RESUMO
It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.
Assuntos
Hepatopâncreas/enzimologia , Palinuridae/enzimologia , Isoformas de Proteínas/isolamento & purificação , Tripsina/química , Animais , Fenômenos Fisiológicos do Sistema Digestório/fisiologia , Proteólise , Relação Estrutura-AtividadeRESUMO
Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/metabolismo , Palinuridae/enzimologia , Palinuridae/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Relação Dose-Resposta Imunológica , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Imunofluorescência , Brânquias/citologia , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Hemócitos/citologia , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Soros Imunes , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/imunologia , Palinuridae/genética , Conformação Proteica , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.
Assuntos
Substituição de Aminoácidos , Palinuridae/enzimologia , Tripsina/química , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Sistema Digestório/metabolismo , Dados de Sequência Molecular , Palinuridae/genética , Alinhamento de Sequência , Especificidade por Substrato , Tripsina/genéticaRESUMO
Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/imunologia , Palinuridae/enzimologia , Palinuridae/genética , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Hemócitos/imunologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Palinuridae/classificação , Palinuridae/imunologia , Alinhamento de SequênciaRESUMO
Changes in major digestive enzymes through developmental and molt stages were studied for the spiny lobster Panulirus argus. There were significant positive relationships between specific activity of trypsin and amylase enzymes and lobster size, whereas esterase and lipase specific activities decreased as lobsters aged. No relationship was found between amylase/trypsin ratio and lobster size. Positive trends were found, however, for trypsin/lipase and amylase/lipase ratios. Results suggest that changes in enzyme activity respond to the lobsters' physiological needs for particular dietary components although multivariate analysis suggested that enzyme activities could be not totally independent of diet. On the other hand, the pattern of changes of major enzyme activities through molt cycle was similar for most enzymes studied. Following molt, trypsin, chymotrypsin, amylase, and lipase activities gradually increased to maximal levels at late intermolt (C4) and premolt (D). There were no variations in the electrophoretic pattern of digestive enzymes through developmental and molt stages and thus, it is demonstrated that regulation is exerted quantitatively rather than qualitatively. Further studies on the effect of other intrinsic and extrinsic factors on digestive enzyme activities are needed to fully understand digestive abilities and regulation mechanisms in spiny lobsters.
Assuntos
Amilases/metabolismo , Esterases/metabolismo , Lipase/metabolismo , Palinuridae/enzimologia , Tripsina/metabolismo , Animais , Tamanho Corporal , Digestão , Palinuridae/crescimento & desenvolvimentoRESUMO
We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.
Assuntos
Sistema Digestório/enzimologia , Palinuridae/enzimologia , Animais , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Enzimas/metabolismo , Suco Gástrico/enzimologia , Concentração de Íons de Hidrogênio , Inibidores de Proteases/farmacologia , TemperaturaRESUMO
Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.
Assuntos
Hemocianinas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Palinuridae/enzimologia , 2-Propanol/farmacologia , Animais , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Catecóis/metabolismo , Quimotripsina/metabolismo , Dopamina/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Hemocianinas/química , Hemocianinas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Levodopa/metabolismo , Peso Molecular , Monofenol Mono-Oxigenase/química , Palinuridae/química , Percloratos/farmacologia , Compostos de Sódio/farmacologia , Dodecilsulfato de Sódio/química , Especificidade por Substrato , Tripsina/metabolismoRESUMO
The prophenoloxidase activating system plays a major role in the defense mechanism of arthropods. In the present study, the phenoloxidase activity and its location in the hemolymph of the spiny lobster Panulirus argus is presented. Phenoloxidase activity was observed in the hemocyte lysate supernatant (HLS) and plasma after their incubation with trypsin. Higher amounts of trypsin were required to activate the HLS prophenoloxidase, due to the presence of a trypsin inhibitor in this fraction. Activation of prophenoloxidase was found when HLS was incubated with calcium, with an optimal pH between 7.5 and 8. This spontaneous activity is due to the prophenoloxidase activating enzyme, a serine proteinase that activates the prophenoloxidase once calcium ions were available. SDS was able to induce phenoloxidase activity in plasma and hemocyte fractions. Prophenoloxidase from HLS occurs as an aggregate of 300kDa. Electrophoretic studies combining SDS-PAGE and native PAGE indicate that different proteins produced the phenoloxidase activity found in HLS and plasma. Thus, as in most crustaceans, Panulirus argus contains a prophenoloxidase activating system in its hemocyte, comprising at least the prophenoloxidase activating enzyme and the prophenoloxidase. Finally, it is suggested that phenoloxidase activity found in plasma is produced by hemocyanin.
Assuntos
Monofenol Mono-Oxigenase/metabolismo , Palinuridae/enzimologia , Animais , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Hemócitos/enzimologia , Monofenol Mono-Oxigenase/sangue , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Fatores de Tempo , Tripsina/metabolismoRESUMO
In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.