RESUMEN
Mannose-binding lectin (MBL) activates the complement system lectin pathway and subsequent inflammatory mechanisms. The incidence and outcome of many human diseases, such as brain ischemia and infections, are associated with and influenced by the activity and serum concentrations of MBL in body fluids. To quantify MBL levels, tests based on ELISA are used, requiring several incubation and washing steps and lengthy turnaround times. Here, we aimed to develop a nanoplasmonic assay for direct MBL detection in human serum at the point of care. Our assay is based on gold nanorods (GNRs) functionalized with mannose (Man-GNRs) via an amphiphilic linker. We experimentally determined the effective amount of sugar linked to the nanorods' surface, resulting in an approximate grafting density of 4 molecules per nm2, and an average number of 11 to 13 MBL molecules binding to a single nanoparticle. The optimal Man-GNRs concentration to achieve the highest sensitivity in MBL detection was 15 µg·mL-1. The specificity of the assay for MBL detection both in simple buffer and in complex pooled human sera was confirmed. Our label-free biosensor is able to detect MBL concentrations as low as 160 ng·mL-1 within 15 min directly in human serum via a one-step reaction and by using a microplate reader. Hence, it forms the basis for a fast, noninvasive, point-of-care assay for diagnostic indications and monitoring of disease and therapy.
Asunto(s)
Técnicas Biosensibles , Oro , Lectina de Unión a Manosa , Sistemas de Atención de Punto , Humanos , Oro/química , Lectina de Unión a Manosa/sangre , Lectina de Unión a Manosa/química , Técnicas Biosensibles/métodos , Nanotubos/química , Manosa/química , Manosa/sangre , Nanopartículas del Metal/químicaRESUMEN
C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.
Asunto(s)
Lectinas Tipo C , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Humanos , Ligandos , Sitios de Unión , Calcio/metabolismo , Calcio/química , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Unión Proteica , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/metabolismo , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/química , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Antígenos CD/química , Antígenos CD/metabolismoRESUMEN
Collagen triple helices are critical in the function of mannan-binding lectin (MBL), an oligomeric recognition molecule in complement activation. The MBL collagen regions form complexes with the serine proteases MASP-1 and MASP-2 in order to activate complement, and mutations lead to common immunodeficiencies. To evaluate their structure-function properties, we studied the solution structures of four MBL-like collagen peptides. The thermal stability of the MBL collagen region was much reduced by the presence of a GQG interruption in the typical (X-Y-Gly)n repeat compared to controls. Experimental solution structural data were collected using analytical ultracentrifugation and small angle X-ray and neutron scattering. As controls, we included two standard Pro-Hyp-Gly collagen peptides (POG)10-13, as well as three more peptides with diverse (X-Y-Gly)n sequences that represented other collagen features. These data were quantitatively compared with atomistic linear collagen models derived from crystal structures and 12,000 conformations obtained from molecular dynamics simulations. All four MBL peptides were bent to varying degrees up to 85o in the best-fit molecular dynamics models. The best-fit benchmark peptides (POG)n were more linear but exhibited a degree of conformational flexibility. The remaining three peptides showed mostly linear solution structures. In conclusion, the collagen helix is not strictly linear, the degree of flexibility in the triple helix depends on its sequence, and the triple helix with the GQG interruption showed a pronounced bend. The bend in MBL GQG peptides resembles the bend in the collagen of complement C1q and may be key for lectin pathway activation.
Asunto(s)
Colágeno , Activación de Complemento , Lectina de Unión a Manosa , Colágeno/química , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/metabolismo , Soluciones/química , Conformación Proteica , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Relación Estructura-Actividad , Estabilidad Proteica , Dispersión del Ángulo Pequeño , Difracción de Neutrones , Ultracentrifugación , Simulación de Dinámica Molecular , Cristalografía por Rayos X , DocilidadRESUMEN
The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.
Asunto(s)
Proteínas del Helminto/farmacología , Lectina de Unión a Manosa/farmacología , Aglutinación , Secuencia de Aminoácidos , Animales , Bacterias/citología , Bacterias/efectos de los fármacos , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Fasciola/química , Fasciola/genética , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Hemaglutinación/efectos de los fármacos , Humanos , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/metabolismo , Alineación de Secuencia , Streptococcus/citología , Streptococcus/efectos de los fármacosRESUMEN
Agaricus bisporus mannose-binding protein (Abmb) was discovered as part of mushroom tyrosinase (PPO3) complex. Apart from its presence, nothing is known about its function or activity in the mushroom. The protein is evolutionarily related to lectins with ß-trefoil fold, which are glucose or galactose (and their derivatives) binding proteins. Abmb is also recently showed to display the typical agglutination activity of lectin when in complex with PPO3; this further supports Abmb similarity to its structural homologs from lectin with ß-trefoil fold. However, Abmb has no affinity towards glucose or galactose but for mannose, thus its binding to the sugar may be different from its homologs. To date, the natural ligand of Abmb is unknown and the structure of Abmb in the presence of a ligand is not available. Therefore, the mannose-binding site of Abmb was predicted using molecular docking, which was consulted with the information from its structural homologs. This conservative approach would prevent over-speculation. The mannose-binding site of Abmb is likely located in the same region to that of Abmb structural homologs but with a shift in position due to the presence of additional surface loop. In addition, benefiting from the information from an in vitro study on Abmb sugar specificity, the mannose poses suggested that the sugar might interact with the side chains of Arg15, Thr45, Gln48, Asp49, Asp51 and Arg51. Most of these residues were equally present in Abmb structural homologs despite variation of their positions in the amino acid sequence. The variation probably originates from alteration of its amino acid sequence during evolution.
Asunto(s)
Agaricus/química , Proteínas Fúngicas/química , Lectina de Unión a Manosa/química , Pliegue de Proteína , Sitios de Unión , Conformación Proteica en Lámina betaRESUMEN
The biomolecular recognition of D-mannose-binding lectin from Artocarpus heterophyllus (ArtinM) by Horseradish Peroxidase (HRP) mediated by glycosylation allows their application in a multitude of biological systems. The present work describes the use of molecular dynamics (MD) to assess the Gibbs free energy associated with the formation of a ArtinM-HRP conjugate mediated by a glycosylation molecule. For the enthalpy term, we applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method and for the vibrational entropy term, we use the quasi-harmonic approximation. Our results show that, even without glycosylation, the binding free energy between ArtinM and HRP is - 196.154 kJmol- 1, an extremely high affinity with low selectivity, originated mainly through the van der Waals energy terms. The binding free energy between ArtinM and the glycosylated HRP (gHRP) was calculated at - 66.156 kJmol- 1, an absolute and considerably lower value, however, originated from electrostatic energy terms, which increases the selectivity of molecular recognition. Our work has shown that the HRP active site region has a high affinity and low selectivity for other biomolecules. The presence of glycosylation plays a role in increasing this selectivity for this region. Thus, we conclude that performing mutagenesis of amino acid residues near the entrance of the catalytic site, can improve the activity of non-glycosylated HRPs. This illustrates new insights that can be applied to carbohydrate-based immunochemistry.
Asunto(s)
Artocarpus/metabolismo , Lectina de Unión a Manosa/metabolismo , Simulación de Dinámica Molecular , Glicosilación , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Lectina de Unión a Manosa/química , Lectinas de Plantas , TermodinámicaRESUMEN
YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.
Asunto(s)
Proteínas Arqueales/química , Euryarchaeota/enzimología , Methanobacteriaceae/enzimología , Methanocaldococcus/enzimología , Oxidorreductasas/química , Tioamidas/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Euryarchaeota/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Cinética , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/metabolismo , Methanobacteriaceae/genética , Methanocaldococcus/genética , Modelos Moleculares , Mutación , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Tioamidas/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) is an infectious disease caused by a virus called "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)." In the majority of patients, infection with COVID-19 may be asymptomatic or may cause only mild symptoms. However, in some patients, there can also be immunological problems, such as macrophage activation syndrome (CSS) that results in cytokine storm syndrome (CSS) and acute respiratory distress syndrome (ARDS). Comprehension of host-microbe communications is the critical aspect in the advancement of new therapeutics against infectious illnesses. Endogenous animal lectins, a class of proteins, may perceive non-self glycans found on microorganisms. Serum mannose-binding lectin (sMBL), as a part of the innate immune framework, recognizes a wide range of microbial microorganisms and activates complement cascade via an antibody-independent pathway. Although the molecular basis for the intensity of SARS-CoV-2 infection is not generally understood, scientific literature indicates that COVID-19 is correlated with unregulated activation of the complement in terms of disease severity. Disseminated intravascular coagulation (DIC), inflammation, and immune paralysis contribute to unregulated complement activation. Pre-existing genetic defects in MBL and their association with complement play a major role in immune response dysregulation caused by SARS-CoV-2. In order to generate anti-complement-based therapies in Covid-19, an understanding of sMBL in immune response to SARS-CoV-2 and complement is therefore essential. This review highlights the role of endogenous sMBL and complement activation during SARS-CoV-2 infection and their therapeutic management by various agents, mainly plant lectins, since antiviral mannose-binding plant lectins (pMBLs) offer potential applications in the prevention and control of viral infections.
Asunto(s)
Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , COVID-19/metabolismo , Proteínas del Sistema Complemento/metabolismo , Lectina de Unión a Manosa/metabolismo , Antivirales/farmacología , COVID-19/sangre , COVID-19/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Lectina de Unión a Manosa/sangre , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/deficiencia , SARS-CoV-2/efectos de los fármacosRESUMEN
The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.
Asunto(s)
Aeromonas hydrophila/inmunología , Cíclidos/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Lectina de Unión a Manosa/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus agalactiae/inmunología , Animales , Cíclidos/microbiología , Femenino , Enfermedades de los Peces/microbiología , Proteínas de Peces/química , Proteínas de Peces/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/veterinaria , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Infecciones Estreptocócicas/veterinariaRESUMEN
Agaricus bisporus mannose-binding protein (Abmb) was discovered as part of the mushroom tyrosinase (PPO3) complex, but its function in the mushroom has remained obscure. The protein has a ß-trefoil structure that is common for Ricin-B-like lectins. Indeed, its closest structural homologs are the hemagglutinin components of botulinum toxin (HA-33) and the Ricin-B-like lectin from Clitocybe nebularis (CNL), both of which bind galactose, and actinohivin, a recently discovered mannose-binding lectin from actinomycetes. Here we show that Abmb is evolutionarily related to them, which are lectins with a ß-trefoil fold. We also show for the first time that Abmb can exhibit typical lectin agglutination activity but only when in the complex with mushroom tyrosinase. This is unexpected and unique because the two proteins are not evolutionarily related and have different activities. Lectin and tyrosinase major role in defense mechanism as well as Abmb and PPO3 gene regulation during the early stages of the development of mushroom fruiting bodies suggested that Abmb has likely a function in defense against bacterial infection and/or insect-induced damage.
Asunto(s)
Agaricus/química , Proteínas Fúngicas/química , Lectinas/química , Lectina de Unión a Manosa/química , Agaricus/genética , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Lectinas/genética , Lectina de Unión a Manosa/genética , Modelos Moleculares , Filogenia , Conformación Proteica en Lámina betaRESUMEN
The mushroom Agaricus bisporus secretes biologically active compounds and proteins with benefits for human health. Most reported proteins from A. bisporus are tyrosinases and lectins. Lectins are of therapeutic or pharmaceutical interest. To date, only limited information is available on A. bisporus lectins and lectin-like proteins. No therapeutic products derived from A. bisporus lectin (ABL) are available on the market despite its extensive exploration. Recently, A. bisporus mannose-binding protein (Abmb) was discovered. Its discovery enriches the information and increases the interest in proteins with therapeutic potential from this mushroom. Furthermore, the A. bisporus genome reveals the possible occurrence of other lectins in this mushroom that may also have therapeutic potential. Most of these putative lectins belong to the same lectin groups as ABL and Abmb. Their relationship is discussed. Particular attention is addressed to ABL and Abmb, which have been explored for their potential in medicinal or pharmaceutical applications. ABL and Abmb have anti-proliferative activities toward cancer cells and a stimulatory effect on the immune system. Possible scenarios for their use in therapy and modification are also presented.
Asunto(s)
Agaricus/química , Lectinas/genética , Lectina de Unión a Manosa/genética , Monofenol Monooxigenasa/genética , Agaricus/genética , Genoma Fúngico/genética , Humanos , Lectinas/uso terapéutico , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/uso terapéutico , Monofenol Monooxigenasa/químicaRESUMEN
Lectins have the ability to bind specific carbohydrates and they have potential applications as medical and pharmacological agents. The unique structure and usefulness of red algal lectin have been reported, but these lectins are limited to a few marine algal groups. In this study, a novel mannose-binding lectin from Grateloupia chiangii (G. chiangii lectin, GCL) was purified using antiviral screens and affinity chromatography. We characterized the molecular weight, agglutination activity, hemagglutination activity, and heat stability of GCL. To determine the carbohydrate specificity, a glycan microarray was performed. GCL showed strong binding affinity for Maltohexaose-ß-Sp1 and Maltoheptaose-ß-Sp1 with weak affinity for other monosaccharides and preferred binding to high-mannan structures. The N-terminal sequence and peptide sequence of GCL were determined using an Edman degradation method and LC-MS/MS, and the cDNA and peptide sequences were deduced. GCL was shown to consist of 231 amino acids (24.9 kDa) and the N-terminus methionine was eliminated after translation. GCL possessed a tandem repeat structure of six domains, similar to the other red algal lectins. The mannose binding properties and tandem repeat structure of GCL may confer it the potential to act as an antiviral agent for protection against viral infection.
Asunto(s)
Proteínas Algáceas/química , Antivirales/química , Lectina de Unión a Manosa/química , Rhodophyta/química , Proteínas Algáceas/metabolismo , Proteínas Algáceas/farmacología , Animales , Antivirales/metabolismo , Antivirales/farmacología , Perros , Pruebas de Hemaglutinación , Caballos , Células de Riñón Canino Madin Darby , Lectina de Unión a Manosa/metabolismo , Lectina de Unión a Manosa/farmacología , Unión Proteica , Rhodophyta/metabolismo , Ovinos , Virosis/tratamiento farmacológico , Virus/efectos de los fármacosRESUMEN
Agaricus bisporus mannose binding protein (Abmb) demonstrates permeability to epithelial monolayer barrier of the intestine, resistance to gastrointestinal tract conditions and to proteolysis therefore it holds potential as a drug carrier for oral route administration. Abmb also display antiproliferative activity to breast cancer cells and stimulation of immune system thus could potentially be also developed for therapeutic purpose. It is not immunogenic or toxic thereby safe for use. In this paper we further provide evidence that Abmb also lacks of agglutinating activity despite sharing high structural homology to lectins. Abmb is thereby the only mannose specific binding protein that is not member of lectin family. This evidence provides further support on the use of Abmb as pharmaceutical or medicinal agent. Its molecular globularity that may contribute to its lack of agglutination capacity was also evaluated.
Asunto(s)
Agaricus/metabolismo , Proteínas Fúngicas/farmacología , Lectinas/farmacología , Lectina de Unión a Manosa/farmacología , Animales , Eritrocitos/efectos de los fármacos , Eritrocitos/inmunología , Proteínas Fúngicas/administración & dosificación , Proteínas Fúngicas/química , Hemaglutinación/efectos de los fármacos , Hemaglutinación/inmunología , Pruebas de Hemaglutinación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas/administración & dosificación , Lectinas/química , Lectina de Unión a Manosa/administración & dosificación , Lectina de Unión a Manosa/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
Asunto(s)
Acanthamoeba castellanii/metabolismo , Hongos/patogenicidad , Lectina de Unión a Manosa/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/microbiología , Acanthamoeba castellanii/ultraestructura , Animales , Candida albicans/patogenicidad , Candida albicans/ultraestructura , Concanavalina A/metabolismo , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/ultraestructura , Histoplasma/patogenicidad , Histoplasma/ultraestructura , Interacciones Huésped-Patógeno , Larva/microbiología , Lepidópteros/microbiología , Manosa/química , Manosa/metabolismo , Lectina de Unión a Manosa/química , Espectrometría de Masas , Microscopía Electrónica de Rastreo , Paracoccidioides/patogenicidad , Paracoccidioides/ultraestructura , Saccharomyces cerevisiae/patogenicidad , Saccharomyces cerevisiae/ultraestructura , Factores de Tiempo , Imagen de Lapso de Tiempo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Mannose-binding lectin (MBL) is a pattern recognition receptor (PRR) that plays an important role in the innate immune response. In this study, a novel mannose-binding lectin was cloned from the swimmimg crab Portunus trituberculatus (designated as PtMBL). The complete cDNA of PtMBL gene was 1208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gill with sexually dimorphic expression. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca2+-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d-galactose and d-mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. These results together indicate that PtMBL could serve as not only a PRR in immune recognition but also a potential antibacterial protein in the innate immune response of crab.
Asunto(s)
Braquiuros/genética , Braquiuros/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Femenino , Perfilación de la Expresión Génica , Masculino , Lectina de Unión a Manosa/química , Micrococcus luteus/fisiología , Filogenia , Pichia/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Vibrio alginolyticus/fisiologíaRESUMEN
The complement system has been implicated in several kidney diseases, such as antibody-mediated rejection after kidney transplantation. Antibody-depletion techniques allow successful ABO- and/or HLA-incompatible transplantation. Considering the IgG removal, the use of semi-selective immunoadsorption (IA) has been advocated. However, because of results on incomplete IgM depletion, the adjunctive use of membrane filtration (MF) has been proposed to enhance the removal of macromolecules and to interfere with complement activation. This secondary endpoint analysis of a recently published randomized, controlled, cross-over trial was designed to investigate the effect of combined treatment IA + MF compared to IA alone on complement depletion. Two treatment sequences, a single session of IA + MF followed by IA (and vice versa), were analyzed with regard to C5b-9, properdin, and mannose-binding lectin (MBL) levels. Neither IA alone nor IA + MF provoked complement activation as demonstrated by stable low levels of C5b-9 after the procedure as compared to the previous. The combined treatment substantially lowered properdin (77% vs. 26% reduction, P < 0.0001) as well as MBL concentrations (81% vs. 11% reduction, P < 0.0001). Recovery of properdin and MBL levels appears to be longer after IA alone compared to IA + MF. Depletion of properdin and MBL levels may have potential clinical implications in the setting of kidney transplantation.
Asunto(s)
Eliminación de Componentes Sanguíneos/métodos , Inmunoglobulina M/inmunología , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/cirugía , Trasplante de Riñón/métodos , Membranas Artificiales , Sistema del Grupo Sanguíneo ABO , Adsorción , Adulto , Incompatibilidad de Grupos Sanguíneos , Activación de Complemento , Proteínas del Sistema Complemento/inmunología , Estudios Cruzados , Femenino , Humanos , Masculino , Lectina de Unión a Manosa/química , Persona de Mediana Edad , Properdina/inmunologíaRESUMEN
Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)-i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl ß-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.
Asunto(s)
Chlorophyta/química , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetidas en Tándem , Secuencia de Aminoácidos , Animales , Carbohidratos/química , Pruebas de Hemaglutinación , Caballos , Lectina de Unión a Manosa/aislamiento & purificación , Lectinas de Plantas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Ovinos , SolubilidadRESUMEN
The core α1-6 fucosylation-specific lectin from a mushroom Pholiota squarrosa (PhoSL) is a potential tool for precise diagnosis of cancers. This lectin consists of only 40 amino acids and can be chemically synthesized. We showed here that a synthesized PhoSL peptide formed a trimer by gel filtration and chemical cross-linking assays, and determined a structure of the PhoSL trimer by NMR. The structure possesses a ß-prism motif with a three-fold rotational symmetry, where three antiparallel ß-sheets are tightly connected by swapping of ß-strands. A triad of Trp residues comprises the structural core, forming NH-π electrostatic interactions among the indole rings. NMR analysis with an excess amount of fucose revealed the structural basis for the molecular recognition. Namely, fucose deeply enters a pocket formed at a junction of ß-sheet edges, with the methyl group placed at the bottom. It forms a number of hydrophobic and hydrogen-bonding interactions with PhoSL residues. In spite of partial similarities to the structures of other functionally related lectins, the arrangement of the antiparallel ß-sheets in the PhoSL trimer is novel as a structural scaffold, and thus defines a novel type of lectin structure.
Asunto(s)
Fucosa/metabolismo , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/metabolismo , Fragmentos de Péptidos/química , Pholiota/metabolismo , Multimerización de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Fucosa/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Manosa/química , Manosa/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Conformación ProteicaRESUMEN
Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Genetic factors associated with immune response contribute to infection development and disease. M. leprae has the capacity to invade Schwann cells in the peripheral nervous system and cause neuropathy. However, while the responsible molecular mechanisms remain to be fully unveiled, they have begun being elucidated. We studied genetic variants Myelin Protein Zero (MPZ), a major structural component of the myelin sheath, and Mannose Binding Lectin 2 (MBL2), a protein involved in immune response, in 112 family groups of 114 leprosy patients using PCR-RFLP, aiming to calculate the association and allelic transmission of variants associated in first, second and third-degree relatives. Polymorphisms found in MPZ and MBL2 showed association with leprosy. Different probabilities for allelic transmission were found for first and second-degree relatives, a fact that is important to take into account when evaluating risk in contacts of leprosy patients. Structural analysis allows the study of putative amino acids and their possible effect on protein structure and function, as well as on the assembly of a protein homotetramer. Our results suggest that the identified MPZ and MBL2 gene mutations are associated with leprosy in a Colombian population, which correlates with MPZ and MBL2 protein function, and increase the risk of M. leprae infection in leprosy-patients' family members. Additionally, structural analyses were carried out specifically for MPZ protein using information available in databases, and analyzing the substitutions in wildtype and mutant protein. The results show significant structural changes, which may be associated to infection and pathogenicity.
Asunto(s)
Lepra , Lectina de Unión a Manosa , Proteína P0 de la Mielina , Adulto , Colombia , Femenino , Humanos , Lepra/genética , Lepra/inmunología , Masculino , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Persona de Mediana Edad , Modelos Moleculares , Proteína P0 de la Mielina/química , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/inmunología , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Complement receptor type 1 (CR1) is a multi modular membrane receptor composed of 30 homologous complement control protein modules (CCP) organized in four different functional regions called long homologous repeats (LHR A, B, C, and D). CR1 is a receptor for complement-opsonins C3b and C4b and specifically interacts through pairs of CCP modules located in LHR A, B, and C. Defense collagens such as mannose-binding lectin (MBL), ficolin-2, and C1q also act as opsonins and are involved in immune clearance through binding to the LHR-D region of CR1. Our previous results using deletion variants of CR1 mapped the interaction site for MBL and ficolin-2 on CCP24-25. The present work aimed at deciphering the interaction of C1q with CR1 using new CR1 variants concentrated around CCP24-25. CR1 bimodular fragment CCP24-25 and CR1 CCP22-30 deleted from CCP24-25 produced in eukaryotic cells enabled to highlight that the interaction site for both MBL and C1q is located on the same pair of modules CCP24-25. C1q binding to CR1 shares with MBL a main common interaction site on the collagen stalks but also subsidiary sites most probably located on C1q globular heads, contrarily to MBL.