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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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Hz formation activity in the presence or absence of maltose in vitro.Ctl - hemin; Mal - Maltose; CF - chromatographic fraction; CF + Mal → H - chromatographic fraction + maltose and hemin added 4 h after starting assay; CF + H → Mal - chromatographic fraction + hemin and maltose added 4 h 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 of aggregated heme, during 24 h, for 8 µg protein. The results are the mean and standard deviation of one experiment run in triplicate. The experiment where maltose was added before hemin was significantly different from that with protein alone or that with hemin being added first *(P<0.05).
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pone-0006966-g002: Hz formation activity in the presence or absence of maltose in vitro.Ctl - hemin; Mal - Maltose; CF - chromatographic fraction; CF + Mal → H - chromatographic fraction + maltose and hemin added 4 h after starting assay; CF + H → Mal - chromatographic fraction + hemin and maltose added 4 h 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 of aggregated heme, during 24 h, for 8 µg protein. The results are the mean and standard deviation of one experiment run in triplicate. The experiment where maltose was added before hemin was significantly different from that with protein alone or that with hemin being added first *(P<0.05).

Mentions: The action of competitive inhibitors of α-glucosidase (DEPC, castanospermine and erythritol) inhibiting the Hz formation suggests that the same site can be involved in both activities. In order to address this question, the Hz formation activity in the presence of both maltose (30 mM), an α-glucosidase substrate, and hemin was tested. Ion exchange chromatographic fraction containing α-glucosidase activity was firstly incubated with maltose for 4 hours. After this time, hemin was added but Hz formation did not occur (Figure 2). Primary incubation of chromatographic fraction with acetate buffer containing hemin, followed by the addition of maltose, did not interfere in the efficiency to sustain Hz formation (Figure 2). The results show that the binding of maltose to the enzyme blocked the Hz formation activity. Since the binding of maltose and hemin are mutually exclusive processes, the simplest explanation is that both share a common binding site.


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 activity in the presence or absence of maltose in vitro.Ctl - hemin; Mal - Maltose; CF - chromatographic fraction; CF + Mal → H - chromatographic fraction + maltose and hemin added 4 h after starting assay; CF + H → Mal - chromatographic fraction + hemin and maltose added 4 h 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 of aggregated heme, during 24 h, for 8 µg protein. The results are the mean and standard deviation of one experiment run in triplicate. The experiment where maltose was added before hemin was significantly different from that with protein alone or that with hemin being added first *(P<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734994&req=5

pone-0006966-g002: Hz formation activity in the presence or absence of maltose in vitro.Ctl - hemin; Mal - Maltose; CF - chromatographic fraction; CF + Mal → H - chromatographic fraction + maltose and hemin added 4 h after starting assay; CF + H → Mal - chromatographic fraction + hemin and maltose added 4 h 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 of aggregated heme, during 24 h, for 8 µg protein. The results are the mean and standard deviation of one experiment run in triplicate. The experiment where maltose was added before hemin was significantly different from that with protein alone or that with hemin being added first *(P<0.05).
Mentions: The action of competitive inhibitors of α-glucosidase (DEPC, castanospermine and erythritol) inhibiting the Hz formation suggests that the same site can be involved in both activities. In order to address this question, the Hz formation activity in the presence of both maltose (30 mM), an α-glucosidase substrate, and hemin was tested. Ion exchange chromatographic fraction containing α-glucosidase activity was firstly incubated with maltose for 4 hours. After this time, hemin was added but Hz formation did not occur (Figure 2). Primary incubation of chromatographic fraction with acetate buffer containing hemin, followed by the addition of maltose, did not interfere in the efficiency to sustain Hz formation (Figure 2). The results show that the binding of maltose to the enzyme blocked the Hz formation activity. Since the binding of maltose and hemin are mutually exclusive processes, the simplest explanation is that both share a common binding site.

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