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Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: an evolutionary history.

Mury FB, da Silva JR, Ferreira LS, dos Santos Ferreira B, de Souza-Filho GA, de Souza-Neto JA, Ribolla PE, Silva CP, do Nascimento VV, Machado OL, Berbert-Molina MA, Dansa-Petretski M - PLoS ONE (2009)

Bottom Line: Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities.Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes.The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, Rio de Janeiro, Brazil.

ABSTRACT

Background: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/principal findings: Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/significance: Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

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Related in: MedlinePlus

Hz formation and α-glucosidase activities in the presence or absence of inhibitors in vitro.A. C. and D. Hz formation. B. α-glucosidase activity. Ctl - hemin; PE - protein extract of midgut epithelium; PE + Eri - protein extract + erythritol; PE + DEPC - protein extract + diethypyrocarbonate; PE + Cs - protein extract + castanospermine; PE + AB - protein extract + anti D. peruvianus α-glucosidase antibody; Ctl + AB - hemin + antibody; Ctl + Cs - hemin + castanospermine; Ctl + DEPC - hemin + diethypyrocarbonate; PE → AB - protein extract + antibody 10 hours after starting assay; PE → DEPC - protein extract + diethypyrocarbonate 10 hours after starting assay; PE 100°C - protein extract boiled for 10 min before starting assay; PE → 100°C - protein extract boiled 10 hours after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in materials and methods. Hz formation activity was expressed as nmol heme aggregated in 24 h for 15 µg protein extract. The assays of α-glucosidase activity were determined using a colorimetric method. Unless otherwise indicated, activity was expressed as nmol ρ-nitrofenolate released in 1 min. Results shown are means ±SEM (n = 4) of two experiments run in triplicate. The experiments with inhibitors were significantly different from protein extract alone *(P<0.05).
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pone-0006966-g001: Hz formation and α-glucosidase activities in the presence or absence of inhibitors in vitro.A. C. and D. Hz formation. B. α-glucosidase activity. Ctl - hemin; PE - protein extract of midgut epithelium; PE + Eri - protein extract + erythritol; PE + DEPC - protein extract + diethypyrocarbonate; PE + Cs - protein extract + castanospermine; PE + AB - protein extract + anti D. peruvianus α-glucosidase antibody; Ctl + AB - hemin + antibody; Ctl + Cs - hemin + castanospermine; Ctl + DEPC - hemin + diethypyrocarbonate; PE → AB - protein extract + antibody 10 hours after starting assay; PE → DEPC - protein extract + diethypyrocarbonate 10 hours after starting assay; PE 100°C - protein extract boiled for 10 min before starting assay; PE → 100°C - protein extract boiled 10 hours after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in materials and methods. Hz formation activity was expressed as nmol heme aggregated in 24 h for 15 µg protein extract. The assays of α-glucosidase activity were determined using a colorimetric method. Unless otherwise indicated, activity was expressed as nmol ρ-nitrofenolate released in 1 min. Results shown are means ±SEM (n = 4) of two experiments run in triplicate. The experiments with inhibitors were significantly different from protein extract alone *(P<0.05).

Mentions: Erythritol (100 mM) and castanospermine (30 µM), specific inhibitors of α-glucosidase, Diethypyrocarbonate (DEPC) (10 mM), which react with and modify histidine residues, and a polyclonal antibody raised against α-glucosidase from D. peruvianus (1∶2500) were used in order to investigate the correlation between the α-glucosidase and Hz formation activities (Figure 1A and 1B). Erythritol is a competitive inhibitor of the α-glucosidase assayed with NPαGlu [23]; only one molecule of these inhibitors is able to bind at putative sub-sites 1 or 2 of this enzyme. Here, the effect of erythritol and castanospermine on α-glucosidase activity and Hz formation were tested. The results show that both activities were very sensitive to these agents. DEPC is used to reveal mechanistic differences among α-glucosidases from mammalian, plant and yeast cells [24]–[26]. For the R. prolixus α-glucosidase, DEPC strongly inhibited this enzyme as well as the Hz formation, suggesting that histidine residue (s) at or close to the catalytic domain of the enzyme to be important for both activities. An antibody raised against α-glucosidase of the phytophagous hemipteran D. peruvianus, which also successfully recognizes the enzyme in the PMM of the R. prolixus midgut [20], was able to drastically inhibit Hz formation and α-glucosidase activities in vitro (Figure 1A and 1B). The next set of experiments was designed to evaluate whether α-glucosidase acts as a nucleation site for the process of Hz formation. To test this hypothesis, the Hz formation assay was carried out using the midgut protein extract from insects previously fed on blood. Additions of the D. peruvianus anti-α-glucosidase antibody (Figure 1C) or DEPC (Figure 1D), 10 hours after the onset of the experiment, were not able to inhibit the hemozoin formation, since it had already been nucleated. As a corroborative datum, a 10 min boiling step of the protein extract preceding the start of Hz formation assay reduced its ability to initiate Hz nucleation. Moreover, boiling the assay mixture 10 hours after the onset of the assay did not interfere in the continuing occurrence of the Hz formation process (Figure 1D). These results suggest that the enzyme is implied in the process of Hz formation, particularly on nucleation step, since specific α-glucosidase inhibitors were able to interfere in the Hz formation.


Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: an evolutionary history.

Mury FB, da Silva JR, Ferreira LS, dos Santos Ferreira B, de Souza-Filho GA, de Souza-Neto JA, Ribolla PE, Silva CP, do Nascimento VV, Machado OL, Berbert-Molina MA, Dansa-Petretski M - PLoS ONE (2009)

Hz formation and α-glucosidase activities in the presence or absence of inhibitors in vitro.A. C. and D. Hz formation. B. α-glucosidase activity. Ctl - hemin; PE - protein extract of midgut epithelium; PE + Eri - protein extract + erythritol; PE + DEPC - protein extract + diethypyrocarbonate; PE + Cs - protein extract + castanospermine; PE + AB - protein extract + anti D. peruvianus α-glucosidase antibody; Ctl + AB - hemin + antibody; Ctl + Cs - hemin + castanospermine; Ctl + DEPC - hemin + diethypyrocarbonate; PE → AB - protein extract + antibody 10 hours after starting assay; PE → DEPC - protein extract + diethypyrocarbonate 10 hours after starting assay; PE 100°C - protein extract boiled for 10 min before starting assay; PE → 100°C - protein extract boiled 10 hours after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in materials and methods. Hz formation activity was expressed as nmol heme aggregated in 24 h for 15 µg protein extract. The assays of α-glucosidase activity were determined using a colorimetric method. Unless otherwise indicated, activity was expressed as nmol ρ-nitrofenolate released in 1 min. Results shown are means ±SEM (n = 4) of two experiments run in triplicate. The experiments with inhibitors were significantly different from protein extract alone *(P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2734994&req=5

pone-0006966-g001: Hz formation and α-glucosidase activities in the presence or absence of inhibitors in vitro.A. C. and D. Hz formation. B. α-glucosidase activity. Ctl - hemin; PE - protein extract of midgut epithelium; PE + Eri - protein extract + erythritol; PE + DEPC - protein extract + diethypyrocarbonate; PE + Cs - protein extract + castanospermine; PE + AB - protein extract + anti D. peruvianus α-glucosidase antibody; Ctl + AB - hemin + antibody; Ctl + Cs - hemin + castanospermine; Ctl + DEPC - hemin + diethypyrocarbonate; PE → AB - protein extract + antibody 10 hours after starting assay; PE → DEPC - protein extract + diethypyrocarbonate 10 hours after starting assay; PE 100°C - protein extract boiled for 10 min before starting assay; PE → 100°C - protein extract boiled 10 hours after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in materials and methods. Hz formation activity was expressed as nmol heme aggregated in 24 h for 15 µg protein extract. The assays of α-glucosidase activity were determined using a colorimetric method. Unless otherwise indicated, activity was expressed as nmol ρ-nitrofenolate released in 1 min. Results shown are means ±SEM (n = 4) of two experiments run in triplicate. The experiments with inhibitors were significantly different from protein extract alone *(P<0.05).
Mentions: Erythritol (100 mM) and castanospermine (30 µM), specific inhibitors of α-glucosidase, Diethypyrocarbonate (DEPC) (10 mM), which react with and modify histidine residues, and a polyclonal antibody raised against α-glucosidase from D. peruvianus (1∶2500) were used in order to investigate the correlation between the α-glucosidase and Hz formation activities (Figure 1A and 1B). Erythritol is a competitive inhibitor of the α-glucosidase assayed with NPαGlu [23]; only one molecule of these inhibitors is able to bind at putative sub-sites 1 or 2 of this enzyme. Here, the effect of erythritol and castanospermine on α-glucosidase activity and Hz formation were tested. The results show that both activities were very sensitive to these agents. DEPC is used to reveal mechanistic differences among α-glucosidases from mammalian, plant and yeast cells [24]–[26]. For the R. prolixus α-glucosidase, DEPC strongly inhibited this enzyme as well as the Hz formation, suggesting that histidine residue (s) at or close to the catalytic domain of the enzyme to be important for both activities. An antibody raised against α-glucosidase of the phytophagous hemipteran D. peruvianus, which also successfully recognizes the enzyme in the PMM of the R. prolixus midgut [20], was able to drastically inhibit Hz formation and α-glucosidase activities in vitro (Figure 1A and 1B). The next set of experiments was designed to evaluate whether α-glucosidase acts as a nucleation site for the process of Hz formation. To test this hypothesis, the Hz formation assay was carried out using the midgut protein extract from insects previously fed on blood. Additions of the D. peruvianus anti-α-glucosidase antibody (Figure 1C) or DEPC (Figure 1D), 10 hours after the onset of the experiment, were not able to inhibit the hemozoin formation, since it had already been nucleated. As a corroborative datum, a 10 min boiling step of the protein extract preceding the start of Hz formation assay reduced its ability to initiate Hz nucleation. Moreover, boiling the assay mixture 10 hours after the onset of the assay did not interfere in the continuing occurrence of the Hz formation process (Figure 1D). These results suggest that the enzyme is implied in the process of Hz formation, particularly on nucleation step, since specific α-glucosidase inhibitors were able to interfere in the Hz formation.

Bottom Line: Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities.Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes.The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, Rio de Janeiro, Brazil.

ABSTRACT

Background: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/principal findings: Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/significance: Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

Show MeSH
Related in: MedlinePlus