title: Glyceraldehyde-3-phosphate dehydrogenase is a mediator of hemocyte-spreading behavior and molecular target of immunosuppressive factor CrV1.
abstract: Cellular immunity is accompanied by hemocyte-spreading behavior, which undergoes cytoskeletal rearrangement. Polydnaviral factor CpBV-CrV1 can inhibit the hemocyte-spreading behavior and suppress host immune response of Plutella xylostella. However, host target molecule of CpBV-CrV1 that inhibits the hemocyte behavior has not been identified yet. This study used a pull-down approach to identify the target molecule of CpBV-CrV1. A protein bound to CpBV-CrV1 was co-precipitated and identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by LC-MS/MS analysis. RNA interference (RNAi) specific to GAPDH of P. xylostella was found to be able to inhibit the hemocyte-spreading behavior, while RNAi treatments with other glycolytic genes had no effect on the spreading behavior. An addition of recombinant CpBV-CrV1 on hemocyte monolayer interrupted the association between GAPDH and α-tubulin in the cytoplasm. Overlay of mutant proteins (Y492A or Y501A with tyrosine to alanine at putative GAPDH-binding site) of CpBV-CrV1 on hemocyte monolayer revealed that they could enter hemocytes unlike a mutant in the N-terminal coiled-coil domain. However, they failed to inhibit the hemocyte-spreading behavior without any binding affinity to GAPDH. These results suggest that GAPDH plays a critical role in hemocyte-spreading behavior during immune challenge as a molecular target of viral factor CpBV-CrV1.
authors.affiliation: Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea.
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea. Electronic address: hosanna@andong.ac.kr.
authors.forename: Yonggyun
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D005455:Fluorescent Antibody Technique; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000276:immunology*; D006434:Hemocytes / Q000276:immunology*; D006790:Host-Parasite Interactions / Q000502:physiology*; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology / Q000276:immunology* / Q000821:virology*; D016297:Mutagenesis, Site-Directed; D018071:Polydnaviridae / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D014764:Viral Proteins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
keywords: Coiled-coil domain; CrV1; GAPDH; Plutella xylostella; Polydnavirus