Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

Abstract

BackgroundHematophagous 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 FindingsHz 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/SignificanceHerein the Hz formation is shown to be associated to an α-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.

On the physico-chemical and physiological requirements of hemozoin formation promoted by perimicrovillar membranes in Rhodnius prolixus midgut

“…1C suggest that three distinct mechanisms are involved in crystal growth, in such a way that in each phase the rates of crystal growth are distinct. Conceivably, in early periods (0e6 h), nucleation of the first heme crystals could be supported by the recently described a-glucosidase activity (Mury et al, 2009), which may be consistent with spots of heme-peroxidase staining within the PMVM as shown by Silva et al (2007). Then, a second highly efficient process, acts between 6 h and 24 h. Possibly, during this period, extension of small Hz crystals could be promoted by the hydrophilicehydrophobic interfaces provided by lipids from PMVM.…”

“…Silva et al, 2007 have shown that development of PMVM changes during blood digestion, in which maximum PMVM levels are detected seven days after a blood meal . They also demonstrate that PMVM levels are correlated with a-glucosidase activity and it seems that part of the heme crystallization activity is due to the nucleation action of a-glucosidase on Hz formation (Mury et al, 2009). Regarding the role of insect aging, since R. prolixus is an obligatory hematophagous insect, it is not surprising that all the insect's stages are capable of producing this heme crystal.…”

“…In the triatomine insect R. prolixus Hz formation is promoted by midgut PMVM (Oliveira et al, 1999(Oliveira et al, , 2000a(Oliveira et al, , 2005Silva et al, 2007). Very recently, Hz formation was shown to be associated with an a-glucosidase activity in such a way that it may participate in the nucleation process of nascent Hz crystals (Mury et al, 2009). In the present work, we describe some of the physico-chemical and physiological parameters that regulate PMVM-induced heme crystallization in R. prolixus.…”

“…However, Hz formation driven by PMVM-derived lipids in that work was significantly lower compared to the activity supported by native PMVM. Also, there is recent compelling evidence that an enzymatic marker of PMVM, the a-glucosidase activity, is involved in Hz formation by this structure possibly at the nucleation step (Mury et al, 2009).…”

Biomimetic Approaches to Understanding the Mechanism of Haemozoin Formation

“…The process is promoted by PMVM, with the lipids playing a major role (Stiebler et al, 2010a). A protein, -glucosidase may also play a role in haemozoin formation in this organism (Mury et al, 2009). …”