2024-03-29T05:51:42Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/835252022-11-17T02:08:08Zhdl_2115_20049hdl_2115_141Production of two recombinant insulin-like growth factor binding protein-1 subtypes specific to salmonidsHasegawa, RyuyaMiura, TakutoKaneko, NobutoKizaki, RyousukeOishi, GakutoTanaka, HanaeSato, MoeShimizu, MunetakaWhole genome duplicationTetraploidyPituitary cellsGrowth hormone releaseSubfunction partitioning660Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.ElsevierJournal Articleapplication/pdfhttp://hdl.handle.net/2115/83525https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/83525/1/HaseForHUSCAP.pdf0016-6480General and Comparative Endocrinology2991136062020-12-01enginfo:doi/10.1016/j.ygcen.2020.113606© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 licenseauthor