Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut.

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.

The role of lipoxygenase products on the endocytosis of yolk proteins in insects: participation of cAMP.

The participation of eicosanoids and second messengers in the regulation of endocytosis by the ovaries was investigated using the uptake of Rhodnius heme binding protein (RHBP) as an experimental model. The rate of RHBP uptake decreased up to 40% in the presence of BWA4C and NDGA, 5 and 12-lipoxygenase inhibitors, respectively, suggesting the involvement of lipoxygenase products in endocytosis regulation. Addition of Leukotriene B4 (LTB(4); one product of the 5 lipoxygenase pathway) increased in vitro the uptake of RHBP by 30%. The content of cAMP in the Rhodnius' ovaries were monitored after treatment with different eicosanoids and inhibitors of eicosanoids synthesis. The amount of cAMP decreased in the presence of indomethacin (by 50%), while treatment with PGE(2) induced an increase of 85% of this messenger in the ovaries. The presence of LTB(4) in the medium inhibited in 60% the content of cAMP in the ovaries, while BWA4C induced a 100% increase of this messenger in the ovaries. Addition of 1 microM DBcAMP in the medium resulted in a 30% decrease in the rate of RHBP uptake. Taken together, these data show that cyclooxygenase and lipoxygenase products participate in the control of protein internalization by modulation of cAMP levels.

On the biosynthesis of Rhodnius prolixus heme-binding protein.

The biosynthesis of Rhodnius prolixus heme-binding protein (RHBP), which is present in the hemolymph and oocytes of Rhodnius prolixus, was investigated. Fat bodies of female insects incubated in vitro with 14C-leucine were able to synthesize and secrete 14C-RHBP to the culture medium. Titrtion of synthesized RHBP with hemin showed that the protein secreted by the fat bodies is bound to heme, despite the presence of apo-RHBP in the hemolymph. The sequence of the RHBP cDNA encodes a pre-protein of 128 amino acids with no significant homology to any known protein. Northern-blot assays revealed that RHBP expression was limited to fat bodies. The levels of both RHBP mRNA and secreted protein increased in response to blood meal. In addition, the time-course of RHBP secretion in vitro paralleled mRNA accumulation observed in vivo. The inhibition of the de novo heme biosynthesis by treatment of fat bodies with succinyl acetone (SA), an irreversible inhibitor of delta-aminolevulinic acid-dehydratase, led to a significant decrease of heme-RHBP secretion. Nevertheless, the levels of RHBP mRNA were not modified by SA treatment, suggesting that the heme availability is involved in a post-transcriptional control of the RHBP synthesis.

Crystallization and preliminary X-ray study of haem-binding protein from the bloodsucking insect Rhodnius prolixus.

Rhodnius haem-binding protein (RHBP) from the bloodsucking insect Rhodnius prolixus, a 15 kDa protein, has been crystallized using polyethylene glycol as a precipitant. X-ray diffraction data have been collected at a synchrotron source. The crystals belong to the space group P4(1(3))2(1)2, with unit-cell parameters a = b = 64.98, c = 210.68 A, and diffract beyond 2.6 A resolution.

A heme-binding aspartic proteinase from the eggs of the hard tick Boophilus microplus.

An aspartic proteinase that binds heme with a 1:1 stoichiometry was isolated and cloned from the eggs of the cattle tick Boophilus microplus. This proteinase, herein named THAP (tick heme-binding aspartic proteinase) showed pepstatin-sensitive hydrolytic activity against several peptide and protein substrates. Although hemoglobin was a good substrate for THAP, low proteolytic activity was observed against globin devoid of the heme prosthetic group. Hydrolysis of globin by THAP increased as increasing amounts of heme were added to globin, with maximum activation at a heme-to-globin 1:1 ratio. Further additions of heme to the reaction medium inhibited proteolysis, back to a level similar to that observed against globin alone. The addition of heme did not change THAP activity toward a synthetic peptide or against ribonuclease, a non-hemeprotein substrate. The major storage protein of tick eggs, vitellin (VT), the probable physiological substrate of THAP, is a hemeprotein. Hydrolysis of VT by THAP was also inhibited by the addition of heme to the incubation media. Taken together, our results suggest that THAP uses heme bound to VT as a docking site to increase specificity and regulate VT degradation according to heme availability.

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus.

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus.

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.