MBL has a dual role during parasite-host cell interaction as lectin complement pathway activator and as binding molecule to invade the host cell. We used two polarized strains of T. cruzi, R4 (susceptible) and 860 (resistant) strains, to investigate the role of MBL in complement-mediated lysis. Interestingly R4, but not 860 metacyclic strain, markedly increases the invasion of host cells, suggesting that MBL drives the invasion process while the parasite deactivates the Lectin complement pathway.

Heteromeric Complexes of Native Collectin Kidney 1 and Collectin Liver 1 Are Found in the Circulation with MASPs and Activate the Complement System

The complement system is an important part of the innate immune system. The complement cascade may be initiated downstream of the lectin activation pathway upon binding of mannan-binding lectin, ficolins, or collectin kidney 1 (CL-K1, alias CL-11) to suitable microbial patterns consisting of carbohydrates or acetylated molecules. During purification and characterization of native CL-K1 from plasma, we observed that collectin liver 1 (CL-L1) was copurified. Based on deglycosylation and nonreduced/reduced two-dimensional SDS-PAGE, we detected CL-K1 and CL-L1 in disulfide bridge-stabilized complexes. Heteromeric complex formation in plasma was further shown by ELISA and transient coexpression. Judging from the migration pattern on two-dimensional SDS-PAGE, the majority of plasma CL-K1 was found in complex with CL-L1. The ratio of this complex was in favor of CL-K1, suggesting that a heteromeric subunit is composed of one CL-L1 and two CL-K1 polypeptide chains. We found that the complex bound to mannan-binding lectin-associated serine proteases (MASPs) with affinities in the nM range in vitro and was associated with both MASP-1/-3 and MASP-2 in plasma. Upon binding to mannan or DNA in the presence of MASP-2, the CL-L1-CL-K1 complex mediated deposition of C4b. In favor of large oligomers, the activity of the complex was partly determined by the oligomeric size, which may be influenced by an alternatively spliced variant of CL-K1. The activity of the native heteromeric complexes was superior to that of recombinant CL-K1. We conclude that CL-K1 exists in circulation in the form of heteromeric complexes with CL-L1 that interact with MASPs and can mediate complement activation.

Co-Complexes of MASP-1 and MASP-2 Associated with the Soluble Pattern-Recognition Molecules Drive Lectin Pathway Activation in a Manner Inhibitable by MAp44

The lectin pathway of complement is an integral component of innate immunity. It is activated upon binding of mannan-binding lectin (MBL) or ficolins (H-, L-, and M-ficolin) to suitable ligand patterns on microorganisms. MBL and ficolins are polydisperse homo-oligomeric molecules, found in complexes with MBL-associated serine proteases (MASP-1, -2, and -3) and MBL-associated proteins (MAp19 and MAp44). This scenario is far more complex than the well-defined activation complex of the classical pathway, C1qC1r(2)C1s(2), and the composition of the activating complexes of the lectin pathway is ill defined. We and other investigators recently demonstrated that both MASP-1 and MASP-2 are crucial to lectin pathway activation. MASP-1 transactivates MASP-2 and, although MASP-1 also cleaves C2, MASP-2 cleaves both C4 and C2, allowing formation of the C3 convertase, C4bC2a. Juxtaposition of MASP-1 and MASP-2 during activation must be required for transactivation. We previously presented a possible scenario, which parallels that of the classical pathway, in which MASP-1 and MASP-2 are found together in the same MBL or ficolin complex. In this study, we demonstrate that, although MASPs do not directly form heterodimers, the addition of MBL or ficolins allows the formation of MASP-1-MASP-2 co-complexes. We find that such co-complexes have a functional role in activating complement and are present in serum at varying levels, impacting on the degree of complement activation. This raises the novel possibility that MAp44 may inhibit complement, not simply by brute force displacement of MASP-2 from MBL or ficolins, but by disruption of co-complexes, hence impairing transactivation. We present support for this contention.

Mechanism of MBL inhibiting the LPS-induced DC maturation

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.

The salivary scavenger and agglutinin binds MBL and regulates the lectin pathway of complement in solution and on surfaces

The salivary scavenger and agglutinin (SALSA), also known as gp340, salivary agglutinin and deleted in malignant brain tumor 1, is a 340-kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A, and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan-binding lectin (MBL) as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit Candida albicans-induced complement activation. Thus, SALSA has a dual complement activation modifying function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid phase. These activities are mediated via a direct interaction with MBL. This suggests that SALSA could target the innate immune responses to certain microorganisms and simultaneously limit complement activation in the fluid phase.

Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells

Mannan-binding lectin (MBL) plays a key role in the lectin pathway of complement activation and can influence cytokine expression. Toll-like receptor 4 (TLR4) is expressed extensively and has been demonstrated to be involved in lipopolysaccharide (LPS)-induced signaling. We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-κB (NF-κB) activity by using the monocytoid cell line THP-1. We then investigated the possible mechanisms underlying any observed regulatory effect. Using ELISA and reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we found that at both the protein and mRNA levels, treatment with MBL suppresses LPS-induced tumor-necrosis factor (TNF)-α and IL-12 production in THP-1 cells. An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-κB DNA binding and translocation in THP-1 cells. While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations, this binding occurred optimally in response to supraphysiological calcium concentrations. This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody (HTA125). Activation of THP-1 cells by LPS treatment resulted in increased MBL binding. We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner, and this interaction could attenuate the binding of LPS to cell surfaces. Taken together, these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways. These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.

Specific interaction of hepatitis C virus glycoproteins with mannan binding lectin inhibits virus entry

Mannan-binding lectin (MBL) is a soluble innate immune protein that binds to glycosylated targets. MBL acts as an opsonin and activates complement, contributing to the destruction and clearance of infecting microorganisms. Hepatitis C virus (HCV) encodes two envelope glycoproteins E1 and E2, expressed as non-covalent E1/E2 heterodimers in the viral envelope. E1 and E2 are potential ligands for MBL. Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment, the full-length E1/E2 heterodimer, expressed in vitro, and assess the effect of this interaction on virus entry. A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent, saturating binding of MBL to HCV glycoproteins. Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL. MBL binds to E1/E2 representing a broad range of virus genotypes. MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles (HCVpp) bearing E1/E2 from a wide range of genotypes. HCVpp were neutralized to varying degrees. MBL was also shown to neutralize an authentic cell culture infectious virus, strain JFH-1 (HCVcc). Furthermore, binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2. In conclusion, MBL interacts directly with HCV glycoproteins, which are present on the surface of the virion, resulting in neutralization of HCV particles.

Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot.

The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-D-dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection.

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

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

Protective role of mannan-binding lectin in a murine model of invasive pulmonary aspergillosis

Innate immune molecules such as lung collectins and serum pentraxins have evolved as important host defence proteins against Aspergillus fumigatus, a medically important opportunistic fungal pathogen. Mannan-binding lectin (MBL), an opsonin and lectin complement pathway activator, constitutes another vital player of innate immunity against several pathogenic organisms in the serum. Studies have reported significant binding of MBL to A. fumigatus; however, the protective role of MBL against A. fumigatus-mediated invasive disease remains elusive. Henceforth, we investigated the contribution of externally administered recombinant human (rh) MBL towards anti-fungal defence in invasive pulmonary aspergillosis (IPA) by in vivo and in vitro studies. In murine models of IPA with corticosteroid-induced immunosuppression, rhMBL-treated mice showed 80% survival compared to untreated IPA mice with no survivors. Treated IPA mice also showed a marked increase in tumour necrosis factor (TNF)-alpha and interleukin (IL)-1alpha and a significant decrease in pulmonary fungal hyphae and IL-10. In vitro, rhMBL-bound A. fumigatus conidia showed a dose-dependent increase in the deposition of C4b, the first product of the lectin pathway. There was an enhanced uptake of A. fumigatus conidia by the polymorphonuclear cells (PMNs) in the presence of rhMBL that increased further in the presence of MBL supplemented with MBL-deficient serum. However, an increase in the oxidative burst of PMNs and A. fumigatus killing were observed only when MBL was supplemented with MBL-deficient serum. The study suggests a therapeutic role of ex vivo-administered MBL in host defence against aspergillosis, possibly through MBL-mediated complement activation and other protective mechanisms aimed both directly at the pathogen, and indirectly through modulation of the host inflammatory responses.

Recognition ofCandida albicansby Mannan‐Binding Lectin In Vitro and In Vivo

Mannan-binding lectin (MBL) is a component of the innate immune system. The goal of the present study was to evaluate binding of MBL to Candida albicans in vitro and in vivo and to assess the impact of MBL treatment on host resistance. The results showed a variable and often discontinuous pattern of binding to individual yeast cells. MBL bound to cells grown at 37 degrees C but not to cells grown at 23 degrees C. The putative MBL ligand was constitutively present on yeast cells grown at 23 degrees C, but the ligand was masked on such cells, such that MBL could not bind. C. albicans yeasts and hyphae in infected tissue bound MBL. Finally, parenteral administration of MBL increased resistance of mice to hematogenously disseminated candidiasis. These results suggest that MBL is an important component of innate resistance to candidiasis and that MBL therapy may be a means to prevent disseminated candidiasis in high-risk patients.

Interaction of Mannan Binding Lectin with α2 Macroglobulin via Exposed Oligomannose Glycans: A CONSERVED FEATURE OF THE THIOL ESTER PROTEIN FAMILY?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.

Detection of an Autologous Ligand for Mannan‐Binding Lectin on Human B Lymphocytes

Mannan-binding lectin (MBL) is a collectin and a major soluble pattern-recognition protein. MBL can distinguish self from nonself and altered self using its C-type carbohydrate recognition domain and may also interact via its collagen-like region with autologous cells. Recently, it was found that MBL could bind to adherent cells (monocytes) and dendritic cells in a specific and sugar-sensitive manner. We have now investigated the interaction of MBL with fresh human peripheral blood cells and report binding to B lymphocytes and natural killer cells. The binding to B lymphocytes was studied in detail and was compared with the binding of MBL to monocytes and dendritic cells. Binding of MBL to B cells was evident at physiological MBL and calcium concentrations but was optimal at supraphysiological MBL concentrations. It was readily inhibited by autologous serum, mannan, mannose, GlcNAc and (to a lesser extent) galactose but not by C1q. A similar, but not identical, inhibition profile was observed with dendritic cells, but monocytes were not sensitive to mannose or mannan. We conclude that MBL is capable of binding to differently glycosylated ligands on several autologous cell types via its carbohydrate-recognition domain. We speculate that this could have functional significance at extravascular sites, but perhaps only in individuals possessing MBL genotypes conferring MBL sufficiency.

Mannan-binding-lectin-associated serine proteases, characteristics and disease associations

Mannan-binding lectin (MBL)-associated serine proteases (MASPs) circulate in plasma as zymogens in complexes with MBL and with L- and H-ficolin. Upon binding of MBL or ficolin to pathogen-associated molecular patterns, the MASPs are activated. MASP-2 can now cleave C4 and C2 to generate the C3 convertase, C4bC2b. The functions of the other two MASPs, MASP-1 and MASP-3 have not been elucidated. MASP-1 can cleave C2, and with low efficiency also C3, and may serve a function through direct C3 activation. No natural substrate for MASP-3 has been identified. MBL deficiency, occurring at a frequency of about 10%, is the most common congenital immunodeficiency and is associated with susceptibility to infections and autoimmune disorders. Inherited MASP-2 deficiency has been described as the result of a mutation causing the exchange of aspartic acid with a glycine at position 105, a position in the first domain, CUB1, involved in calcium binding. This mutation abolishes the binding to MBL and ficolins, and deprives MASP-2 of functional activity. The index case suffered from recurrent severe infections and autoimmune reactions. The gene frequency of the mutation among Caucasians is 3.6%. It is not found in Chinese, who present a different mutation also associated with MASP-2 deficiency.

Poster session 7: complement diagnosis and therapeutic intervention

The mannan-binding lectin (MBL) pathway of complement activation is part of the innate immune defense. The binding of MBL to microbial carbohydrates activates the MBL-associated serine proteases (MASPs), which recruit the complement factors, C4 and C2, to generate the C3 convertase or directly activate C3. We present a phylogenetically highly conserved member of the MBL complex, MASP-3, which is generated through alternative splicing of the MASP-1/3 gene. The designation of MASP-3 as a protease is based on homology to known MASPs. Different MBL oligomers were found to have distinct MASP composition and biological activities. MASP-1, MAp19, and direct C3-cleaving activity are associated with smaller oligomers whereas MASP-3 is found together with MASP-2 on larger oligomers. MASP-3 downregulate the C4 and C2 cleaving activity of MASP-2.

The binding of MBL to common bacteria in infectious diseases of children

To purify Mannan-binding lectin (MBL) from human serum and detect its binding ability to several kinds of bacteria common in infectious diseases of children. MBL was purified from human serum by affinity chromatography on mannan-Sepharose 4B column. Its binding ability to eight species, 97 strains of bacteria was detected by enzyme-linked lectin assay (ELLA). MBL has different binding ability to bacteria and shows strong binding ability to Klebsiella ornithinolytica and Escherichia coli, but shows relatively lower binding ability to Staphylococcus haemolyticus, Enterobacter cloacae and Staphylococcus epidermidis. To different isolates of Klebsiella pneumoniae, Haemophilus influenzae and Staphylococcus aureus, MBL shows quite different binding ability. MBL has different binding ability to different bacteria, and has relatively stronger binding ability to Gram-negative bacteria. Its binding ability to different isolates of certain kinds of bacteria is quite different.