RESUMO
In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fucα1,2 (GalNAcα1,3) Galß1,4) showed increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins with similar specificity; healthy cervical tissue was negative for MeA. Trypsin treatment of recognized tissues abolished MeA's recognition;moreover, interaction of MeA was inhibited with oligosaccharides from mucin. As demonstrated by Western blot of 2-D electrophoresis, MeA recognized ten glycoproteins in the range from 122 to 42 kDa in cervical cancer lysates. The LC-ESI-MS/MS analysis of the MeAs' recognized peptides revealed that the latter matched mainly with the amino acid sequences of lamin A/C, vimentin, elongation factor 2, keratin 1, and beta actin. Our results suggest that MeA recognizes a complex of over-expressed O-glycosidically-linked proteins that play a relevant role in cervical cancer's invasive capacity. O-glycosylation participates in the disassembly of intercellular junctions favoring cancer progression.
Assuntos
Aglutininas/metabolismo , Glicoproteínas/metabolismo , Lectinas de Plantas/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Glicosilação , Histocitoquímica , Humanos , Immunoblotting , Espectrometria de Massas , Sus scrofa , Tripsina/metabolismoRESUMO
A 66-kDa lectin (OmA) was purified from the serum of the Yucatan peninsula endemic octopus (Octopus maya) by a single step affinity chromatography on glutaraldehyde-fixed stroma from rat erythrocytes. OmA corresponds to 0.8% of the total circulating protein in the hemolymph; it is composed of three equal subunits of 22kDa each, and 7.4% of linked carbohydrates. The amino acids' composition indicated that agglutinin contained mainly aspartic and glutamic acids, and cysteine and methionine were identified in minor proportion. OmA agglutinates mainly rat, guinea pig, and rabbit erythrocytes, and this activity is partially inhibited by galactosamine, melobiose, galacturonic acid, mannose, and methyl alpha and beta galactosides. Hemagglutinating activity is not dependent on divalent cations, such as Ca(2+), Mg(2+), or Mn(2+). The OmA subunits showed no identity for any lectin in databases but partial identity with the type A hemocyanin from Octopus dolfleini hemolymph; the main similarities are related to tyrosinase domains and copper A and B sites that conform to the oxygen-binding site of hemocyanin.
Assuntos
Aglutininas/sangue , Aglutininas/química , Lectinas/sangue , Lectinas/química , Octopodiformes/metabolismo , Aglutininas/isolamento & purificação , Aminoácidos/análise , Animais , Cromatografia de Afinidade , Lectinas/isolamento & purificação , Ratos , Análise de Sequência de ProteínaRESUMO
Crustacean aquaculture represents a major industry in tropical developing countries. As a result of high culture densities and increasing extension of aquaculture farms, the presence of diseases has also increased, inducing economic losses. Invertebrates, which lack adaptive immune systems, have developed defense systems that respond against antigens on the surface of potential pathogens. The defense mechanisms of crustaceans depend completely on the innate immune system that is activated when pathogen-associated molecular patterns are recognized by soluble or by cell surface host proteins, such as lectins, antimicrobial, clotting, and pattern recognition proteins, which, in turn, activate cellular or humoral effector mechanisms to destroy invading pathogens. This work is aimed at presenting the main characteristics of the crustacean proteins that participate in immune defense by specific recognition of carbohydrate containing molecules, i.e. glycans, glycolipids, glycoproteins, peptidoglycans, or lipopolysaccharides from Gram-negative and Gram-positive bacteria, viruses, or fungi. We review some basic aspects of crustacean effector defense processes, like agglutination, encapsulation, phagocytosis, clottable proteins, and bactericidal activity, induced by these carbohydrate-driven recognition patterns.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Catecol Oxidase/imunologia , Crustáceos/imunologia , Precursores Enzimáticos/imunologia , Lectinas/imunologia , Fagocitose/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Catecol Oxidase/metabolismo , Crustáceos/metabolismo , Precursores Enzimáticos/metabolismo , Imunidade Inata , Lectinas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
We determined the cross-reactivity of a monoclonal antibody against the Macrobrachium rosenbergii lectin with proteins in the hemolymph from Procambarus clarkii (Pc), Procambarus americanus (Pa), Litopenaeus setiferus (Ls), and Pseudothelphusa americana (Psa). Crustaceans' hemolymph agglutinated erythrocytes from rat, mouse, guinea pig, and rabbit. Decapods' hemolymph hemagglutinating activity was inhibited by N-acetylated carbohydrates as well as by antibodies. Western blot assays indicated that the antibodies recognized two main proteins of 97.5 and 80.9 kDa in all hemolymphs studied; moreover, ELISA assays indicated that, in PSa, 7.2% of total proteins showed crossreactivity with antibodies in Pa, Pc, and Lc hemolymphs represented 4.2, 3.1%, and 2.5%, respectively. Our results suggested that antibodies recognize the lectin active site in the crustacean species tested; we propose the use of antibodies as an immunological marker for lectin identification and quantification among crustaceans.
Assuntos
Anticorpos Monoclonais/imunologia , Hemolinfa/metabolismo , Lectinas/imunologia , Animais , Cromatografia de Afinidade , Crustáceos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Humanos , Lectinas/sangue , Lectinas/isolamento & purificaçãoRESUMO
Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.
Assuntos
Alérgenos/química , Peptídeos Catiônicos Antimicrobianos/química , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Hipersensibilidade ao Látex/imunologia , Lectinas de Plantas/química , Alérgenos/imunologia , Sequência de Aminoácidos , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Epitopos de Linfócito B/imunologia , Humanos , Imunoglobulina E/sangue , Dados de Sequência Molecular , Lectinas de Plantas/imunologia , Conformação Proteica , Alinhamento de SequênciaRESUMO
The GlcNAc-specific adhesin from Mannheimia haemolytica (MhA) has been shown to participate in pathogenicity of mannheimiosis due to its capacity to adhere to tracheal epithelial cells and activate the oxidative burst of bovine neutrophils. In this work, we purified the MhA receptor from bovine neutrophils (MhAr) by affinity chromatography on MhA-Sepharose. The MhAr, which corresponded to approximately 2% of the protein from cell lysate, is a glycoprotein mainly composed of Glu, Ala, Ser, Gly, and Asp, without cysteine. The glycan portion, which corresponds to 20% by weight, is composed of GalNAc, GlcNAc, Man, Gal, and NeuAc. The receptor is a 165-kDa glycoprotein, as determined by molecular sieve chromatography under native conditions; SDS-PAGE analysis shows a heterodimer of 83 and 80 kDa subunits. This work suggests that the GlcNAc-containing receptor plays a relevant role by activating bovine neutrophils through non-opsonic mechanisms.
Assuntos
Acetilglucosamina/metabolismo , Adesinas Bacterianas/metabolismo , Mannheimia haemolytica/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/isolamento & purificação , Acetilglucosamina/imunologia , Adesinas Bacterianas/imunologia , Animais , Bovinos , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Ativação de Neutrófilo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Explosão RespiratóriaRESUMO
Hemagglutinin-neuraminidase (HN) from porcine rubulavirus La Piedad Michoacan (RvpLPM) is one of the most antigenic proteins known, and is responsible for virus-host cell interaction. We analyzed the amino acid sequence of HN, using computer-assisted techniques to identify B cell epitopes. From a pool of 18 possible antigenic peptides, we evaluated the antigenicity of the 2 peptides with the highest scores and the 1 with lowest score. Antibodies from RvpLPM-infected pigs recognized the synthesized HN-A, HN-B, and HN-R peptides (optical density [OD]: 0.33 +/- 0.02 for HN-A, 0.20 +/- 0.02 for HN-B, and 0.07 +/- 0.01 for HN-R); bovine serum albumin-coupled HN-A and HN-B induced rabbit anti-RvpLPM antibodies (OD: 0.39 +/- 0.01 for HN-A and 0.35 +/- 0.02 for HN-B). Loop 5 from the outer membrane protein, OmpC, from Salmonella typhi was replaced with HN-B; this protein was then expressed in Escherichia coli UH302. BALB/c mice were challenged intraperitoneally or orogastrically with the fusion protein expressed in E. coli and murine antibodies obtained from both types of administration inhibited virus-hemagglutinating activity, as did the antibodies from RvpLPM-infected swine. These results suggest that HN-A and HN-B are peptides involved in RvpLPM cell carbohydrate recognition, and could therefore be considered potential targets for vaccine and diagnostic procedures development.
Assuntos
Epitopos de Linfócito B/imunologia , Proteína HN/imunologia , Peptídeos/imunologia , Infecções por Rubulavirus/imunologia , Rubulavirus/imunologia , Algoritmos , Animais , Mapeamento de Epitopos , Proteína HN/química , Testes de Inibição da Hemaglutinação , Hemaglutinação por Vírus , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Infecções por Rubulavirus/virologia , Software , SuínosRESUMO
Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.
Assuntos
Neutrófilos/metabolismo , Receptores Mitogênicos/sangue , Aglutininas do Germe de Trigo/metabolismo , Sequência de Aminoácidos , Animais , Granulócitos/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Subunidades Proteicas , Receptores Mitogênicos/química , Receptores Mitogênicos/genética , Receptores Mitogênicos/isolamento & purificação , Explosão RespiratóriaRESUMO
In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.
Assuntos
Adesinas Bacterianas/farmacologia , Doenças dos Bovinos/microbiologia , Bovinos/sangue , Mannheimia haemolytica/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Pasteurelose Pneumônica/imunologia , Explosão Respiratória/efeitos dos fármacos , Acetilglucosamina/imunologia , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/isolamento & purificação , Animais , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Citometria de Fluxo/veterinária , Imuno-Histoquímica/veterinária , Iodoacetamida/farmacologia , Mannheimia haemolytica/isolamento & purificação , Ativação de Neutrófilo/imunologia , Neutrófilos/microbiologia , Pasteurelose Pneumônica/microbiologia , Piroxicam/farmacologia , Explosão Respiratória/imunologia , Superóxido Dismutase/farmacologiaRESUMO
We determined the effect of low molecular weight components (LMWC) from healthy juvenile and adult Macrobrachium rosenbergii hemolymph on lectin activity and oxidative burst (OB) in hemocytes. In an attempt to identify the LMWC that affect the lectin's hemagglutinating activity or oxidative burst, we determined the hemolymph carbohydrates and free amino acids (FAA) concentration. The LMWC (<2000 Da) were obtained after dialysis of the hemolymph. Our results showed that LMWC from juveniles exerted a greater inhibition on lectin than LMWC from adult hemolymph. Production of superoxide radicals by hemocytes was lower in the presence of juvenile (p<0.05) as compared to adult LMWC. FAA composition of the hemolymph and of LMWC from adults showed higher proportion of alanine (which corresponded to 25% of total FAA) and proline (>20%); whereas, in juveniles, the main FAA identified were glycine (>40%) and alanine (26%). N-Acetyl-D-glucosamine (GlcNAc) was the main sugar residue in the hemolymph and LMWC from juveniles; its concentration was 2.4 times higher than glucose (Glc), whereas, in adults, Glc was the main free sugar residue. Our results suggest that the proportion of FAA and carbohydrates in the hemolymph of M. rosenbergii seems to be correlated with the maturation process; furthermore, the high proportion of free GlcNAc and glycine regulate, in the juvenile stage, lectin activity and cellular oxidative mechanisms, respectively.
Assuntos
Aminoácidos/sangue , Carboidratos/sangue , Hemolinfa/metabolismo , Lectinas/metabolismo , Palaemonidae/metabolismo , Aminoácidos/química , Animais , Carboidratos/química , Eritrócitos/fisiologia , Água Doce , Hemaglutinação , Hemócitos/metabolismo , Ratos , Ratos Wistar , Explosão Respiratória , Superóxidos/metabolismoRESUMO
Using a spectrophotometric NBT reduction assay and phagocytosis, we identified that production of superoxide anions and phagocytic activity of hemocytes from Macrobrachium rosenbergii were significantly higher in the presence of rat, rabbit, and chicken erythrocytes than with human, pig, or horse erythrocytes. Hemocytes stimulated with MrL, MrLMab, or PMA increased 4.7, 5.1, and 6.1 fold, respectively, the oxidative response as compared to non-stimulated hemocytes. MrLMab together with MrL increased 5.7 fold the oxidative capacity of hemocytes as compared to non-stimulated cells. These effects were inhibited with 100 mM GalNAc, GlcNAc, or Neu5Ac and 0.2 microM of sialylated submaxillary gland mucin and fetuin. Piroxicam inhibited (P < 0.05) the production of O(2)(-) induced by MrL, whereas iodoacetamide inhibited the effect of MrLMAb (P < 0.05) in a dose-dependent manner. Our results suggest that MrLMab might activate the oxidative burst through the metabolism of glucose as opposed to MrL which utilizes NADPH-independent mechanisms, very probably through pro-inflammatory metabolites.
Assuntos
Membrana Celular/química , Crustáceos/metabolismo , Hemócitos/metabolismo , Lectinas/metabolismo , Soro/química , Animais , Carboidratos/farmacologia , Membrana Celular/imunologia , Eritrócitos/metabolismo , Glicoproteínas/farmacologia , Hemócitos/imunologia , Humanos , Lectinas/imunologia , Microscopia Confocal , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/fisiologia , Soro/imunologia , Superóxidos/metabolismoRESUMO
Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-D-galactosamine (GalNAc). In this work, we determined a value of 1.0 x 10(-2) M for the association constant of ALL for GalNAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro O-glycosylation reaction using microsomal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, kappa light chain (5D4) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GalNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GalNAc residue at Thr in #2, nor others (with more than two GalNAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GalNAc residues when they are spaced out in glycan structures, whereas GalNAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GalNAc-containing motif found in different O-glycoproteins.
Assuntos
Glicopeptídeos/química , Glicoproteínas/química , Lectinas de Plantas/química , Acetilgalactosamina/química , Amaranthus , Sequência de Aminoácidos , Animais , Eletroforese Capilar , Feminino , Glicopeptídeos/metabolismo , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Espectrometria de Fluorescência , Timo/metabolismoRESUMO
In invertebrates, lectins play relevant roles in innate immunity; however, their regulatory mechanisms have not been identified yet. In this work, we purified, by gel filtration and affinity chromatography, lectin aggregates circulating in the hemolymph of the freshwater prawn Macrobrachium rosenbergii and compared their physicochemical properties with a previously described lectin (MrL). High-molecular weight MrL aggregates (MrL-I) lack hemagglutinating activity and showed bands of 62.1, 67.1 and 81.4 kDa, whereas MrL-III, which corresponds to MrL, showed hemagglutinating activity and is constituted by a single 9.6-kDa band as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. MrL-I and MrL-III showed similar amino acid composition but different carbohydrates concentration. Edman degradation indicated NH2-terminal sequence of five amino acids for the 9.6-kDa MrL-III (DVPLL/A) and eleven for the main 81.4-kDa band identified in MrL-I (DVPLL/AXKQQQD); analysis by MALDI-TOF indicated a different tryptic pattern for MrL-I and MrL-III. MrL-I was recognized by monoclonal antibodies against MrL-III. Circular dichroism indicated that the secondary structure in both proteins is similar and contains 23% of beta-sheet and 24% of alpha-helix. Our results suggest that differential posttranslational processes that favor aggregation are involved in regulating the activity of the lectin.