RESUMO
Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.
Assuntos
Bacteriófagos , Biblioteca de Peptídeos , Anticorpos Monoclonais , Bacteriófagos/genética , Bioprospecção , Técnicas de Visualização da Superfície Celular/métodos , Proteínas de MembranaRESUMO
Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed ß-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc11-22[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone 15N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc11-22[Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited excellent proteolytic stability against trypsin and chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase. We show that sCon-Vc11-22[NNN12-14] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 µM while maintaining the SDH fold. This study serves as a starting point in utilizing the SDH fold as a peptide scaffold.
Assuntos
Conotoxinas/química , Peptídeos Cíclicos/química , Engenharia de Proteínas , Proteínas Supressoras da Sinalização de Citocina/química , Conotoxinas/genética , Conotoxinas/metabolismo , Cisteína/química , Cistina/química , Epitopos , Humanos , Cinética , Isótopos de Nitrogênio , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Conformação Proteica em Folha beta , Dobramento de Proteína , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
The malaria vaccine candidate merozoite surface protein 2 (MSP2) has shown promise in clinical trials and is in part responsible for a reduction in parasite densities. However, strain-specific reductions in parasitaemia suggested that polymorphic regions of MSP2 are immuno-dominant. One strategy to bypass the hurdle of strain-specificity is to bias the immune response towards the conserved regions. Two mouse monoclonal antibodies, 4D11 and 9H4, recognise the conserved C-terminal region of MSP2. Although they bind overlapping epitopes, 4D11 reacts more strongly with native MSP2, suggesting that its epitope is more accessible on the parasite surface. In this study, a structure-based vaccine design approach was applied to the intrinsically disordered antigen, MSP2, using a crystal structure of 4D11 Fv in complex with its minimal binding epitope. Molecular dynamics simulations and surface plasmon resonance informed the design of a series of constrained peptides that mimicked the 4D11-bound epitope structure. These peptides were conjugated to keyhole limpet hemocyanin and used to immunise mice, with high to moderate antibody titres being generated in all groups. The specificities of antibody responses revealed that a single point mutation can focus the antibody response towards a more favourable epitope. This structure-based approach to peptide vaccine design may be useful not only for MSP2-based malaria vaccines, but also for other intrinsically disordered antigens.
RESUMO
Bv8, a 77-residue protein isolated from frogs, is the prototypic member of the prokineticin family of cytokines. Prokineticins (PKs) have only recently been identified in vertebrates (including humans), and they are believed to be involved in a number of key physiological processes, such as angiogenesis, neurogenesis, nociception, and tissue development. We used a combination of Boc solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited full activity in a human neuroblastoma cell line and rat dorsal root ganglion (DRG) neurons. The 3D structure of the synthetic protein was determined by using NMR spectroscopy and it was found to be homologous with that of mamba intestinal toxin 1, which is the only other known prokineticin structure. Analysis of a truncated mutant lacking five residues at the N terminus that are critical for receptor binding and activation showed no perturbation to the core protein structure. Together with the functional data, this suggests that receptor binding is likely to be a highly cooperative process possibly involving major allosterically driven structural rearrangements. The facile and efficient synthesis presented here will enable preparation of unique chemical analogues of prokineticins, which should be powerful tools for modulating the structure and function of prokineticins and their receptors, and studying the many physiological processes that have been linked to them.
Assuntos
Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anuros/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/síntese química , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismoRESUMO
Cnidarians are one of the oldest known animal lineages (ca. 700 million years), with a unique envenomation apparatus to deliver a potent mixture of peptides and proteins. Some peptide toxins from cnidarian venom have proven therapeutic potential. Here, we use a transcriptomic/proteomic strategy to identify sequences with similarity to known venom protein families in the tentacles of the endemic Australian 'speckled anemone' (Oulactis sp.). Illumina RNASeq data were assembled de novo. Annotated sequences in the library were verified by cross-referencing individuals' transcriptomes or protein expression evidence from LC-MS/MS data. Sequences include pore-forming toxins, phospholipases, peptidases, neurotoxins (sodium and potassium channel modulators), cysteine-rich secretory proteins and defensins (antimicrobial peptides). Fewer than 4% of the sequences in the library occurred across the three individuals examined, demonstrating high sequence variability of an individual's arsenal. We searched for actinoporins in Oulactis sp. to assess sequence similarity to the only described toxins (OR-A and -G) for this genus and examined the domain architecture of venom-related peptides and proteins. The novel putative actinoporin of Oulactis sp. has a greater similarity to other species in the Actiniidae family than to O. orientalis. Venom-related sequences have an architecture that occurs in single, repeat or multi-domain combinations of venom-related (e.g. ShK-like) and non-venom (e.g. whey acid protein) domains. This study has produced the first transcriptomes for an endemic Australian sea anemone species and the genus Oulactis, while identifying nearly 400 novel venom-related peptides and proteins for future structural and functional analyses and venom evolution studies.
Assuntos
Venenos de Cnidários/química , Anêmonas-do-Mar/química , Transcriptoma , Animais , Cromatografia Líquida , Venenos de Cnidários/genética , Proteoma/análise , Anêmonas-do-Mar/genética , Espectrometria de Massas em TandemRESUMO
We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.
RESUMO
ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21-24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.
Assuntos
Venenos de Cnidários/química , Canal de Potássio Kv1.1/antagonistas & inibidores , Canal de Potássio Kv1.3/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/química , Sequência de Aminoácidos/genética , Animais , Doenças Autoimunes/tratamento farmacológico , Venenos de Cnidários/genética , Venenos de Cnidários/farmacologia , Humanos , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.1/ultraestrutura , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.3/ultraestrutura , Conformação Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/genética , Bloqueadores dos Canais de Potássio/farmacologia , Anêmonas-do-Mar/químicaRESUMO
Transient interactions in which binding partners retain substantial conformational disorder play an essential role in regulating biological networks, challenging the expectation that specificity demands structurally defined and unambiguous molecular interactions. The monoclonal antibody 6D8 recognises a completely conserved continuous nine-residue epitope within the intrinsically disordered malaria antigen, MSP2, yet it has different affinities for the two allelic forms of this antigen. NMR chemical shift perturbations, relaxation rates and paramagnetic relaxation enhancements reveal the presence of transient interactions involving polymorphic residues immediately C-terminal to the structurally defined epitope. A combination of these experimental data with molecular dynamics simulations shows clearly that the polymorphic C-terminal extension engages in multiple transient interactions distributed across much of the accessible antibody surface. These interactions are determined more by topographical features of the antibody surface than by sequence-specific interactions. Thus, specificity arises as a consequence of subtle differences in what are highly dynamic and essentially non-specific interactions.
RESUMO
Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.
Assuntos
Venenos de Cnidários/química , Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Dobramento de Proteína , Anêmonas-do-Mar/química , Animais , Venenos de Cnidários/farmacologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Consistent with their diverse pharmacology, peptides derived from venomous animals have been developed as drugs to treat disorders as diverse as hypertension, diabetes and chronic pain. Melanoma has a poor prognosis due in part to its metastatic capacity, warranting further development of novel targeted therapies. This prompted us to examine the anti-melanoma activity of the spider peptides gomesin (AgGom) and a gomesin-like homolog (HiGom). AgGom and HiGom dose-dependently reduced the viability and proliferation of melanoma cells whereas it had no deleterious effects on non-transformed neonatal foreskin fibroblasts. Concordantly, gomesin-treated melanoma cells showed a reduced G0/G1 cell population. AgGom and HiGom compromised proliferation of melanoma cells via activation of the p53/p21 cell cycle check-point axis and the Hippo signaling cascade, together with attenuation of the MAP kinase pathway. We show that both gomesin peptides exhibit antitumoral activity in melanoma AVATAR-zebrafish xenograft tumors and that HiGom also reduces tumour progression in a melanoma xenograft mouse model. Taken together, our data highlight the potential of gomesin for development as a novel melanoma-targeted therapy.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Melanoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Xenoenxertos , Camundongos , Transplante de Neoplasias , Resultado do Tratamento , Peixe-ZebraRESUMO
Centipedes are venomous arthropods responsible for a significant number of non-lethal human envenomations. Despite this, information about the composition and function of their venom contents is scarce. In this study, we have used a 'structure to function' proteomic approach combining two-dimensional chromatography (2D-LC), electrospray ionization quadrupole/time-of-flight mass spectrometry (ESI-Q-TOF/MS), N-terminal sequencing and similarity searching to better understand the complexities of the venoms from two Brazilian centipede species: Scolopendra viridicornis nigra and Scolopendra angulata. Comparisons between the LC profiles and the mass compositions of the venoms of the two species are provided. The observed molecular masses ranged from 3019.62 to 20996.94Da in S. viridicornis nigra (total: 62 molecular masses) and from 1304.73 to 22639.15Da in S. angulata (total: 65 molecular masses). Also, the N-termini of representatives of 10 protein/peptide families were successfully sequenced where nine of them showed no significant similarity to other protein sequences deposited in the Swiss-Prot database. A screening for insecto-toxic activities in fractions from S. viridicornis venom has also been performed. Six out of the 12 tested fractions were responsible for clear toxic effects in house flies. This work demonstrates that centipede venoms might be a neglected but important source of new bioactive compounds.
Assuntos
Venenos de Artrópodes/química , Artrópodes , Sequência de Aminoácidos , Animais , Venenos de Artrópodes/toxicidade , Cromatografia Líquida , Dípteros/efeitos dos fármacos , Feminino , Masculino , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Testes de ToxicidadeRESUMO
Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored antigen on the malaria parasite Plasmodium falciparum. MSP2 induces an immune response in the context of natural infections and vaccine trials, and these responses are associated with protection from parasite infection. Recombinant MSP2 is highly disordered in solution but antigenic analyses suggest that it is more ordered on the merozoite surface. We have shown previously that the interaction of recombinant full-length MSP2 with lipid surfaces induces a conformational change in the conserved N-terminal region of MSP2, which contributes to epitope masking in this region. To explore the impacts of lipid interactions on the conformation and antigenicity of the conserved C-terminal region of MSP2, a construct corresponding to this domain, MSP2172-221 , was designed. NMR studies indicate that many residues in MSP2172-221 interact with DPC micelles, including some in epitopes recognised by C-terminal-specific monoclonal antibodies, but, in contrast to the MSP2 N-terminus, there is no indication of stable helical conformation. The binding affinities of a panel of monoclonal antibodies indicate that MSP2172-221 is antigenically similar to full-length MSP2 and show that liposome conjugation alters the antigenicity in a manner that may mimic native MSP2 on the merozoite surface. These findings highlight the impact of lipid interactions on the conformation and antigenicity of MSP2172-221 and will assist in the design of recombinant MSP2 immunogens for use as malaria vaccine candidates. DATABASES: Resonance assignments are available in the BioMagResBank (BMRB) database under the accession number 27134.
Assuntos
Antígenos de Protozoários/imunologia , Proteínas de Protozoários/imunologia , Antígenos de Protozoários/química , Ensaio de Imunoadsorção Enzimática , Lipossomos/química , Espectroscopia de Ressonância Magnética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/químicaRESUMO
In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.
Assuntos
Aminoácidos/análise , Venenos de Moluscos/química , Peptídeos Cíclicos/química , Animais , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Caramujos/química , Canais de Cátion TRPC/metabolismo , TranscriptomaRESUMO
Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Epitopos/genética , Epitopos/imunologia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Calorimetria , Cristalografia por Raios X , Epitopos/química , Vacinas Antimaláricas/química , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/química , Ressonância de Plasmônio de SuperfícieRESUMO
HsTX1[R14A] is a potent and selective Kv1.3 channel blocker peptide with the potential to treat autoimmune diseases. Given the typically poor oral bioavailability of peptides, we evaluated pulmonary administration of HsTX1[R14A] in rats as an alternative route for systemic delivery. Plasma concentrations of HsTX1[R14A] were measured by liquid chromatography coupled with tandem mass spectrometry in rats receiving intratracheal administration of HsTX1[R14A] in solution (1-4 mg/kg) or a mannitol-based powder (1 mg/kg) and compared with plasma concentrations after intravenous administration (2 mg/kg). HsTX1[R14A] stability in rat plasma and lung tissue was also determined. HsTX1[R14A] was more stable in plasma than in lung homogenate, with more than 90% of the HsTX1[R14A] remaining intact after 5 h, compared with 40.5% remaining in lung homogenate. The terminal elimination half-life, total clearance, and volume of distribution of HsTX1[R14A] after intravenous administration were 79.6 ± 6.5 min, 8.3 ± 0.6 mL/min/kg, and 949.8 ± 71.0 mL/kg, respectively (mean ± SD). After intratracheal administration, HsTX1[R14A] in solution and dry powder was absorbed to a similar degree, with absolute bioavailability values of 39.2 ± 5.2% and 44.5 ± 12.5%, respectively. This study demonstrated that pulmonary administration is a promising alternative for systemically delivering HsTX1[R14A] for treating autoimmune diseases.
Assuntos
Doenças Autoimunes/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Canal de Potássio Kv1.3/antagonistas & inibidores , Pulmão/metabolismo , Bloqueadores dos Canais de Potássio/metabolismo , Venenos de Escorpião/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Pulmão/efeitos dos fármacos , Masculino , Bloqueadores dos Canais de Potássio/administração & dosagem , Ratos , Ratos Sprague-Dawley , Venenos de Escorpião/administração & dosagem , Resultado do TratamentoRESUMO
MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum. Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro. We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Imunoglobulina G/biossíntese , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/classificação , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/genética , Ensaios Clínicos como Assunto , Sequência Conservada , Proteção Cruzada , Mapeamento de Epitopos , Epitopos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Merozoítos/química , Merozoítos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium falciparum/química , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , VacinaçãoRESUMO
Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues.
Assuntos
Venenos de Anfíbios/química , Anuros/fisiologia , Tetrodotoxina/análogos & derivados , Tetrodotoxina/isolamento & purificação , Venenos de Anfíbios/toxicidade , Animais , Relação Dose-Resposta a Droga , Camundongos , Estrutura Molecular , Tetrodotoxina/toxicidadeRESUMO
Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum. MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Protozoários/química , Epitopos/química , Vacinas Antimaláricas/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Humanos , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologiaRESUMO
Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.
Assuntos
Venenos de Anfíbios/química , Anuros/metabolismo , Neurotoxinas/análise , Tetrodotoxina/análise , Extratos de Tecidos/química , Animais , Brasil , Cromatografia Líquida de Alta Pressão , Feminino , Fígado/química , Longevidade/efeitos dos fármacos , Camundongos , Neurotoxinas/toxicidade , Ovário/química , Pele/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tetrodotoxina/análogos & derivados , Tetrodotoxina/toxicidade , Testes de ToxicidadeRESUMO
11-oxoTTX is an analogue 4-5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H(+)), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.