On the mechanisms involved in biological heme crystallization

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.     

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that areincorporated from the lumen by the posterior midgut epithelium ofRhodnius prolixus and the biosynthesis of lipids. We alsodemonstrate that neutral lipids (NL) are transferred to the haemolymphaticlipophorin (Lp) and that phospholipids remain in the tissue in which they areorganised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at theluminal side of isolated midguts of R. prolixus females wasreadily absorbed and was used to synthesise phospholipids (80%) and NL (20%).The highest incorporation of 3H-palmitic acid was on the first day after a bloodmeal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by thetissue decreased in the presence of Lp in the incubation medium. The metabolicfates of 3H-lipids synthesised by the posterior midgut were followed and it wasobserved that DG was the major lipid released to Lp particles. However, themajority of phospholipids were not transferred to Lp, but remained in thetissue. The phospholipids that were synthesised and accumulated in the posteriormidgut were found to be associated with Rhodnius luminalcontents as structural components of PMMs.     

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 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.