On the biosynthesis of Rhodnius prolixus heme-binding protein (original) (raw)
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Heme biosynthesis and oogenesis in the blood-sucking bug, Rhodnius prolixus
Insect Biochemistry and Molecular Biology, 2001
We have previously shown that the pathway of porphyrin synthesis operates in the blood feeding triatomine bug Rhodnius prolixus but not in the cattle tick Boophilus microplus. In the present paper we studied the correlation between heme synthesis and egg development in Rhodnius. There is a sharp increase heme biosynthetic capability in the fat body (160%) and in the ovaries (360%) in response to a blood meal, as evaluated from the activity of the enzyme δ-aminolevulinate dehydratase (EC 4.2.1.24). The in vivo inhibition of ALA-D by succinyl acetone results in a dose dependent decrease of oviposition. Oviposition is recovered when porphobilinogen, the product of the impaired reaction, is added to the succinyl acetone enriched blood. Taken together, these results show that heme biosynthesis is a fundamental event to vitellogenic females. The demand for heme in this metabolic juncture cannot be supplied by the heme eventually absorbed during blood digestion and associated with Rhodnius heme binding protein (RHBP), which is then incorporated into growing oocytes. Inhibition of heme biosynthesis results in lower levels of RHBP in the hemolymph, suggesting that the synthesis of this protein is controlled by heme availability.
Insect Biochemistry and Molecular Biology, 2002
We have previously shown that Rhodnius prolixus' eggs and hemolymph are pink due to the presence of the hemeprotein Rhodnius heme-binding protein (RHBP). In the hemolymph it functions as an antioxidant. Nevertheless, its function in eggs has not been determined. Here we present evidence that RHBP is a source of heme for embryonic development. RHBP content decreases during embryogenesis, but the total heme content of eggs remains unchanged. Biliverdin, the product of heme degradation, is not detectable in late embryos. The activity of the heme-synthesizing pathway is low throughout embryogenesis and rises sharply after nymphs' hatching. Heme-radiolabeled eggs were produced and, at the day of hatching, nymphs were dissected. The presence of radiolabeled heme in their carcass is an indication that heme reutilization is occurring. The only animal known to reutilize heme in significant levels is the cattle tick Boophilus microplus, which cannot synthesize its own heme. Diversely, Rhodnius can synthesize its own heme but, in the context of embryogenesis, heme demand seems to be supplied by the programmed release of heme form RHBP. This behavior indicates that in Rhodnius, we might have a highly unusual profile: heme is both synthesized and reutilized.
Uptake of Rhodnius heme‐binding protein (RHBP) by the ovary of Rhodnius prolixus
Archives of Insect Biochemistry and Physiology, 1998
The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from oocyte was labeled with 125 I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [ 125 I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [ 125 I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [ 125 I]RHBP was linear at least up to 60 min. The uptake was dependent on [ 125 I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [ 125 I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed. Arch. Insect *Abbreviations used: apo-RHBP = RHBP free of heme; BSA = Bovine Serum Albumin; heme-RHBP = RHBP associated with heme; [ 125 I]RHBP = 125 I -labeled RHBP; Lp = lipophorin; Mvg = microvitellogenin; PBS = phosphate buffered saline; RCBP = Rhodnius calcium-binding protein; rRHBP = reconstituted heme-RHBP; RHBP = Rhodnius heme-binding protein; Vg = vitellogenin; Vt = vitellin.
Journal of Insect Physiology, 1994
The hemolymph from the kissing bug, Rhodnius prolixus, contains two major proteins, i.e. storage protein (SP) and lipophorin (LP) in all stages of both sexes, and egg yolk main protein precursor vitellogenin (Vg) in the adult females. We studied changes in the profiles of SP, LP and Vg in the hemolymph and their synthetic activities during nymphal and adult development. Bugs were labeled in vivo with [35S]methionine, hemolymph was separated by native or SDS-PAGE, and visualized with Coomassie blue and fluorography. SP and LP are detected in the hemolymph of both sexes throughout life. Vg appears only in the hemolymph of females both immediately after adult emergence and after engorgement in starved adults. SP is observed both in the fat body and the ovary throughout the nymph and adult stages. Vg (or Vn) is detected in the adult fat body and ovary. SP synthesis is detected in every stage of development and reproduction. The synthetic activity of SP increases after engorgement and decreases during starvation in both nymphs and adults. In autogenous and blood-fed reproductive female adults SP synthesis is reduced except during a short time after emergence and after engorgement. The level of LP synthesis is lower than that of SP and Vg, but detected during the entire life span. In adult females, Vg synthesis is superior to SP synthesis both after emergence and after engorgement. Synthesis of both SP and Vg are detected in the fat body and the ovary. Both SP and LP synthesis are induced in decapitated adult females and males after JH treatment, whereas no protein synthesis is detected in acetone treated animals. Vg synthesis increases greatly after 4 days of JH treatment in adult females and is maintained at a high level for at least 1 week.
Insect Biochemistry, 1988
Abstraet--Hemolymph juvenile hormone-binding protein (JHBP) is synthesized and secreted from fat body in the adult female cockroach, Leucophaea maderae. The data in this paper suggest it is initially secreted from the fat body as a larger peptide whereas data in the accompanying paper demonstrate that JHBP is apolipophorin I. Using media from cultures of fat body maintained in vitro, a JH-binding component was found that is JH III saturable, has a K D of 1.5 x 10 -8 M, binds JH III > JH II > JH I, and has a sedimentation value of 6.5S on high salt sucrose gradients. Each of these properties is identical to those of the JHBP extracted from the hemolymph. To identify the protein that bound JH, media proteins were photoatiinity labeled with 10-[ 10, I 1-3H]epoxyfarnesyl diazoacetate ([3H]EFDA). The results revealed that two media proteins bound [aH]EFDA in the absence of JH III, but not in the presence of 100-fold excess JH III. The molecular weights of the two media peptides were estimated by SDS-PAGE to be 275,000 and 220,000.
Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient bhematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. b-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPCmediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to b-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, b-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.
2010
Triatomine insects are obligatory blood-feeders that detoxify most of the hemoglobin-derived heme through its crystallization into hemozoin (Hz). Previous evidence demonstrates the key role of midgut perimicrovillar membranes (PMVM) on heme crystallization in triatomines. Here, we investigated some of the physico-chemical and physiological aspects of heme crystallization induced by Rhodnius prolixus PMVM. Hz formation in vitro proceeded optimally at pH 4.8 and 28 C, apparently involving three kinetically distinct mechanisms along this process. Furthermore, the insect feeding status and age affected PMVM-induced heme crystallization whereas pharmacological blockage of PMVM formation by azadirachtin, reduced hemoglobin digestion and Hz formation in vivo. Mössbauer spectrometry analyses of R. prolixus midgut showed that Hz represents the only measurable iron species found four days after a blood meal. Autocatalytic heme crystallization to Hz is revealed to be an inefficient process and this conversion is further reduced as the Hz concentration increases. Also, PMVM-derived lipids were able to induce rapid Hz formation, regardless of the diet composition. These results indicate that PMVM-driven Hz formation in R. prolixus midgut occurs at physiologically relevant physico-chemical conditions and that lipids derived from this structure play an important role in heme crystallization.
Journal of Insect Physiology, 1964
The haemolymph protein concentration of active females with developing ovaries is relatively low. It increases at the beginning and decreases towards the end of each reproduction cycle. In allatectomized or castrated females the haemolymph proteins accumulate excessively, the concentration being 3.5 to 4 times higher than that of active females. Implantation of a corpus allatum into allatectomized females induces ovarian development and causes a decrease of the haemolymph protein concentration to the level found in active females. Cardiac-allatectomized or diapausing females showed also an increased haemolymph protein concentration, although it never reached the high values observed in allatectomized females. The haemolymph protein concentration of males was constantly low.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1997
The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca 2ϩ . The hormones stimulate both the influx and the efflux of Ca 2ϩ , the higher influx probably causing an increase in intracellular [Ca 2ϩ ]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca 2ϩ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown. comp biochem physiol 117B;4:463-474, 1997. © 1997 Elsevier Science Inc.