Identification and characterization of globin genes from two lepidopteran insects, Bombyx mori and Samia cynthia ricini Shinpei Kawaoka a , Susumu Katsuma a , Yan Meng a , Nobumitsu Hayashi a , Kazuei Mita b , Toru Shimada a, ⁎ a Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan b National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan abstract article info Article history: Received 25 June 2008 Received in revised form 1 November 2008 Accepted 4 November 2008 Available online 14 November 2008 Received by J.N. Volff Keywords: Insect Bombyx mori Samia cynthia ricini Globin We describe the characterization of hemoglobin-like genes from two lepidopteran insects, Bombyx mori (Bmglobin) and Samia cynthia ricini (Scglobin). Bmglobin and Scglobin are predicted to be intracellular proteins and contain amino acids required for heme and oxygen binding. Expression profiles of two lepidopteran globins, especially Bmglobin, were different from that of other insect globins. Although other insect globins are mainly associated with the tracheal system, Bmglobin was expressed almost exclusively in the Malpighian tubules, and the strongest signal for Scglobin was detected in the fat body. Furthermore, biochemical fractionation analysis revealed that both Bmglobin and Scglobin were localized in the cytoplasm. These results suggest that each lepidopteran globin has a distinct role in the tissues in which it is expressed and that the functions of insect globins are more divergent than previously thought. © 2008 Elsevier B.V. All rights reserved. 1. Introduction For most animals, a successful gas exchange mechanism is critical for survival. The aerobic metabolism of most animals requires a sufficient supply of oxygen to the internal tissues. Small heme- proteins called hemoglobins serve as transporters and storage compartments for oxygen and contribute to the aerobic metabolism. Hemoglobins are either monomeric or polymeric proteins and typically consist of a subunit of about 150 amino acids, which contains eight α-helical segments named helices A–H that form a characteristic 3-over-3 α-helical sandwich structure (Dickerson and Geis, 1983; Berg et al., 2002; Burmester and Hankeln, 2007). The binding of O 2 is mediated by an iron ion, which is coordinated by a porphyrin group and the proximal histidine located at helix-position F8 (i.e., 8th amino acid of helix F). In insects, gas exchange is mediated mainly by trachea, which enables the diffusion of O 2 to their inner tissues. This is because respiratory proteins such as hemoglobins have been regarded as unnecessary for most insects. Only a few insects that live under low- oxygen conditions, such as in aqueous environments, have been reported to have true hemoglobins (Weber and Vinogradov, 2001). However, recently, several studies have demonstrated that hemo- globin-like proteins are part of the standard repertoire of an insect genome (Burmester and Hankeln, 2007). In Drosophila, three hemo- globin-like genes (glob1, glob2, and glob3) have been characterized (Burmester and Hankeln, 1999; Hankeln et al., 2002; Burmester et al., 2006). Hemoglobin-like genes have also been characterized from Apis mellifera (Hankeln et al., 2006), Anopheles gambiae (Burmester et al., 2007), and Aedes aegypti (Burmester et al., 2007). Although the amino acid sequences of these hemoglobin genes are highly diverse, the amino acids required for heme- and oxygen-binding are conserved. The insect hemoglobins recently identified are intracellular proteins and are associated mainly with the tracheal system, suggesting that they might be involved in respiration (Burmester and Hankeln, 2007). In this study, we characterize two lepidopteran hemoglobin-like genes from Bombyx mori (Bmglobin) and Samia cynthia ricini (Scglobin) and show that the lepidopteran globins are different from other insect globins, especially in terms of their expression patterns. 2. Materials and methods 2.1. Insects and cell lines Larvae of the inbred silkworm strain p50T, which are maintained at the University of Tokyo, were reared as described previously (Kawaoka et al., 2008). BmN cells were cultured at 27 °C in IPL-41 medium (Gibco) supplemented with 10% fetal bovine serum. Larvae of the Vietnamese strain of the eri-silkworm S. c. ricini were reared as described previously (Meng et al., 2008). Gene 431 (2009) 33–38 Abbreviations: cDNA, DNA complementary to RNA; ORF, open reading frame; PCR, polymerase chain reaction; ROS, reactive oxygen species. ⁎ Corresponding author. Tel.: +81 3 5841 8130; fax: +81 3 5841 8011. E-mail address: shimada@ss.ab.a.u-tokyo.ac.jp (T. Shimada). 0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.11.004 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene