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

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 α-glucosidase was investigated. Hz formation was inhibited by specific α-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 α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-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 α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. Conclusions/Significance: Herein the Hz formation is shown to be associated to an a-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-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. © 2009 Mury et al

Analysis of α-glucosidase genes using Blastx.

<p>Analysis of α-glucosidase genes using Blastx.</p

Hz formation activity in the presence or absence of maltose <i>in vitro</i>.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006966#s4" target="_blank">materials and methods</a>. 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 *(<i>P</i><0.05).</p

Physiological effects of dsRNA-mediated silencing of α-glucosidase.

<p>The insects were injected with 2 µL of 100 mM PBS pH 7.4 or dsLacZ (controls) and dsα-glu (2 or 10 µg/female); mortality and oviposition were monitored 4 days after feeding. In all panels, results are means ±SEM (n = 70). The insects injected with 10 µg dsα-glu, analyzed 4 days feeding, were significantly different from control insects injected with both PBS and dsLacZ and also analyzed 4 days after feeding ^*(<i>P</i><0.05).</p>a<p>Access number to GenBank database.</p>b<p>Function expected according the Blastx sequence results similarity in GenBank database.</p>c<p>Protein Data Bank.</p