title: Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.
abstract: In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans.
authors.affiliation: Faculty of Science, Health, Education and Engineering, GeneCology Research Centre, University of the Sunshine Coast, Queensland 4558, Australia.
authors.forename: Joseph
authors.initials: J
authors.lastname: Aizen
authors.affiliation: Faculty of Science, Health, Education and Engineering, GeneCology Research Centre, University of the Sunshine Coast, Queensland 4558, Australia.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Chandler
authors.affiliation: Fisheries and Aquaculture, Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania, Australia.
authors.forename: Quinn P
authors.initials: QP
authors.lastname: Fitzgibbon
authors.affiliation: Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Amir
authors.initials: A
authors.lastname: Sagi
authors.affiliation: Fisheries and Aquaculture, Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania, Australia.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Battaglene
authors.affiliation: Faculty of Science, Health, Education and Engineering, GeneCology Research Centre, University of the Sunshine Coast, Queensland 4558, Australia.
authors.forename: Abigail
authors.initials: A
authors.lastname: Elizur
authors.affiliation: Faculty of Science, Health, Education and Engineering, GeneCology Research Centre, University of the Sunshine Coast, Queensland 4558, Australia. Electronic address: tventura@usc.edu.au.
authors.forename: Tomer
authors.initials: T
authors.lastname: Ventura
mesh_terms: D000728:Androgens / Q000378:metabolism*; D000818:Animals; D008297:Male; D033521:Palinuridae / Q000235:genetics*; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D012733:Sex Differentiation; D046468:Sexual Development; D013737:Testis / Q000254:growth & development*
chemical_list: D000728:Androgens; D020794:Receptor Protein-Tyrosine Kinases
keywords: Decapod; Insulin-like androgenic gland hormone; Phosphorylation; Spiny lobster; Tyrosine kinase receptor