The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme

Abstract

Full-length cDNA clones encoding two types of hemopexin-like protein, mWap65-1 and mWap65-2, were isolated from the HNI inbred line of medaka Oryzias latipes. The deduced amino acid sequence of mWap65-2 resembled mammalian hemopexins more closely than that of mWap65-1. Histidine residues required for the high affinity of hemopexins for hemes were conserved in mWap65-2, but not in mWap65-1. Surprisingly, mWap65-1, but not mWap65-2, showed heme-binding ability as revealed by hemin-agarose affinity chromatography, even though mWap65-1 lacked the essential histidine residues. Furthermore, RT-PCR analysis of different tissues demonstrated that the transcripts of mWap65-2 were restricted to liver, whereas those of mWap65-1 were found in various tissues including liver, eye, heart and brain. Quantitative RT-PCR revealed that transcripts of mWap65-2 were expressed earlier than those of mWap65-1 during ontogeny. However, the accumulated mRNA levels of both mWap65-1 and mWap65-2 did not differ significantly in fish acclimated to either 10°C or 30°C for 5 weeks. These characteristics suggest that the two proteins have different physiological functions and that mWap65-2 is not a hemopexin.

Characterisation of the warm acclimated protein gene (wap65) in the Antarctic plunderfish (Harpagifer antarcticus)

“…This gene set has also been shown to be induced in response to immunological stimulus using LPS and hypoxia in C. auratus (Kikuchi et al, 1997;Gracey et al, 2001). However, there is no elevation in the expression of either of the two wap65 isoforms to induction by LPS in O. latipes (Hirayama et al, 2004) or to environmental temperature increases in O. latipes and T. rubripes (Hirayama et al, 2003). Indeed, so far, the correlation of increased expression levels of wap65 with increased environmental temperature have only been identified in the Cypriniformes, specifically the wap65-1 isoform.…”

“…Wap65-2 has a much more restricted tissue distribution (mainly liver), compared to wap65-1, which is present in multiple tissues in O. latipes and Takifugu (Hirayama et al, 2003;2004). In spite of the presence of the two conserved histidine residues proposed to be essential for haem binding, other fish orthologues of Wap65-2, which also contain these residues, do not show an affinity for haem (Hirayama et al, 2004). There is sufficient conservation of gene sequence between H. antarcticus wap65-2 and O. latipes wap65-2 to suggest that the two genes are functional orthologues.…”

“…Further analyses on other fish species (Oncorhynchus mykiss, Ictalurus punctatus, Danio rerio, Oryzias latipes and Takifugu rubripes) have shown that in contrast to the single isoform found in mammals, wap65 is present in duplicate in fish, presumably as a result of the teleost whole genome duplication event (Amores et al, 1998). These fish isoforms display different affinities for haem, tissue distributions and development patterns (Hirayama et al, 2003;2004;Nakaniwa et al, 2005). In contrast to the eurythermal examples quoted earlier, some animals exist within a very narrow environmental temperature envelope.…”

Environmental stress-related gene expression and blood physiological responses in olive flounder (Paralichthys olivaceus) exposed to osmotic and thermal stress

“…The oligosaccharides attached to this protein promote glycoprotein folding, contribute to proteinÁprotein interactions, regulate other ligand recognition processes, and stabilize the protein, making it resistant to proteolysis (Helenius and Aebi 2004;Ohtsubo and Marth 2006). Up to now, the role of the oligosaccharide in this glycoprotein has been unclear; however, Satoh et al (1994) reported that N-linked glycosylation is an essential part of the high affinity of human hemopexin for heme, and Hirayama et al (2004) reported that fish Wap65 binds heme.…”

“…Wap65 was first detected as a glycoprotein in the plasma of goldfish (Kikuchi et al 1993) and has since been identified in medaka (Oryzias latipes), channel catfish (Ictalurus punctatus), swordtail fish (Xiphophorus hellerii), and fugu (Takifigu rubripes) (Kikuchi et al 1995;Kinoshita et al 2001;Hirayama et al 2003Hirayama et al , 2004Nakaniwa et al 2005;Aliza et al 2008). Teleost Wap65 shares significant sequence identity with mammalian hemopexin and has been suggested to play roles in iron homeostasis, antioxidant activity, bacteriostatic defense, nerve regeneration, transfer of heme, and the expression of genes known to promote cell survival (Delanghe and Langlois 2001).…”