On the biosynthesis of Rhodnius prolixus heme-binding protein
Introduction
Hematophagous insects ingest a large amount of blood in a single meal. The digestion of blood produces a high concentration of heme in the midgut lumen. Free heme is a powerful generator of reactive oxygen species, which damage biological systems through oxidation of lipids, proteins and DNA (Tappel, 1955; Gutteridge and Smith, 1988; Vincent, 1989). Therefore, the utilization of blood as a source of nutrients constitutes an oxidative challenge to these insects.
Rhodnius prolixus, a hematophagous hemipteran, has several mechanisms involved in the protection against heme-induced oxidative injury (Graça-Souza et al., 1997, Paes and Oliveira, 1999, Paes et al., 2001, Oliveira et al., 2002). One of these mechanisms involves the metabolism of the hemolymphatic Rhodnius prolixus heme-binding protein (RHBP) (Oliveira et al., 1995). Apo-RHBP circulates in the hemolymph and can bind a heme molecule that eventually crosses the midgut barrier and reaches the hemocoel. Once bound to RHBP, heme is no longer capable of catalyzing the generation of free radicals (Dansa-Petretski et al., 1995).
In adult female insects, RHBP is taken up from the hemolymph by the growing oocytes through a receptor-mediated endocytosis, and targeted to constitute the yolk granules (Machado et al., 1998). In contrast to the hemolymph, where about half of the RHBP molecules can be found as apoproteins (apo-RHBP, i.e. without heme), the oocyte RHBP is saturated with heme (heme-RHBP) (Oliveira et al., 1995) and, therefore cannot play a role as an antioxidant.
During embryogenesis, egg RHBP is degraded and the released heme molecules are incorporated into the hemeproteins synthesized by the embryos (Braz et al., 2002). A reduction of heme availability, produced by changing the diet or by inhibiting its de novo biosynthesis, promotes a decrease in the number of eggs laid by the insect (Machado et al., 1998, Braz et al., 2001). Furthermore, lower levels of total RHBP in the hemolymph are observed when endogenous heme synthesis is diminished, suggesting that heme is important for RHBP production (Braz et al., 2001).
Here, we describe the cloning of the RHBP cDNA and presented some aspects of the biosynthesis of RHBP during the oogenesis of R. prolixus. A possible role of heme in the control of RHBP synthesis and secretion is discussed.
Section snippets
Animals
Insects were taken from a colony of R. prolixus maintained at 28 °C and 70% relative humidity. The insects used were normal mated females fed on rabbit blood at 3-week intervals.
RHBP purification
RHBP was purified from chorionated oocytes as described by Oliveira et al. (1995).
Antibody production
Purified RHBP was emulsified in complete Freund’s adjuvant and injected subcutaneously into the back of white rabbits. A booster of protein in incomplete Freund’s adjuvant was given after 3 weeks. Antiserum was obtained from blood
In vitro RHBP biosynthesis and secretion by Rhodnius fat body
In most insects, vitellogenin (VG) and other yolk proteins are synthesized by fat body and secreted to the hemolymph (Kunkel and Nordin, 1985). When incubated in a culture medium containing 14C-leucine, fat bodies dissected from females of R. prolixus were able to synthesize and secrete several 14C-labelled proteins (Fig. 1, lanes 1 and 2). 14C-RHBP was immunoprecipitated from the culture medium, using an anti-RHBP serum (Fig. 1, lane 3), demonstrating that the fat body is a site of RHBP
Discussion
In R. prolixus females, RHBP is taken up from the hemolymph by the growing oocytes through receptor-mediated endocytosis, being addressed to constitute the yolk granules (Machado et al., 1998). Furthermore, apo-RHBP present in the hemolymph can bind free heme that is produced by blood digestion, avoiding heme-induced oxidative damage (Dansa-Petretski et al., 1995). In this work, we cloned and studied the expression of RHBP after a blood meal in R. prolixus.
Similar to most of the yolk proteins
Acknowledgements
We wish to express our gratitude to Heloisa S.L. Coelho for her expert assistance in HPLC experiments; Liliam S.C. Gomes and Rosane O.M.M. Costa for their excellent technical assistance, José de Souza Lima Jr. and Litiane M. Rodrigues for maintaining our colony of Rhodnius prolixus.
This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Coordenação de Aperfeiçoamento do pessoal de Nível Superior (CAPES), Financiadora de
References (37)
- et al.
The action of juvenile hormone on follicle cells of Rhodnius prolixus in vitro: the effect of colchicine and cytochalasin B
Gen. Comp. Endocrinol.
(1977) - et al.
Heme-hemopexin-mediated induction of metallothionein gene expression
J. Biol. Chem.
(1992) - et al.
Heme biosynthesis and oogenesis in the blood-sucking bug Rhodnius prolixus
Insect Biochem. Mol. Biol.
(2001) - et al.
Rhodnius prolixus heme-binding protein (RHBP) is a heme source for embryonic development in the blood-sucking bug Rhodnius prolixus
Insect Biochem. Mol. Biol.
(2002) - et al.
Purification, partial characterization, and cloning of nitric oxide-carrying, heme protein (nitrophorin) from salivary glands of the blood-sucking insect Rhodnius prolixus
J. Biol. Chem.
(1995) - et al.
Antioxidant role of Rhodnius prolixus heme-binding protein
J. Biol. Chem.
(1995) - et al.
Indirect control of yolk protein genes by 20-Hydroxyecdisone in the fat body of the mosquito Aedes aegypti
Insect Biochem. Mol. Biol.
(1995) - et al.
Juvenile hormone activation of gene transcription in locust fat body
Insect Biochem. Mol. Biol.
(1996) - et al.
Regulating the fate of mRNA: the control of cellular iron metabolism
Cell
(1993) - et al.
Endocrine control of vitellogenin synthesis and vitellogenesis in Triatoma proctacta
J. Insect Physiol.
(1977)
A heme-binding protein from hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus
J. Biol. Chem.
On the pro-oxidant effects of haemozoin
FEBS Lett.
Haem as a multifunctional regulator
TIBS
Post-translational processing of myeloperoxidase is regulated by the availability of heme
Arch. Biochem. Biophys.
Cell Biology of Heme
Am. J. Med. Sci.
Expression of cytochrome P450 genes of the CYP4 family in midgut and fat body of the Tobacco Hornworm, Manduca sexta
Arch. Biochem. Biophys.
Unsaturated lipid oxidation catalyzed by hematin compounds
J. Biol. Chem.
Rapid and efficient isolation of transferrin and ferritin from Manduca sexta
Insect Biochem. Mol. Biol.
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