RESUMEN
Complement factor D (FD) is a serine protease that plays an essential role in the activation of the alternative pathway (AP) by cleaving complement factor B (FB) and generating the C3 convertases C3(H2 O)Bb and C3bBb. FD is produced mainly from adipose tissue and circulates in an activated form. On the contrary, the other serine proteases of the complement system are mainly synthesized in the liver. The activation mechanism of FD has long been unknown. Recently, a serendipitous discovery in the mechanism of FD activation has been provided by a generation of Masp1 gene knockout mice lacking both the serine protease MASP-1 and its alternative splicing variant MASP-3, designated MASP-1/3-deficient mice. Sera from the MASP-1/3-deficient mice had little-to-no lectin pathway (LP) and AP activity with circulating zymogen or proenzyme FD (pro-FD). Sera from patients with 3MC syndrome carrying mutations in the MASP1 gene also had circulating pro-FD, suggesting that MASP-1 and/or MASP-3 are involved in activation of FD. Here, we summarize the current knowledge of the mechanism of FD activation that was finally elucidated using the sera of mice monospecifically deficient for MASP-1 or MASP-3. Sera of the MASP-1-deficient mice lacked LP activity, but those of the MASP-3-deficient mice lacked AP activity with pro-FD. This review illustrates the pivotal role of MASP-3 in the physiological activation of the AP via activation of FD.
Asunto(s)
Factor D del Complemento , Vía Alternativa del Complemento , Humanos , Animales , Ratones , Factor D del Complemento/genética , Factor D del Complemento/metabolismo , Vía Alternativa del Complemento/fisiología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Proteínas del Sistema Complemento , Ratones NoqueadosRESUMEN
INTRODUCTION: Chagas disease (CD), caused by Trypanosoma cruzi, is a major public health issue worldwide affecting 6-7 million people, mainly in Latin America. The complement system plays a crucial role in host immune defense against T. cruzi infection and during the chronic phase of CD; however, the role of the MBL-associated serine protease 1 (MASP1) gene encoding MASP-1, MASP-3, and MAp44 complement proteins has not yet been reported in CD. This study investigated the possible association between MASP1 gene polymorphisms and MASP-3 protein serum levels in chronic CD and its clinical forms. METHODS: Five polymorphisms of MASP1 gene regulatory regions were genotyped in 214 patients with CD and 197 healthy controls (rs7609662 G>A, rs13064994 C>T, rs72549262 C>G, rs1109452 C>T and rs850314 G>A). MASP-3 serum levels were assessed in 70 patients and 66 healthy controls. Clinical data, serum levels of complement proteins (ficolin-2, ficolin-3 and MBL) and inflammatory markers (pentraxin-3 and hsCRP) were also included in the analyses. RESULTS: A significant association of the MASP1 GC_CCA haplotype with CD (padj= 0.002; OR 3.17 [1.19-8.39]) and chronic chagasic cardiomyopathy (CCC) (padj= 0.013; OR 4.57 [1.37-15.16] was observed. MASP-3 and pentraxin-3 levels were positively correlated in the patients (rho = 0.62; p = 0.0001). MASP-3 levels were not associated with MASP1 polymorphisms or CD and its clinical forms. Furthermore, no correlation was observed between MASP-3 levels and that of ficolin-2, ficolin-3, MBL and hsCRP. CONCLUSION: Our findings suggest a possible role for the MASP1 GC_CCA haplotype in susceptibility to chronic CD and CCC clinical forms.
Asunto(s)
Enfermedad de Chagas , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Proteína C-Reactiva , Enfermedad de Chagas/genética , Proteínas del Sistema Complemento , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Polimorfismo GenéticoRESUMEN
The complement system plays an important role in host defense and is activated via three different activation pathways. We have previously reported that mannose-binding lectin-associated serine protease (MASP)-3, unlike its splicing variant MASP-1, circulates in an active form and is essential for the activation of the alternative pathway (AP) via the activation of complement factor D (FD). On the other hand, like MASP-1 and MASP-2 of the lectin pathway (LP), MASP-3 forms a complex with the pattern recognition molecules (PRMs) of the LP (LP-PRMs). Both MASP-1 and MASP-2 can be activated efficiently when the LP-PRMs complexed with them bind to their ligands. On the other hand, it remains unclear how MASP-3 is activated, or whether complex formation of MASP-3 with LP-PRMs is involved in activation of MASP-3 or its efficiency in the circulation. To address these issues, we generated wild-type (WT) and four mutant recombinant mouse MASP-3 proteins fused with PA (human podoplanin dodecapeptide)-tag (rmMASP-3-PAs), the latter of which have single amino acid substitution for alanine in the CUB1 or CUB2 domain responsible for binding to LP-PRMs. The mutant rmMASP-3-PAs showed significantly reduced in-vivo complex formation with LP-PRMs when compared with WT rmMASP-3-PA. In the in-vivo kinetic analysis of MASP-3 activation, both WT and mutant rmMASP-3-PAs were cleaved into the active forms as early as 30 minutes in the circulation of mice, and no significant difference in the efficiency of MASP-3 cleavage was observed throughout an observation period of 48 hours after intravenous administration. All sera collected 3 hours after administration of each rmMASP-3-PA showed full restoration of the active FD and AP activity in MASP-3-deficient mouse sera at the same levels as WT mouse sera. Unexpectedly, all mutant rmMASP-3-PAs showed faster clearance from the circulation than the WT rmMASP-3-PA. To our knowledge, the current study is the first to show in-vivo kinetics of MASP-3 demonstrating rapid activation and clearance in the circulation. In conclusion, our results demonstrated that the complex formation of MASP-3 with LP-PRMs is not required for in-vivo activation of MASP-3 or its efficiency, but may contribute to the long-term retention of MASP-3 in the circulation.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Animales , Lectina de Unión a Manosa de la Vía del Complemento/fisiología , Proteínas del Sistema Complemento , Humanos , Cinética , Lectinas/genética , Lectinas/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Ratones , Mutación , Proteínas Recombinantes/metabolismoRESUMEN
Proteins destined for secretion - after removal of the signal sequence - often undergo further proteolytic processing by proprotein convertases (PCs). Prohormones are typically processed in the regulated secretory pathway, while most plasma proteins travel though the constitutive pathway. The complement system is a major proteolytic cascade in the blood, serving as a first line of defense against microbes and also contributing to the immune homeostasis. Several complement components, namely C3, C4, C5 and factor I (FI), are multi-chain proteins that are apparently processed by PCs intracellularly. Cleavage occurs at consecutive basic residues and probably also involves the action of carboxypeptidases. The most likely candidate for the intracellular processing of complement proteins is furin, however, because of the overlapping specificities of basic amino acid residue-specific proprotein convertases, other PCs might be involved. To our surprise, we have recently discovered that processing of another complement protein, mannan-binding lectin-associated serine protease-3 (MASP-3) occurs in the blood by PCSK6 (PACE4). A similar mechanism had been described for the membrane protease corin, which is also activated extracellularly by PCSK6. In this review we intend to point out that the proper functioning of the complement system intimately depends on the action of proprotein convertases. In addition to the non-enzymatic components (C3, C4, C5), two constitutively active complement proteases are directly activated by PCs either intracellularly (FI), or extracellularly (MASP-3), moreover indirectly, through the constitutive activation of pro-factor D by MASP-3, the activity of the alternative pathway also depends on a PC present in the blood.
Asunto(s)
Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Proproteína Convertasas , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Proproteína Convertasas/metabolismo , ProteolisisRESUMEN
Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1-2 µg/ml among healthy individuals. The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D. In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82-25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8-15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.
Asunto(s)
Factor D del Complemento , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Activación de Complemento , Convertasas de Complemento C3-C5 , Factor D del Complemento/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento , Humanos , Lectinas/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismoRESUMEN
The serine protease complement factor D is fundamental in the activation of the complement system. In addition, it was the first adipokine described (named Adipsin) and shown to improve beta cell function in diabetes. As part of an amplification loop of complement activation, factor D is a rate-limiting enzyme, and its accessibility contributes to the potency of complement activation. The dogma has been that conversion of the zymogen form, profactor D, to mature factor D occurred during secretion by adipocytes by uncharacterized proteases. However, recent findings demonstrated that the serine protease MASP-3 of the lectin pathway of the complement system mediated this conversion, suggesting that pattern recognition of pathogen/danger-associated molecular patterns could be a prior requirement for all complement activation. To facilitate studies addressing this hypothesis, we have developed monoclonal antibodies specific for human profactor D without binding to mature factor D. We demonstrate their applications in accessing the conversion of profactor D into mature factor D and in measuring levels of profactor D.
Asunto(s)
Factor D del Complemento , Lectina de Unión a Manosa de la Vía del Complemento , Anticuerpos , Factor D del Complemento/metabolismo , Proteínas del Sistema Complemento , Precursores Enzimáticos , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la ManosaRESUMEN
The Malpuech, Michels, Mingarelli, Carnevale (3MC) syndrome is a rare, autosomal recessive genetic- disorder associated with mutations in the MASP1/3, COLEC1,1 or COLEC10 genes. The number of 3MC patients with known mutations in these three genes reported so far remains very small. To date, 16 mutations in MASP-1/3, 12 mutations in COLEC11 and three in COLEC10 associated with 3MC syndrome have been identified. Their products play an essential role as factors involved in the activation of complement via the lectin or alternative (MASP-3) pathways. Recent data indicate that mannose-binding lectin-associated serine protease-1 (MASP-1), MASP-3, collectin kidney-1 (collectin-11) (CL-K1), and collectin liver-1 (collectin-10) (CL-L1) also participate in the correct migration of neural crest cells (NCC) during embryogenesis. This is supported by relationships between MASP1/3, COLEC10, and COLEC11 gene mutations and the incidence of 3MC syndrome, associated with craniofacial abnormalities such as radioulnar synostosis high-arched eyebrows, cleft lip/palate, hearing loss, and ptosis.
Asunto(s)
Anomalías Múltiples/genética , Colectinas/genética , Anomalías Craneofaciales/genética , Cardiopatías Congénitas/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Discapacidades del Desarrollo/genética , Humanos , MutaciónRESUMEN
Introduction: Defining mechanisms governing the diffusion from blood to cerebrospinal fluid is central to understanding immune function in the central nervous system. Objective: To describe the dynamics of diffusion of the lectin pathway components from blood to cerebrospinal fluid. Methods: It was organized the information available in PubMed database and of papers from journals, and abstract books from international congresses belongs mainly to Cuban authors all about the lectin pathway of complement including manan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP2), and of other components like MASP3, Map44 as regulatory components and the different starters like MBL, ficolins and CLLK. Results: All the lectin pathways component are blood derived proteins but at the same time it could be synthesized intrathecally. Most of the protein can be transferred from blood to cerebrospinal fluid in different aggregation forms and some of them can be described as a consuming curve. The control mechanism of regulation the lectin pathway can be followed by molecules as MASP3 and Map44. Conclusions: The under- constructed lectin pathway of the complement system required not only the available information in different journals. It had to be completed by reviewing the congress abstract book and congress website of the last years(AU)
Asunto(s)
Humanos , Líquido Cefalorraquídeo/fisiologíaRESUMEN
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.
Asunto(s)
Infecciones Bacterianas/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Infecciones por Protozoos/inmunología , Virosis/inmunología , Infecciones Bacterianas/genética , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/patología , Lectina de Unión a Manosa de la Vía del Complemento/genética , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Polimorfismo Genético , Infecciones por Protozoos/genética , Infecciones por Protozoos/parasitología , Infecciones por Protozoos/patología , Transducción de Señal , Virosis/genética , Virosis/patología , Virosis/virologíaRESUMEN
The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.
Asunto(s)
Anomalías Múltiples/enzimología , Blefaroptosis/enzimología , Anomalías Craneofaciales/enzimología , Craneosinostosis/enzimología , Criptorquidismo/enzimología , Cristalografía por Rayos X/métodos , Anomalías del Ojo/enzimología , Cardiopatías Congénitas/enzimología , Luxación Congénita de la Cadera/enzimología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Estrabismo/enzimología , Músculos Abdominales/anomalías , Músculos Abdominales/enzimología , Discapacidades del Desarrollo/enzimología , Activación Enzimática , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.