Characterization of the interaction between L-ficolin/p35 and mannan-binding lectin-associated serine proteases-1 and -2

Ficolins are oligomeric lectins comprising a collagen-like and a fibrinogen-like domain, with a binding specificity for N-acetylglucosamine. It has been reported recently that L-ficolin/P35 associates with mannan-binding lectin (MBL)-associated serine proteases (MASP-1 and -2) and MBL-associated protein 19 (MAp19) in serum and forms complexes able to activate complement. Using surface plasmon resonance spectroscopy we have shown that recombinant MASP-1 and -2, their N-terminal CUB1 (module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1)-epidermal growth factor (EGF)-CUB2 and CUB1-EGF segments, and MAp19 bind to immobilized L-ficolin/P35 in the presence of Ca(2+) ions. Comparable K(d) values were obtained for the full-length proteases and their CUB1-EGF-CUB2 segments (9.2 and 10 nM for MASP-1 and 4.6 and 5.4 nM for MASP-2, respectively), whereas higher values were obtained for the CUB1-EGF segments (26.7, 15.6, and 14.3 nM for MASP-1, MASP-2, and MAp19). These values are in the same range as those determined for the interaction of these proteins with MBL. Binding was Ca(2+) dependent and was only partly sensitive to EDTA for MASP-1, MASP-2, and MASP-2 CUB1-EGF-CUB2. Half-maximal binding was obtained at comparable Ca(2+) concentrations for MASP-1 and MASP-2 (0.45 and 0.47 micro M, respectively), their CUB1-EGF-CUB2 segments (0.37 and 0.72 micro M), and their CUB1-EGF segments (0.31 and 0.79 micro M). These values are lower than those determined in the case of MBL, indicating a difference between MBL and L-ficolin/P35 with respect to the Ca(2+) dependence of their interaction with the MASPs. Preincubation of the MASPs with soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35 and, conversely, suggesting that these lectins compete with each other for binding to the MASPs in vivo.     

Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, and the MBL-associated protein MAp19

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.     

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.     

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.     

Proteolytic activities of two types of mannose-binding lectin-associated serine protease

Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.     

The X-ray structure of human mannan-binding lectin-associated protein 19 (MAp19) and its interaction site with mannan-binding lectin and L-ficolin

MAp19 is an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB1-epidermal growth factor (EGF) segment of MASP-2, plus four additional residues at its C-terminal end. Like full-length MASP-2, it forms Ca(2+)-dependent complexes with mannan-binding lectin (MBL) and L-ficolin. The x-ray structure of human MAp19 was solved to a resolution of 2.5 A. It shows a head to tail homodimer held together by interactions between the CUB1 module of one monomer and the EGF module of its counterpart. A Ca(2+) ion bound to each EGF module stabilizes the dimer interfaces. A second Ca(2+) ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu(52), Asp(60), Asp(105), Ser(107), Asn(108), and a water molecule. Compared with its counterpart in human C1s, the N-terminal end of the MAp19 CUB1 module contains a 7-residue extension that forms additional inter-monomer contacts. To identify the residues involved in the interaction of MAp19 with MBL and L-ficolin, point mutants were generated and their binding ability was determined using surface plasmon resonance spectroscopy. Six mutations at Tyr(59), Asp(60), Glu(83), Asp(105), Tyr(106), and Glu(109) either strongly decreased or abolished interaction with both MBL and L-ficolin. These mutations map a common binding site for these proteins located at the distal end of each CUB1 module and stabilized by the Ca(2+) ion.     

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

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

The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 +/- 170 Da (MBL-I) and 304,899 +/- 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 +/- 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable K(D) values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.     

The rat and mouse homologues of MASP-2 and MAp19, components of the lectin activation pathway of complement

Recently, we described two novel constituents of the multimolecular initiation complex of the mannan-binding lectin (MBL) pathway of complement activation, a serine protease of 76 kDa, termed MASP-2, and a MASP-2 related plasma protein of 19 kDa, termed MAp19. Upon activation of the MBL/MASPs/MAp19 complex, MASP-2 cleaves the fourth complement component C4, while the role of MAp19 within the MBL/MASP-1/MASP-2/MAp19 complex remains to be clarified. In humans, the mRNA species encoding MASP-2 (2.6 kb) and MAp19 (1.0 kb) arise by an alternative polyadenylation/splicing mechanism from a single structural MASP-2 gene. Here, we report the complete primary structures of the rat homologue of MASP-2 and of rat and mouse MAp19. We show that both MASP-2 and MAp19 are part of the rat MBL pathway activation complex and demonstrate their exclusively hepatic biosynthesis. Southern blot and PCR analyses of rat genomic DNA indicate that as in humans, rat MASP-2 and MAp19 are encoded by a single structural gene.     

Mannan-binding lectin (MBL)-associated plasma protein present in human urine inhibits calcium oxalate crystal growth

Mannan-binding lectin (MBL)-associated plasma protein (MAp19) is an alternatively spliced form of MBL-associated serine protease-2, a component of a complement activation cascade. We observed that MAp19 is excreted in human urine. Interestingly, the amount of MAp19 was higher in urine of renal cell carcinoma patients than healthy people. Pretreatment of urine dialysate with 50 mM EDTA increased the recovery of MAp19, suggesting that MAp19 is a calcium-binding protein. The recombinant MAp19 showed a strong inhibition of calcium oxalate crystal growth in vitro in a concentration-dependent manner. Thus, we conclude that MAp19 plays a role in the inhibition of calcium oxalate renal stone formation.     

MBL调控MASP激活补体系统

甘露聚糖结合凝集素(MBL;或甘露聚糖结合蛋白,MBP)是由相同的多肽链组成的寡聚物,它通过结合细胞表面的碳水化合物能够有效地识别侵入体内的多种致病微生物,并激活补体来杀灭病原微生物。MBL能与血清中其相关蛋白酶(MASP)结合,MASP包含3个丝氨酸蛋白酶MASP-1、MASP-2、MASP-3和非酶蛋白MAp19。研究显示,MBL通过2种机理调控MASP-2的活性,在先天性免疫中具有重要的作用。本文简要综述MBL调控MASP激活补体的作用机理。     

Composition of the lectin pathway of complement in Gallus gallus: absence of mannan-binding lectin-associated serine protease-1 in birds

The lectin pathway of complement is activated by multimolecular complexes that recognize and bind to microbial polysaccharides. These complexes comprise a multimeric carbohydrate recognition subunit (either mannan-binding lectin (MBL) or a ficolin), three MBL-associated serine proteases (MASP-1, -2, and -3), and MAp19 (a truncated product of the MASP-2 gene). In this study we report the cloning of chicken MASP-2, MASP-3, and MAp19 and the organization of their genes and those for chicken MBL and a novel ficolin. Mammals usually possess two MBL genes and two or three ficolin genes, but chickens have only one of each, both of which represent the undiversified ancestors of the mammalian genes. The primary structure of chicken MASP-2 is 54% identical with those of the human and mouse MASP-2, and the organization of its gene is the same as in mammals. MASP-3 is even more conserved; chicken MASP-3 shares approximately 75% of its residues with human and Xenopus MASP-3. It is more widely expressed than other lectin pathway components, suggesting a possible function of MASP-3 different from those of the other components. In mammals, MASP-1 and MASP-3 are alternatively spliced products of a single structural gene. We demonstrate the absence of MASP-1 in birds, possibly caused by the loss of MASP-1-specific exons during phylogeny. Despite the lack of MASP-1-like enzymatic activity in sera of chicken and other birds, avian lectin pathway complexes efficiently activate C4.     

Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.     

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.     

Mannose-binding lectin serine proteases and associated proteins of the lectin pathway of complement: Two genes, five proteins and many functions?

The lectin pathway of the complement system is activated following the binding of carbohydrate-based ligands by recognition molecules such as mannose-binding lectin (MBL) or ficolins. Engagement of the recognition molecules causes activation of associated MBL-associated serine proteases or MASPs, which in turn activate downstream complement molecules to activate the system. Two MASP genes are alternatively spliced during expression to yield 5 proteins, including three proteases (MASP-1, -2 and -3) and two truncated proteins, MAp19 and MAp44. Here we discuss what is currently known about these proteins in terms of their structure and function. MASP-2 is autoactivated following the initial binding events of the pathway and is able to subsequently activate the C4 and C2 substrates required to activate the rest of the pathway. MASP-1 is able to augment MASP-2 activation, but also appears to play other roles, although the physiological significance of these is not yet clear. The roles of the truncated Map19 and Map44 proteins and the MASP-3 protease are currently unknown. The proteases form an interesting sub-family of proteins that clearly should be the focus of future research in order to establish their biological roles.This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

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.     

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.     

Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2

Mannan-binding lectin (MBL)-associated serine proteases (MASP-1, -2, and -3) are homologous modular proteases that each associate with MBL and L- and H-ficolins, which are oligomeric serum lectins involved in innate immunity. To investigate its physicochemical, interaction, and enzymatic properties, human MASP-3 was expressed in insect cells. Ultracentrifugation analysis indicated that rMASP-3 sedimented as a homodimer (s(20,w) = 6.2 +/- 0.1 S) in the presence of Ca(2+), and as a monomer (s(20,w) = 4.6 +/- 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (K(D) = 2.6 nM) and L-ficolin (K(D) = 7.2 nM). The protease was produced in a single-chain, proenzyme form, but underwent slow activation upon prolonged storage at 4 degrees C, resulting from cleavage at the Arg(430)-Ile(431) activation site. Activation was prevented in the presence of protease inhibitors iodoacetamide and 1,10-phenanthroline but was not abolished upon substitution of Ala for the active site Ser(645) of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser(627)-->Ala in MASP-1 and Ser(618)-->Ala in MASP-2 stabilized the latter in their proenzyme form. Likewise, the MASP-1 and MASP-2 mutants were each activated by their active counterparts, but MASP-3 S645A was not. Activated MASP-3 did not react with C1 inhibitor; had no activity on complement proteins C2, C4, and C3; and only cleaved the N-carboxybenzyloxyglycine-L-arginine thiobenzyl ester substrate to a significant extent. Based on these observations, it is postulated that MASP-3 activation and control involve mechanisms that are different from those of MASP-1 and -2.     

Disease-causing mutations in genes of the complement system

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of “conventional” complement deficiencies with these newly described developmental roles.     

Decoupling of carbohydrate binding and MASP-2 autoactivation in variant mannose-binding lectins associated with immunodeficiency

Mannan-binding lectin (MBL) initiates complement activation by binding to arrays of carbohydrates on the surfaces of pathogenic microorganisms and activating MBL-associated serine proteases (MASPs). Separate point mutations to the collagenous domain of human MBL are associated with immunodeficiency, caused by reduced complement activation by the variant MBLs as well as by lower serum MBL concentrations. In the work reported here, we have used the well characterized rat lectin pathway to analyze the molecular and functional defects associated with two of the variant proteins. Mutations Gly25 --> Asp and Gly28 --> Glu create comparable structural changes in rat MBL but the G28E variant activates complement >10-fold less efficiently than the G25D variant, which in turn has approximately 7-fold lower activity than wild-type MBL. Analysis of mutant MBL . MASP-2 complexes assembled from recombinant components shows that reduced complement activation by both mutant MBLs is caused by failure to activate MASP-2 efficiently on binding to a mannan-coated surface. Disruption of MBL-MASP-2 interactions as well as to changes in oligomeric structure and reduced binding to carbohydrate ligands compared with wild-type MBL probably account for the intermediate phenotype of the G25D variant. However, carbohydrate binding and MASP-2 activation are ostensibly completely decoupled in complexes assembled from the G28E mutant, such that the rate of MASP-2 activation is no greater than the basal rate of zymogen MASP-2 autoactivation. Analogous molecular defects in human MBL probably combine to create the mutant phenotypes of immunodeficient individuals.