On the biosynthesis of Rhodnius prolixus heme-binding protein

Abstract

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. Titration 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.

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.