RESUMO
Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.
Assuntos
Agatoxinas/química , Decápodes/genética , Decápodes/metabolismo , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Transcriptoma , Sequência de Aminoácidos , Animais , Clonagem Molecular , Decápodes/classificação , Especificidade de Órgãos , FilogeniaRESUMO
Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods.
Assuntos
Amidas/química , Nephropidae/fisiologia , Rede Nervosa/fisiologia , Neuropeptídeos/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Nephropidae/genética , Neuropeptídeos/química , Neurotransmissores/química , Neurotransmissores/genética , Alinhamento de SequênciaRESUMO
The SIFamides are a broadly conserved arthropod peptide family characterized by the C-terminal motif -SIFamide. In decapod crustaceans, two isoforms of SIFamide are known, GYRKPPFNGSIFamide (Gly1-SIFamide), which is nearly ubiquitously conserved in the order, and VYRKPPFNGSIFamide (Val1-SIFamide), known only from members of the astacidean genus Homarus. While much work has focused on the identification of SIFamide isoforms in decapods, there are few direct demonstrations of physiological function for members of the peptide family in this taxon. Here, we assessed the effects of Gly1- and Val1-SIFamide on the cardiac neuromuscular system of two closely related species of Cancer crab, Cancer borealis and Cancer irroratus. In each species, both peptides were cardioactive, with identical, dose-dependent effects elicited by both isoforms in a given species. Threshold concentrations for bioactivity are in the range typically associated with hormonal delivery, i.e., 10-9 to 10-8â¯M. Interestingly, and quite surprisingly, while the predicted effects of SIFamide on cardiac output are similar in both C. borealis and C. irroratus, frequency effects predominate in C. borealis, while amplitude effects predominate in C. irroratus. These findings suggest that, while SIFamide is likely to increase cardiac output in both crabs, the mechanism through which this is achieved is different in the two species. Immunohistochemical/mass spectrometric data suggest that SIFamide is delivered to the heart hormonally rather than locally, with the source of hormonal release being midgut epithelial endocrine cells in both Cancer species. If so, midgut-derived SIFamide may function as a regulator of cardiac output during the process of digestion.
Assuntos
Braquiúros/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Neuropeptídeos/farmacologia , Animais , Braquiúros/efeitos dos fármacos , Sistema Digestório/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Neuropeptídeos/químicaRESUMO
Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.
Assuntos
Proteínas de Bactérias/metabolismo , Glicoproteínas/metabolismo , Helicobacter pylori/metabolismo , Proteômica/métodos , Estômago/microbiologia , Sequência de Aminoácidos , Azidas/metabolismo , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Cromatografia Líquida , Glicoproteínas/química , Glicosilação , Helicobacter pylori/patogenicidade , Dados de Sequência Molecular , Nanopartículas , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Coloração e Rotulagem , Frações Subcelulares/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismoRESUMO
Crustaceans have long been used for peptide research. For example, the process of neurosecretion was first formally demonstrated in the crustacean X-organ-sinus gland system, and the first fully characterized invertebrate neuropeptide was from a shrimp. Moreover, the crustacean stomatogastric and cardiac nervous systems have long served as models for understanding the general principles governing neural circuit functioning, including modulation by peptides. Here, we review the basic biology of crustacean neuropeptides, discuss methodologies currently driving their discovery, provide an overview of the known families, and summarize recent data on their control of physiology and behavior.
Assuntos
Crustáceos/química , Neuropeptídeos/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Crustáceos/anatomia & histologia , Crustáceos/fisiologia , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Neuropeptídeos/genéticaRESUMO
The PISCF-allatostatins (Manduca sexta- or C-type allatostatins) are a family of pentadecapeptides characterized by a pyroglutamine blocked N-terminus, an unamidated-PISCF C-terminus, and a disulfide bridge between two internal Cys residues. Several isoforms of PISCF-AST are known, all from holometabolous insects. Using a combination of transcriptomics and mass spectrometry, we have identified the first PISCF-type peptides from a non-insect species. In silico analysis of crustacean ESTs identified several Litopenaeus vannamei (infraorder Penaeidea) transcripts encoding putative PISCF-AST precursors. Translation of these ESTs, with subsequent prediction of their putative post-translational processing, revealed the existence of as many as three PISCF-type peptides, including pQIRYHQCYFNPISCF (disulfide bridging between Cys(7) and Cys(14)). Although none of the predicted isoforms was detected by mass spectrometry in L. vannamei, MALDI-FTMS mass profiling identified an m/z signal corresponding to pQIRYHQCYFNPISCF (disulfide bridge present) in neural tissue from 28 other decapods, which included members of six infraorders (Stenopodidea, Astacidea, Thalassinidea, Achelata, Anomura and Brachyura). Further characterization of the peptide using SORI-CID and chemical derivatization/enzymatic digestion supported the theorized structure. In both the crab Cancer borealis and the lobster Homarus americanus, MALDI-based tissue surveys suggest that pQIRYHQCYFNPISCF is broadly distributed in the nervous system; it was also detected in the posterior midgut caecum. Collectively, our data show that members of the PISCF-AST family are not restricted to the holometabolous insects, but instead may be broadly conserved within the Pancrustacea. Moreover, our data suggest that one highly conserved PISCF-type peptide, pQIRYHQCYFN-PISCF, is present in decapod crustaceans, functioning as a brain-gut paracrine/hormone.
Assuntos
Neuropeptídeos/química , Neuropeptídeos/genética , Penaeidae/genética , Penaeidae/metabolismo , Sequência de Aminoácidos , Animais , Etiquetas de Sequências Expressas , Manduca , Dados de Sequência Molecular , Neuropeptídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Linear oligoenes of 1,6-heptadiynes (derived from dialkyl dipropargylmalonates) with a single basic structure and up to 23 conjugated double bonds were synthesized through Wittig-like reactions between bimetallic Mo-alkylidene compounds and aldehyde-capped oligoenes. The relatively rigid and isomerically pure oligoenes have structures with alternating cis,trans conjugated double bonds in which the cis double bond is part of a cyclopentene ring. Molecular weights have been confirmed through MALDI-MS measurements of samples purified by HPLC. Optical spectra of the purified samples show significant vibronic resolution, even in room temperature samples, and are remarkably similar to those of simple polyenes and carotenoids. Therefore, a systematic investigation of the dependence of the allowed electronic transition energies (electronic origins) on conjugation lengths has become possible. Studies of seven allowed transitions for molecules with 5-23 double bonds (= N) indicate asymptotic convergence (with approximately a 1/N dependence) to a common long polyene limit at approximately 16,000 cm(-1). The convergence of these electronic transitions agrees with theoretical treatments of polyene excited-state energies and is consistent with the absorption spectra of analogous diethyl dipropargylmalonate polymers (1/N approximately 0).
RESUMO
Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY((SO3))DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.
Assuntos
Astacoidea/metabolismo , Nephropidae/metabolismo , Neuropeptídeos/química , Peptídeos/química , Precursores de Proteínas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Peso Molecular , Isoformas de Proteínas/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Two tachykinin-related peptides (TRPs) are known in decapods, APSGFLGMRamide and TPSGFLGMRamide. The former peptide appears to be ubiquitously conserved in members of this taxon, while the latter has been suggested to be a genus (Cancer)- or infraorder (Brachyura)-specific isoform. Here, we characterized a cDNA from the American lobster Homarus americanus (infraorder Astacidea) that encodes both TRPs: six copies of APSGFLGMRamide and one of TPSGFLGMRamide. Mass spectral analyses of the H. americanus supraoesophageal ganglion (brain) and commissural ganglia confirmed the presence of both peptides in these neural tissues; both isoforms were also detected in the midgut. Physiological experiments showed that both APSGFLGMRamide and TPSGFLGMRamide are cardioactive in H. americanus, eliciting identical increases in both heart contraction frequency and amplitude. Collectively, our data represent the first genetic confirmation of TRPs in H. americanus and of TPSGFLGMRamide in any species, demonstrate that TPSGFLGMRamide is not restricted to brachyurans, and show that both this peptide and APSGFLGMRamide are brain-gut isoforms, the first peptides thus far confirmed to possess this dual tissue distribution in H. americanus. Our data also suggest a possible role for TRPs in modulating the output of the lobster heart.
Assuntos
Cardiotônicos/isolamento & purificação , Cardiotônicos/farmacologia , Nephropidae/química , Neuropeptídeos/isolamento & purificação , Taquicininas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Química Encefálica , DNA Complementar/análise , Gânglios dos Invertebrados/química , Coração/efeitos dos fármacos , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taquicininas/química , Taquicininas/farmacologiaRESUMO
We report on the characterization of the native form of an American lobster, Homarus americanus, ß-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to ß-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.
Assuntos
Defensinas/metabolismo , Nephropidae/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Simulação por Computador , Defensinas/química , Defensinas/isolamento & purificação , Dissulfetos/metabolismo , Granulócitos/metabolismo , Hemócitos/metabolismo , Temperatura Alta , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em TandemRESUMO
Photocatalysis directed at the removal of persistent organic pollutants, including pharmaceuticals, has been the subject of intense recent research. Bismuth oxychloride (BiOCl) has emerged as a potential alternative to traditional photocatalysts and has shown competitive removal efficiencies. However, pathways responsible for BiOCl photodegradation have not been well characterized. The present work is the first to determine, using LC-MS/MS analysis, the pathways by which BiOCl removes ibuprofen (IBP) from water. HPLC-DAD and LC-MS/MS analyses show that BiOCl converts IBP to two primary photochemical products, 4-isobutylacetophenone (IBAP) and 1-(4-isobutylphenyl)ethanol (IBPE). The reactivity for BiOCl is attributed to interactions of the carboxylic acid group of IBP with holes in the valence band. Hydroxylated-IBP was not detected in BiOCl photocatalytic degradation experiments which would be expected in a process driven by the formation and reactivity of reactive oxygen species. These data were used to formulate a photocatalytic degradation pathway for IBP and highlight the importance of studying both primary and secondary degradation reactions for photocatalytic studies.
Assuntos
Bismuto/química , Ibuprofeno/química , Luz , Nanoestruturas/química , Nanoestruturas/efeitos da radiação , Poluentes Químicos da Água/química , Catálise , Cromatografia Líquida , Fotólise , Espectrometria de Massas em Tandem , Purificação da Água/métodosRESUMO
Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) has become an important method for identifying peptides in neural tissues. The ultra-high-mass resolution and mass accuracy of MALDI-FTMS, in combination with in-cell accumulation techniques, can be used to advantage for the analysis of complex mixtures of peptides directly from tissue fragments or extracts. Given the diversity within the decapods, as well as the large number of extant species readily available for analysis, this group of animals represents an optimal model in which to examine phylogenetic conservation and evolution of neuropeptides and neuropeptide families. Surprisingly, no large comparative studies have previously been undertaken. Here, we have initiated such an investigation, which encompasses 32 species spanning seven decapod infraorders. Two peptides, APSGFLGMRamide and pQDLDHVFLRFamide, were detected in all species. A third peptide, GYRKPPFNGSIFamide, was detected in all species except members of the Astacidean genus Homarus, where a Val(1) variant was present. Our finding that these peptides are ubiquitously (or nearly ubiquitously) conserved in decapod neural tissues not only suggests important conserved functions for them, but also provides an intrinsic calibrant set for future MALDI-FTMS assessments of other peptides in this crustacean order.
Assuntos
Decápodes/química , Neuropeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Decápodes/classificação , Peso Molecular , Neuropeptídeos/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/químicaRESUMO
In this study, the peptide VYRKPPFNGSIFamide (Val(1)-SIFamide) was identified in the stomatogastric nervous system (STNS) of the American lobster, Homarus americanus, using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). When bath-applied to the stomatogastric ganglion (STG), synthetic Val(1)-SIFamide activated the pyloric motor pattern, increasing both burst amplitude and duration in the pyloric dilator (PD) neurons. To determine the distribution of this novel SIFamide isoform within the lobster STNS and neuroendocrine organs, a rabbit polyclonal antibody was generated against synthetic Val(1)-SIFamide. Whole-mount immunolabeling with this antibody showed that this peptide is widely distributed within the STNS, including extensive neuropil staining in the STG and commissural ganglia (CoGs) as well as immunopositive somata in the CoGs and the oesophageal ganglion. Labeling was also occasionally seen in the pericardial organ (PO), but not in the sinus gland. When present in the PO, labeling was restricted to fibers-of-passage and was never seen in release terminals. Adsorption of the antibody by either Val(1)-SIFamide or Gly(1)-SIFamide abolished all Val(1)-SIFamide staining within the STNS, including the STG neuropil, whereas adsorption by other lobster neuropeptides had no effect on immunolabeling. These data strongly suggest that the staining we report is a true reflection of the distribution of this peptide in the STNS. Collectively, our mass spectrometric, physiological, and anatomical data are consistent with Val(1)-SIFamide serving as a locally released neuromodulator in the lobster STG. Thus, our study provides the first direct demonstration of function for an SIFamide isoform in any species.
Assuntos
Sistema Digestório/inervação , Nephropidae/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso , Sistema Nervoso/metabolismo , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Animais , Sistema Digestório/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , Imuno-Histoquímica/métodos , Técnicas In Vitro , Modelos Neurológicos , Nephropidae/anatomia & histologia , Fenômenos Fisiológicos do Sistema Nervoso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/farmacologia , Sistemas Neurossecretores/metabolismo , Antro Pilórico/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de TempoRESUMO
Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.
Assuntos
Braquiúros/química , Olho/química , Nephropidae/química , Oligopeptídeos/análise , Ácido Pirrolidonocarboxílico/análogos & derivados , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Cátions/análise , Cátions/química , Análise de Fourier , Metais Alcalinos/química , Oligopeptídeos/química , Prótons , Ácido Pirrolidonocarboxílico/análise , Ácido Pirrolidonocarboxílico/químicaRESUMO
Current design limitations and ineffective remediation techniques in wastewater treatment plants have led to concerns about the prevalence of pharmaceutical and personal care products (PPCPs) in receiving waters. A novel photocatalyst, TiO2-doped low-silica X zeolite (TiO2-LSX), was used to study the degradation of the pharmaceutical compound, 17α-ethinylestradiol (EE2). The catalyst was synthesized and characterized using XRD, BET surface analysis, SEM-EDAX, and ICP-OES. The effects of different UV light intensities, initial EE2 concentrations, and catalyst dosages on the EE2 removal efficiency were studied. A higher EE2 removal efficiency was attained with UV-TiO2-LSX when compared with UV-TiO2 or UV alone. The EE2 degradation process followed pseudo-first-order kinetics. A comprehensive empirical model was developed to describe the EE2 degradation kinetics under different conditions using multiple linear regression analysis. The EE2 degradation mechanism was proposed based on molecular calculations, identification of photoproducts using HPLC-MS/MS, and reactive species quenching experiments; the results showed that oxidative degradation pathways initiated by hydroxyl radicals were predominant. This novel TiO2-doped zeolite system provides a promising application for the UV disinfection process in wastewater treatment plants.
Assuntos
Etinilestradiol/química , Titânio/química , Zeolitas/química , Algoritmos , Catálise , Radical Hidroxila , Microscopia Eletrônica de Varredura , Fotoquímica , Espectrometria de Fluorescência , Raios Ultravioleta , Instalações de Eliminação de Resíduos , Eliminação de Resíduos Líquidos , Difração de Raios XRESUMO
Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.
Assuntos
Metanol/química , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Animais , Artefatos , Cromatografia Líquida , Temperatura Baixa , Temperatura Alta , Extração Líquido-Líquido , Metilação , Nephropidae , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The allatostatins comprise three structurally distinct peptide families that regulate juvenile hormone production by the insect corpora allata. A-type family members contain the C-terminal motif -YXFGLamide and have been found in species from numerous arthropod taxa. Members of the B-type family exhibit a -WX(6)Wamide C-terminus and, like the A-type peptides, appear to be broadly conserved within the Arthropoda. By contrast, members of the C-type family, typified by the unblocked C-terminus -PISCF, a pyroglutamine blocked N-terminus, and a disulfide bridge between two internal Cys residues, have only been found in holometabolous insects, i.e. lepidopterans and dipterans. Here, using transcriptomics, we have identified SYWKQCAFNAVSCFamide (disulfide bridging predicted between the two Cys residues), a known honeybee and water flea C-type-like peptide, from the American lobster Homarus americanus (infraorder Astacidea). Using matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), a mass corresponding to that of SYWKQCAFNAVSCFamide was detected in the H. americanus brain, supporting the existence of this peptide and its theorized structure. Furthermore, SYWKQCAFNAVSCFamide was detected by MALDI-FTMS in neural tissues from five additional astacideans as well as 19 members of four other decapod infraorders (i.e. Achelata, Anomura, Brachyura and Thalassinidea), suggesting that it is a broadly conserved decapod peptide. In H. americanus, SYWKQCAFNAVSCFamide is capable of modulating the output of both the pyloric circuit of the stomatogastric nervous system and the heart. This is the first demonstration of bioactivity for this peptide in any species.
Assuntos
Coração/efeitos dos fármacos , Nephropidae/metabolismo , Neuropeptídeos/química , Neurotransmissores/química , Sequência de Aminoácidos , Animais , Biologia Computacional , Sequência Conservada , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Análise de Fourier , Mucosa Gástrica/efeitos dos fármacos , Genômica , Dados de Sequência Molecular , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/farmacologia , Neurotransmissores/isolamento & purificação , Neurotransmissores/farmacologia , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Modulation of neural circuits in the crustacean stomatogastric nervous system (STNS) allows flexibility in the movements of the foregut musculature. The extensive repertoire of such resulting motor patterns in dietary generalists is hypothesized to permit these animals to process varied foods. The foregut and STNS of Pugettia producta are similar to those of other decapods, but its diet is more uniform, consisting primarily of kelp. We investigated the distribution of highly conserved neuromodulators in the stomatogastric ganglion (STG) and neuroendocrine organs of Pugettia, and documented their effects on its pyloric rhythm. Using immunohistochemistry, we found that the distributions of Cancer borealis tachykinin-related peptide I (CabTRP I), crustacean cardioactive peptide (CCAP), proctolin, red pigment concentrating hormone (RPCH) and tyrosine hydroxylase (dopamine) were similar to those of other decapods. For all peptides except proctolin, the isoforms responsible for the immunoreactivity were confirmed by mass spectrometry to be the authentic peptides. Only two modulators had physiological effects on the pyloric circuit similar to those seen in other species. In non-rhythmic preparations, proctolin and the muscarinic acetylcholine agonist oxotremorine consistently initiated a full pyloric rhythm. Dopamine usually activated a pyloric rhythm, but this pattern was highly variable. In only about 25% of preparations, RPCH activated a pyloric rhythm similar to that seen in other species. CCAP and CabTRP I had no effect on the pyloric rhythm. Thus, whereas Pugettia possesses all the neuromodulators investigated, its pyloric rhythm, when compared with other decapods, appears less sensitive to many of them, perhaps because of its limited diet.
Assuntos
Braquiúros/fisiologia , Gânglios dos Invertebrados/citologia , Sistemas Neurossecretores/efeitos dos fármacos , Neurotransmissores/farmacologia , Piloro/inervação , Animais , Braquiúros/efeitos dos fármacos , Eletrofisiologia , Gânglios dos Invertebrados/efeitos dos fármacos , Imuno-Histoquímica , Microscopia de Fluorescência , Piloro/efeitos dos fármacos , Piloro/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , WashingtonRESUMO
Two beta-pigment-dispersing hormone (beta-PDH) isoforms have been identified in several decapod crustaceans, including the crab Cancer productus, but whether these peptides serve common or distinct physiological roles remains to be elucidated. Here we show that the distribution of beta-PDH-like immunoreactivity in the nervous system of C. productus is similar to that found in other brachyurans, suggesting roles as both a circulating hormone and a locally released transmitter for members of this peptide family. cDNAs encoding NSELINSILGLPKVMNDAamide (authentic beta-PDH; here termed Canpr-beta-PDH I) or NSELINSLLGLSRLMNEAamide [corrected](Canpr-beta-PDH II) were cloned. Double in situ hybridization revealed that these two beta-PDH isoforms are differentially distributed within the eyestalk. For example, in most neurons between the medulla interna (MI) and the medulla terminalis (MT), both isoforms appear present; however, in some neurons in this region, mRNA for only one or the other isoform was detected. Likewise, only prepro-beta-pdh I mRNA was detected in the somata of the lamina ganglionaris (LG) and in the brain. By direct tissue mass spectrometry, only Canpr-beta-PDH II was detected in the neurosecretory sinus gland (SG), whereas Canpr-beta-PDH I was found in all other parts of the eyestalk. Collectively, these data suggest distinct functions for each of the C. productus beta-PDHs; Canpr-beta-PDH II appears to be a neurohormone in the SG, whereas Canpr-beta-PDH I may function as a local transmitter/modulator. Our data support the hypothesis that duplication and subsequent mutation of a common neuropeptide gene may underlie the evolution of two differentially distributed transcripts that serve distinct physiological roles.
Assuntos
Braquiúros/metabolismo , Clonagem Molecular/métodos , Expressão Gênica/fisiologia , Peptídeos/genética , Peptídeos/metabolismo , Animais , Braquiúros/ultraestrutura , Análise de Fourier , Espectrometria de Massas , Microscopia Confocal , Sistema Nervoso/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The development of expressed sequence tags (ESTs) for crustacean cDNA libraries and their deposition in publicly accessible databases has generated a rich resource for peptide discovery in this commercially and ecologically important arthropod subphylum. Here, we have conducted in silico searches of these databases for unannotated ESTs encoding putative neuropeptide precursors using the BLAST program tblastn, and have predicted the mature forms of the peptides encoded by them. The primary strategy used was to query the database with known decapod prepro-hormone sequences or, in some instances, insect precursor protein sequences. For neuropeptides for which no prepro-hormones are known, the peptides themselves were used as queries. For those peptides expected to originate from a common precursor, the individual sequences were combined, with each peptide flanked by a dibasic processing site and, if amidated, a glycine residue. Using these approaches, 13 unannotated ESTs encoding putative neuropeptide precursors were found. For example, using the first strategy, putative Marsupenaeus japonicus prepro-hormones encoding B-type allatostatins, neuropeptide F (NPF), and orcokinins were identified. Similarly, several Homarus americanus ESTs encoding putative orcokinin precursors were found. In addition to the decapod prepro-hormones, ESTs putatively encoding a NPF isoform and a red pigment concentrating hormone-like peptide were identified from the cladoceran Daphnia magna, as was one EST putatively encoding multiple tachykinin-related peptides from the isopod Eurydice pulchra. Using the second strategy, we identified a Carcinus maenas EST encoding HIGSLYRamide, a peptide recently discovered via mass spectrometry from Cancer productus. Using mass spectral methods we confirmed that this peptide is also present in Carcinus maenas. Collectively over 50 novel crustacean peptides were predicted from the identified ESTs, providing a strong foundation for future investigations of the evolution, regulation and function of these and related molecules in this arthropod taxon.