Manduca sexta IRP1: molecular characterization and in vivo response to iron

Abstract

Manduca sexta IRP1 was cloned and sequenced. The deduced amino acid sequence of Manduca IRP1 shows high similarity to other IRP1 proteins. The Cys residues required as ligands for the iron sulfur cluster, as well as all residues necessary for aconitase activity are conserved in the insect protein. Purified recombinant Manduca IRP1 binds specifically to transcripts of the iron responsive element (IRE) of Manduca or human ferritin subunit mRNA. Binding activity of the recombinant protein was not influenced by the presence of β-mercaptoethanol. However, IRP/IRE binding activity of cytoplasmic extracts from fat body was decreased by reducing agents in a dose-responsive manner. Fat body IRP1/IRE binding activity was reduced for Manduca sexta larvae injected with low doses of iron, while IRP1 mRNA and protein levels remained stable. At higher iron doses, binding activity increased and stabilized. Hemolymph ferritin levels showed an inverse relationship to IRP1/IRE binding activity. These data suggest that the Manduca IRP1 is likely involved in translational control of ferritin synthesis in a manner similar to that found in vertebrates. However, factors other than iron can influence IRP/IRE interaction and hemolymph ferritin levels in insects.

Introduction

Iron is a required nutrient for all living organisms. Yet, iron can promote the formation of toxic free radicals by the Fenton reaction. Many organisms prevent toxicity, but maintain iron availability, by storing excess iron in ferritin (Theil, 1990, Andrews et al., 1992). Mammalian ferritins have been extensively studied. These proteins consist of 24 subunits configured as a hollow sphere wherein iron is stored.

Lepidopteran ferritin is ∼660 kDa, composed of several subunits and appears structurally similar to the mammalian ferritins (Nichol and Locke, 1989, Pham et al., 1996). Lepidopteran ferritin is abundant in hemolymph, and several lines of evidence imply that fat body is a source of hemolymph ferritin. Early work showed that transferrin-bound iron is taken up by the fat body and reappears in hemolymph associated with a high mass protein (Huebers et al., 1988). The deduced amino acid sequences of ferritin subunits expressed by fat body of Calpodes ethlius (S and G subunits, Nichol and Locke, 1999) and Manduca sexta ferritin light chain homologue [(LCH), Pham et al., 1996] have a leader sequences signaling secretion. The predicted N-terminal sequences of the mature proteins correspond to the amino-terminal residues of hemolymph ferritin subunits. Finally, ferritin can be visualized in the fat body endoplasmic reticulum and secretory system. Iron loading of larvae does not result in an increase in iron-loaded ferritin in fat body, but does increase ferritin in hemolymph (Locke et al., 1991).

To date, the mRNA sequences of all ferritin subunits from Lepidopterans have an iron responsive element (IRE) in the 5′-untranslated region (UTR) (Pham et al., 1996, Nichol and Locke, 1999). The mammalian ferritin mRNA IRE is a well known translational regulatory control site. In mammals, when intracellular iron concentrations are low, iron regulatory proteins, IRP1 and IRP2, bind to the IRE. IRP/IRE interaction blocks recruitment of the small ribosomal subunit to the cap-binding complex preventing ferritin synthesis (Muckenthaler et al., 1998a). When iron becomes available, IRP/IRE interaction declines and ferritin is translated (Hentze and Kuhn, 1996, Eisenstein and Blemings, 1998). IRP2 is rapidly degraded in the presence of increased iron (Iwai et al., 1998, Hanson and Leibold, 1999). However, IRP1 is unique in that when intracellular iron increases, an iron sulfur cluster (4Fe–4S) forms in the protein core that prevents IRP1/IRE interaction (Haile, 1999, Beinert et al., 1996). In mammals, the formation of the iron sulfur cluster allows IRP1 to function as an iron biosensor (Constable et al., 1992). In addition, it permits IRP1 to respond to other cell factors, such as nitric oxide and hydrogen peroxide. These compounds alter iron sulfur cluster formation and retention, and thereby up-regulate IRP1/IRE interaction and down regulate ferritin synthesis (Hanson and Leibold, 1999, Hentze and Kuhn, 1996). When the 4Fe–4S cluster is present, the protein becomes cytoplasmic aconitase. Recent work indicates that cytoplasmic aconitase is important for energy metabolism (Narahari et al., 2000).

Available evidence suggests that the ferritin mRNA IRE of insects could be a functional site for translational control of ferritin synthesis. Lepidopteran hemolymph ferritin subunit messages have a 5′-UTR IRE and the protein increases in response to iron loading (Winzerling et al., 1995). IRP1 proteins are conserved in invertebrates (Huang et al., 1996, Rothenberger et al., 1990, Muckenthaler et al., 1998b), and two IRP1s have been sequenced from Dipterans, Drosophila IRP1A and IRP1B (Muckenthaler et al., 1998b). The deduced amino acid sequences of these proteins display 86% identity, high similarity to the human IRP1, are expressed in all embryonic stages. In addition, studies of the IRE identified in Drosophila succinate dehydrogenase subunit b (Muckenthaler et al., 1998b, Kohler et al., 1995, Melefors, 1996) show that translational control of protein synthesis by IRP/IRE interaction occurs in insects.

We are studying the properties, expression and activity of Lepidopteran IRP1. We report the cloning and sequencing of the M. sexta IRP1. We show that the recombinant M. sexta IRP1 binds specifically to transcripts of the IRE. We found that IRP1 is expressed by M. sexta fat body, and that fat body IRP1 binding activity declines in the presence of β-mercaptoethanol. Neither IRP1 message or protein expression respond to iron administration. In contrast, IRP1/IRE binding activity of fat body declines in a dose-responsive manner at low iron concentrations. However, at higher iron concentrations, binding activity increases. Hemolymph ferritin levels correlate inversely with fat body IRP1/IRE binding activity.