Molecular Characterization and Functional Analysis of the Dipeptidyl Peptidase IV from Venom of the Ectoparasitoid Scleroderma guani

Abstract

Dipeptidyl peptidase IV (DPPIV) is a proline-specific serine peptidase that remains poorly investigated in terms of venom composition. Here, we describe the molecular characteristics and possible functions of DPPIV as a major venom component of the ant-like bethylid ectoparasitoid, Scleroderma guani, named SgVnDPPIV. The SgVnDPPIV gene was cloned, which encodes a protein with the conserved catalytic triads and substrate binding sites of mammalian DPPIV. This venom gene is highly expressed in the venom apparatus. Recombinant SgVnDPPIV, produced in Sf9 cells using the baculovirus expression system, has high enzymatic activity, which can be efficiently inhibited by vildagliptin and sitagliptin. Functional analysis revealed that SgVnDPPIV affects genes related to detoxification, lipid synthesis and metabolism, response to stimuli, and ion exchange in pupae of Tenebrio molitor, an envenomated host of S. guani. The present work contributes towards understanding the role of venom DPPIV involved in the interaction between parasitoid wasp and its host.

Keywords: dipeptidyl peptidase IV; gene expression; parasitoid; transcriptome; venom.

Figure 1

Multiple alignment of SgVnDPPIV and DPPIVs from other insects and humans. Sgua, Scleroderma guani; Tmol, Tenebrio molitor (CAH1368919); Vbas, Vespa basalis (ABG57265); Mdem, Microplitis demolitor (XP_008553451); Hsap, Homo sapiens (P27487). The transmembrane domains of S. guani, V. basalis, M. demolitor, and H. sapiens DPPIVs are marked in red boxes. The signal peptides of T. molitor and V. basalis DPPIVs are indicated in green boxes. The catalytic triads and substrate binding sites are indicated with triangles and solid circles, respectively. The identical and highly conserved residues are shaded in black and grey, respectively.

Figure 2

Phylogenetic tree of DPPIVs from various hymenopteran species. Amino acid sequences of DPPIVs used to construct the phylogenetic tree are listed in Table S2.

Figure 3

Gene expression profiles of SgVnDPPIV detected using quantitative real-time PCR. (A) Expression profiles of SgVnDPPIV gene in various tissues of female adults. He, head; Th, thorax; Ab, abdomen derived of venom apparatus; VA, venom apparatus. (B) Expression profiles of SgVnDPPIV gene at different developmental stages. E, egg; EL, early larvae; LL, late larvae; ML, mature larvae; SL, spinning mature larvae; WP, pupae in white cocoon; YP, pupae in yellow cocoon; BP, pupae in back cocoon; A1–25, adults after 1–25 days emergence. Different superscript letters indicate statistically significant differences among different samples.

Figure 4

Enzymatic assay of recombinant SgVnDPPIV. (A) Expression of SgVnDPPIV in Sf9 cells using the baculovirus expression system. The recombinant SgVnDPPIV was purified and detected with Coomassie Brilliant Blue. (B) Enzymatic activity of gut extract of Tenebrio molitor larvae, venom of Scleroderma guani, and recombinant SgVnDPPIV. (C) Effect of inhibitors on the activity of recombinant SgVnDPPIV. Different superscript letters indicate statistically significant differences among different samples or treatments.

Figure 5

Differentially expressed genes in Tenebrio molitor pupae envenomated by SgVnDPPIV. (A) Volcano plot of differentially expressed genes identified 6 h post SgVnDPPIV injection. (B) Volcano plot of differentially expressed genes identified 24 h post SgVnDPPIV injection. (C) Venn diagram of differentially expressed genes identified 6 h and 24 h post SgVnDPPIV injection.

Figure 6

Gene ontology (GO) enrichment analysis at a broad level of differentially expressed genes in Tenebrio molitor pupae envenomated by SgVnDPPIV. (A) Enriched GO terms and differentially expressed gene number 6 h post SgVnDPPIV injection. (B) Enriched GO terms and differentially expressed gene number 24 h post SgVnDPPIV injection.