Circulating salmon 28- and 22-kDa insulin-like growth factor binding proteins (IGFBPs) are co-orthologs of IGFBP-1

Abstract

Circulating insulin-like growth factor binding proteins (IGFBPs) play pivotal roles in stabilizing IGFs and regulating their availability to target tissues. In the teleost circulation, three major IGFBPs are typically detected by ligand blotting with molecular masses around 20-25, 28-32 and 40-45 kDa. However, their identity is poorly established and often confused. We previously identified salmon 22-and 41-kDa forms as IGFBP-1 and-2b, respectively. In the present study, we cloned the cDNA of 28-kDa IGFBP from Chinook salmon (Oncorhynchus tshawytscha) as well as rainbow trout (O. mykiss) based on the partial N-terminal amino acid sequence of purified protein and identified it as an ortholog of IGFBP-1. Structural and phylogenetic analyses revealed that the 28-kDa IGFBP is more closely related to human IGFBP-1 and zebrafish IGFBP-1a than the previously identified salmon IGFBP-1 (i.e. 22-kDa IGFBP). We thus named salmon 28-and 22-kDa forms as IGFBP-1a and-1b, respectively. Salmon IGFBP-1a contains a potential PEST region involved in rapid protein turnover and phospholylation sites typically found in mammalian IGFBP-1, although the PEST and phospholylation scores are not as high as those of human IGFBP-1. There was a striking difference in tissue distribution patterns between subtypes; Salmon igfbp-1a was expressed in a variety of tissues while igfbp-1b was almost exclusively expressed in the liver, suggesting that IGFBP-1a has more local actions. Direct seawater exposure (osmotic stress) of Chinook salmon parr caused increases in both IGFBP-1s in plasma, while IGFBP-1b appeared to be more sensitive. The presence of two co-orthologs of IGFBP-1 in the circulation in salmon, and most likely in other telesots, provides a good opportunity to investigate subfunction partitioning of duplicated IGFBP-1 during postnatal growth. Keywords insulin-like growth factor binding protein; salmon; identification; gene duplication; subfunction partitioning circulating 28-32-kDa IGFBP in salmonids. 2. Materials and methods 2.1. Serum collection Serum was collected from spawning male Chinook salmon (Oncorhynchus tshawytscha) in the adult return pond on the University of Washington campus, Seattle, WA, in late October and early November. Fish were anesthetized in MS-222. Blood was withdrawn by syringe from the caudal veins, allowed to clot overnight at 4°C and then centrifuged at 1350 g for 30 min. Serum was stored at-80°C until use. One-year-old rainbow trout (Oncorhynchus mykiss) reared at Nanae Freshwater Experimental Station, Hokkaido University (Kameda, Hokkaido, Japan) were injected with cortisol (Sigma-Aldrich, St. Louis, MO) at a dose of 50 µg/g body weight and blood was withdrawn 8 h after injection. Serum was collected as described above and stored at-30°C until use. The experiment was carried out in accordance with the guidelines of Hokkaido University Field Science Center Animal Care and Use Committee. 2.2. Electrophoresis and Western ligand blotting Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a ...

Lack of hormonal stimulation prevents the landlocked Biwa salmon (Oncorhynchus masousubspecies) from adapting to seawater

“…Total RNA was extracted from the tissues as described in Shimizu et al (45). RNA (1.5 g) was reverse-transcribed using SuperScript VILO cDNA Synthesis kit (Invitrogen, Carlsbad, CA) in a 10-l reaction according to the manufacturer's instruction.…”

Cloning, molecular characterization and expression analysis of insulin-like growth factor binding protein-2 (IGFBP-2) cDNA in goldfish, Carassius auratus

“…These results suggested that the inhibitory effect of fish IGFBP-2 mainly do not depend on the RGD and HBD motifs, but by binding to IGFs. Because of the additional genome duplication, the duplicate copies of IGFBP-2 (IGFBP-2a and IGFBP2b) have been reported in many teleosts, including zebra fish, tetraodon, chinook salmon, and Japanese founder (Duan et al 1999;Zhou et al 2008;Rodgers et al 2008;Shimizu et al 2011;Zhang et al 2013). Although the IGFBP-2a and IGFBP-2b shared highly degree identity, they displayed district expression pattern in different tissues, embryogenesis and physiological status Shimizu et al 2011;Zhang et al 2013).…”

“…The similar result was also observed in chinook salmon. The salmon IGFBP-2b had three N-glycosylation sites, and IGFBP-2a had only one (Shimizu et al 2011). However, the N-glycosylation did not exist in all fish IGFBP-2 and the numbers of the putative N-glycosylation sites of other fish IGFBP-2 was 3(zebra fish IGFBP-2a), 1(common carp), 1(tetraodon IGFBP-2a).…”

“…It is also believed that the six IGFBPs are derived from a single protein by three rounds of gene duplication before the emergence of the teleosts (Daza et al 2011). Because of the extra round of gene duplication, duplicate copies of IGFBPs have been found in teleosts, and they display district expression pattern in different tissues and embryogenesis Zhou et al 2008;Wang et al 2009;Dai et al 2010;Shimizu et al 2011;Zhang et al 2013).…”

“…Over the past few years, IGFBP-2 cDNA had been cloned and characterized in many fish species, including zebra fish (Danio rerio) (Duan et al 1999;Zhou et al 2008), goby (Gillichthys mirabilis) (Gracey et al 2001), gilthead sea bream (Sparus aurata) (Funkenstein et al 2002), common carp (Cyprinus carpio) ), yellowtail (Seriola quinqueradiata) (Pedroso et al 2009), orange-spotted grouper (Epinephelus coioides) (Chen et al 2010), chinook salmon (Oncorhynchus tshawytscha) (Shimizu et al 2011), Atlantic croaker (Micropogonias undulatus) (Rahman and Thomas 2011), fine flounder (Paralichthys adspersus) (Safian et al 2012) and Japanese flounder (Paralichthys olivaceus) (Zhang et al 2013). Together these results, it shows that the structure of fish IGFBP-2 is conserved with highly degree to their mammalian counterparts, suggesting its conserved functions.…”