Characterization of a glutamate-cysteine ligase in Bombyx mori

Abstract

Background: Glutamate cysteine ligase (GCL) is a crucial enzyme involved in the synthesis of glutathione (GSH). Despite various studies on glutathione transferase and its essential role in detoxi cation and resistance to oxidative stress, GSH synthesis has not been described in silkworms to date. Silkworms form part of the lepidopterans that are considered as a model of agricultural pests. This study aimed to understand the GSH synthesis through the GCL in silkworms so that insecticides can be formulated to tackle agricultural pests.Methods and Results: Based on the amino acid sequence and phylogenetic tree, this GCL belonged to group 2 and designated bmGCL. Recombinant bmGCL was overproduced and puri ed to ensure homogeneity. Biochemical studies revealed that bmGCL uses ATP and Mg 2+ to ligate glutamate and cysteine, respectively. High expression levels of bmgcl mRNA and GSH were observed in the silkworm fat body after exposure to insecticides and UV-B. Moreover, we found an increase in bmgcl mRNA and GSH content during pupation in the silkworm fat body. Conclusion:In this study, we found GCL in the silkworm Bombyx mori and its biochemical properties were investigated. These observations indicate that bmGCL might play an important role in the resistance to oxidative stress in B. mori.

Characterization of glutathione‐specific gamma glutamyl cyclotransferase (ChaC) in Bombyx mori

“…Human ChaC2 is constitutively expressed (Wang et al, 2017), whereas human ChaC1 is an inducible enzyme (Kaur et al, 2017; Mungrue et al, 2009). Previously, we found that bmGCL mRNA was highly expressed in the tissues (Afrin et al, 2023); however, here we observed that bmChaC and bmGCL mRNAs had the lowest expression levels in the silk glands on Day 3 at the fifth instar. In the tissues, bmChaC and bmGCL are involved in GSH homeostasis, which is essential for protecting tissue function.…”

“…We evaluated the functional characteristics of enzymes crucial for GSH homeostasis in the silkworm B. mori . We previously identified and characterized a bmGCL essential for synthesizing GSH (Afrin et al, 2023). Based on the results of a BLAST search, there is only one GGT and Dug in the silkworm genome.…”

“…Thus, GSH homeostasis could be an essential step for detoxifying insecticides. We previously identified and characterized glutamate‑cysteine ligase (GCL), which can synthesize GSH in B. mori (Afrin et al, 2023). In the present study, we investigated the metabolic mechanism underlying GSH degradation mediated by B. mori ChaC.…”

Involvement of Glutamate Cysteine Ligase Genes in Tolerance to Emamectin Benzoate in Spodoptera frugiperda and Their Putative Regulatory Mechanisms

“…However, yeast and bacteria have only a single Gcl gene. − In insects, studies in Drosophila melanogaster revealed that DmGcl is also composed of a catalytic subunit, DmGclc, and a modifier subunit, DmGclm. , Recently, genes encoding Gclc and Gclm have been identified in Anopheles gambiae and S. litura . , However, only one Gcl gene was found in the silkworm genome . In this study, genes encoding Gclc and Gclm in FAW were cloned by RT-PCR.…”

“…23,41 However, only one Gcl gene was found in the silkworm genome. 42 In this study, genes encoding Gclc and Gclm in FAW were cloned by RT-PCR. Sequence analysis revealed that SfGclc and SfGclm contain a conserved Gcl motif and domain and share high similarity with other insect homologues.…”