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Gérard J Arlaud

Publications and source records attributed to Gérard J Arlaud.

At least 19 recordsLinked to original sources
PubMed2007

Structural insights into the innate immune recognition specificities of L- and H-ficolins.

Innate immunity relies critically upon the ability of a few pattern recognition molecules to sense molecular markers on pathogens, but little is known about these interactions at the atomic level. Human L- and H-ficolins are soluble oligomeric defence proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. The X-ray structures of their trimeric recognition domains, alone and in complex with various ligands, have been solved to resolutions up to 1.95 and 1.7 A, respectively. Both domains have three-lobed structures with clefts separating the distal parts of the protomers. Ca(2+) ions are found at sites homologous to those described for tachylectin 5A (TL5A), an invertebrate lectin. Outer binding sites (S1) homologous to the GlcNAc-binding pocket of TL5A are present in the ficolins but show different structures and specificities. In L-ficolin, three additional binding sites (S2-S4) surround the cleft. Together, they define an unpredicted continuous recognition surface able to sense various acetylated and neutral carbohydrate markers in the context of extended polysaccharides such as 1,3-beta-D-glucan, as found on microbial or apoptotic surfaces.

Acetylation↗
PubMed2006

Modified low density lipoproteins differentially bind and activate the C1 complex of complement.

Several studies suggest that complement plays an important role in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of low density lipoprotein (LDL) to bind and activate C1, the complex that triggers the classical pathway of complement. For this purpose, LDL was both obtained commercially and purified according to an established procedure, and oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were generated from each preparation. Whereas the unmodified LDL and oxLDL samples did not activate C1 in the presence of excess C1 inhibitor, the E-LDL derivatives obtained by sequential treatment of LDL with a protease and then with cholesterol esterase triggered efficient C1 activation under these conditions, with activation levels approximately 60% upon incubation with 1 microM E-LDL for 90 min at 37 degrees C. In agreement with these findings, as shown by surface plasmon resonance spectroscopy (SPR), the C1q recognition subunit of C1 showed no interaction with unmodified LDL but bound to both E-LDL samples with high affinity (K(D)=58-75 nM). More unexpectedly, although they did not trigger direct C1 activation, both oxLDL samples were also efficiently recognized by C1q. Whereas the oxLDL derivative of commercial LDL activated C1 to a significant extent ( approximately 30%) in the presence of C-reactive protein (CRP), much lower activation levels (<10%) were obtained using the oxLDL derivative of purified LDL. As measured by SPR, CRP bound equally well to the oxLDL and E-LDL derivatives obtained from purified LDL. These data provide the first experimental evidence that E-LDL triggers efficient C1 activation under conditions close to the physiological situation, suggesting that activation of the classical complement pathway by this derivative may be a crucial factor in the pathogenesis of atherosclerosis. In contrast, it appears unlikely that oxLDL significantly activates C1 directly or in a CRP-dependent manner in vivo.

Animals↗
PubMed2006

Molecular organization of human Ficolin-2.

Human Ficolin-2 (L-Ficolin) is an oligomeric serum protein consisting of a collagen-like stalk and fibrinogen-like recognition domains. The protein binds to arrays of sugars present on different microorganisms, enhances phagocytosis and promotes activation of the lectin complement pathway. So far the detailed oligomeric structure and composition of human Ficolin-2 has not been determined. Recombinant human Ficolin-2 was expressed in Chinese hamster ovary cells and its structure and biological functions were investigated by gel filtration, sucrose density gradient ultracentrifugation, mass spectrometry and surface plasmon resonance spectroscopy. It was revealed that Ficolin-2 has a high molecular weight due to extensive disulfide bridge formation. It was able to bind to different ligands, interact with mannose-binding lectin associated serine proteases and activate the complement system. Mass values of 807 and 403 kDa were determined corresponding to a 24-mer and a 12-mer of 34.4 kDa polypeptides. However, the 24-mer was unstable and the 12-mer is likely the major functional form of the protein. Our results are consistent with the view that Ficolin-2 is built up by a mixture of stable homodimers and homotrimers. Based on our findings we propose a model in which disulfide bridges located in the N-terminal region of the polypeptides explain the oligomerization pattern of human Ficolin-2.

Animals↗
PubMed2006

Evidence that complement protein C1q interacts with C-reactive protein through its globular head region.

C1q acts as the recognition unit of the first complement component, C1, and binds to immunoglobulins IgG and IgM, as well as to non-Ig ligands, such as C-reactive protein (CRP). IgG and IgM are recognized via the globular head regions of C1q (C1qGR), whereas CRP has been postulated to interact with the collagen-like region (C1qCLR). In the present study, we used a series of nine mAbs to C1q, five directed against C1qGR and four against C1qCLR, to inhibit the interaction of C1q with CRP. The F(ab')(2) of each of the five mAbs directed against C1qGR inhibited binding of C1q to polymerized IgG. These five mAbs also successfully inhibited the interaction of C1q with CRP. Moreover, these five mAbs inhibited C1 activation by CRP as well as by polymerized IgG in vitro. In contrast, none of the four mAbs against C1qCLR inhibited C1q interaction with CRP or IgG, or could reduce activation of complement by CRP or polymerized IgG. These results provide the first evidence that the interaction of C1q with CRP or IgG involves sites located in the C1qGR, whereas sites in the CLR do not seem to be involved in the physiological interaction of C1q with CRP.

Antibodies, Monoclonal↗
PubMed2005

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules.

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

Amino Acid Sequence↗
PubMed2005

Functional role of the linker between the complement control protein modules of complement protease C1s.

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of C (C1). Its catalytic domain comprises two complement control protein (CCP) modules connected by a four-residue linker Gln340-Pro-Val-Asp343 and a serine protease domain. To assess the functional role of the linker, a series of mutations were performed at positions 340-343 of human C1s, and the resulting mutants were produced using a baculovirus-mediated expression system and characterized functionally. All mutants were secreted in a proenzyme form and had a mass of 77,203-77,716 Da comparable to that of wild-type C1s, except Q340E, which had a mass of 82,008 Da, due to overglycosylation at Asn391. None of the mutations significantly altered C1s ability to assemble with C1r and C1q within C1. Whereas the other mutations had no effect on C1s activation, the Q340E mutant was totally resistant to C1r-mediated activation, both in the fluid phase and within the C1 complex. Once activated, all mutants cleaved C2 with an efficiency comparable to that of wild-type C1s. In contrast, most of the mutations resulted in a decreased C4-cleaving activity, with particularly pronounced inhibitory effects for point mutants Q340K, P341I, V342K, and D343N. Comparable effects were observed when the C4-cleaving activity of the mutants was measured inside C1. Thus, flexibility of the C1s CCP1-CCP2 linker plays no significant role in C1 assembly or C1s activation by C1r inside C1 but plays a critical role in C4 cleavage by adjusting positioning of this substrate for optimal cleavage by the C1s active site.

Amino Acid Motifs↗
PubMed2005

Interactions of the extracellular matrix proteoglycans decorin and biglycan with C1q and collectins.

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.

Amino Acid Sequence↗
PubMed2005

Complement protein C1q recognizes a conformationally modified form of the prion protein.

Several studies have suggested the implication of the classical complement pathway in the early stages of prion disease pathogenesis. To explore this hypothesis, surface plasmon resonance spectroscopy was used to test the ability of human C1q to recognize mouse PrP immobilized on a sensor chip. In this configuration, C1q bound avidly to PrP, with a K(D) of 5.4 nM (k(on) = 2.4 x 10(5) M(-1) s(-1); k(off) = 1.3 x 10(-3) s(-1)). The isolated C1q globular domain also bound to immobilized PrP, although with a higher K(D) (238 nM), due to a decreased k(on) (4.2 x 10(3) M(-1) s(-1)). Interaction was strongly enhanced by Cu(2+) ions, with a 10-fold increase in overall binding in the presence of 10 microM CuSO(4), without significant modification of the kinetic parameters. In contrast, using the same technique, no interaction was detected between immobilized C1q and soluble PrP. Likewise, gel filtration and chemical cross-linking analyses yielded no evidence for an interaction between these proteins in solution. Comparative analysis of the antigenic reactivity of soluble and immobilized PrP was performed by ELISA and surface plasmon resonance spectroscopy, respectively, using anti-PrP monoclonal antibodies. This analysis provides evidence that immobilized PrP undergoes a major conformational change in the sequence stretch 141GNDWEDRYYRENMYRYPNQ159 located in its C-terminal globular domain. It is concluded that immobilized PrP undergoes structural modifications that possibly mimic the conformational changes occurring during conversion to the pathological isoform and that C1q represents a natural sensor of these changes. Pathological implications of this recognition property are discussed in light of recent reports.

Amino Acid Sequence↗
PubMed2005

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.

Acetylglucosamine↗
PubMed2005

Mass spectrometry analysis of the oligomeric C1q protein reveals the B chain as the target of trypsin cleavage and interaction with fucoidan.

C1q is a subunit of the C1 complex that triggers activation of the complement classical pathway through recognition and binding of immune complexes. C1q also binds to nonimmune ligands such as the sulfated polysaccharide fucoidan, a potent anticomplementary agent. C1q was submitted for the first time to mass spectrometry analysis, yielding insights into its assembly and its interaction with fucoidan. The MALDI-TOF mass spectrometry technique on membrane allowed partial preservation of noncovalent interactions, allowing precise analysis of its substructure and estimation of the C1q molecular weight at 459520-461883, with an average mass of 460793 g x mol(-1). The disulfide-linked A-B and C-C dimers as well as the noncovalent structural unit (A-B:C)-(C:B-A) were detected, providing experimental support to the C1q model based on covalent and noncovalent associations of six heterotrimers. Trypsin treatment of native C1q led to proteolysis of the B chain only, at a single cleavage site (Arg(109)) located in the globular region. Unlike DNA, fucoidan protected C1q from trypsin cleavage, indicating that this polysaccharide binds to the B moiety of the globular head. Given the involvement of the C1q globular heads in the recognition of IgG, this interaction may account for the observed anticomplementary activity of fucoidan.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

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.

Alanine↗
PubMed2003

Unusual Ca(2+)-calmodulin binding interactions of the microtubule-associated protein F-STOP.

F-STOP is a microtubule-associated protein that stabilizes microtubules in a calmodulin (CaM)-dependent manner. All members of the stable tubule only polypeptide (STOP) family have a central domain that contains nearly identical multiple repeats, and a CaM binding motif is present in multiple copies within this domain. We present here an analysis of this CaM binding interaction and find that it is highly unusual in nature. For this work, we synthesized two model peptides of a single STOP central repeat motif and analyzed their binding to CaM by fluorescence, circular dichroism, infrared and NMR spectroscopy. Both peptides bind to CaM with an affinity of 4 microM, similar to that of the native protein. Results indicate that the peptides bind CaM in an atypical manner. Binding is highly dependent on the concentration of cations, indicating that it is to some extent electrostatic. Further, IR and CD analysis shows that, in contrast to typical CaM binding reactions, CaM does not change in helical structure on binding. NMR mapping confirms that CaM remains in extended conformation on binding a single STOP peptide. Binding of a single peptide to CaM occurs principally in the CaM C-terminal region, and the C-terminal domain of CaM effectively competes for STOP binding. Our results establish that CaM binds STOP in an unusual manner, involving mainly the C-terminus of CaM, thus leaving CaM potentially accessible for another binding partner at the N-terminus. This intriguing possibility could be of physiological importance in F-STOP mediated CaM regulation of microtubule dynamics or stability, specifically during mitosis where CaM and STOP colocalize.

Amino Acid Motifs↗
PubMed2003

The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties.

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 A of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.

C-Reactive Protein↗
PubMed2003

X-ray structure of the Ca2+-binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement.

C1, the complex that triggers the classical pathway of complement, is assembled from two modular proteases C1r and C1s and a recognition protein C1q. The N-terminal CUB1-EGF segments of C1r and C1s are key elements of the C1 architecture, because they mediate both Ca2+-dependent C1r-C1s association and interaction with C1q. The crystal structure of the interaction domain of C1s has been solved and refined to 1.5 A resolution. The structure reveals a head-to-tail homodimer involving interactions between the CUB1 module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca2+ ion is bound to each EGF module and stabilizes both the intra- and inter-monomer interfaces. Unexpectedly, a second Ca2+ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu45, Asp53, Asp98, and two water molecules. These acidic residues and Tyr17 are conserved in approximately two-thirds of the CUB repertoire and define a novel, Ca2+-binding CUB module subset. The C1s structure was used to build a model of the C1r-C1s CUB1-EGF heterodimer, which in C1 connects C1r to C1s and mediates interaction with C1q. A structural model of the C1q/C1r/C1s interface is proposed, where the rod-like collagen triple helix of C1q is accommodated into a groove along the transversal axis of the C1r-C1s heterodimer.

Amino Acid Sequence↗
PubMed2003

A recombinant chimeric epidermal growth factor-like module with high binding affinity for integrins.

Integrins are cell surface receptors involved in numerous pathological processes such as metastasis invasion and abnormal angiogenesis. To target these receptors, the epidermal growth factor (EGF)-like domain of human complement protease C1r was used as a natural scaffold to design chimeric modules containing the RGD motif. Here we report a high yield bacterial expression system and its application to the production of two such modules, EGF-RGD and V2, the latter variant mimicking the RGD-containing domain of disintegrins. These modules were characterized chemically, and their biological activity was investigated by cellular assays using various Chinese hamster ovary cell lines expressing beta1 and beta3 integrins and by surface plasmon resonance spectroscopy. Remarkably, the modifications leading to the V2 variant had differential effects on the interaction with beta3 and beta1 integrins. The disintegrin-like V2 module exhibited enhanced binding affinities compared with EGF-RGD, with KD values of 7.2 nm for alpha5beta1 (a 4-fold decrease) and 3.5 nm for alphavbeta3 (a 1.5-fold decrease), comparable with the values determined for natural integrin ligands. Analysis by NMR spectroscopy also revealed a differential dynamic behavior of the RGD motif in the EGF-RGD and V2 variants, providing insights into the structural basis of their relative binding efficiency. These novel RGD-containing EGF modules open the way to the design of improved variants with selective affinity for particular integrins and their use as carriers for other biologically active modules.

Amino Acid Sequence↗
PubMed2002

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.

Amino Acid Motifs↗