Hypothermal stress induced differential expression profiles of the immune response gene, warm-temperature-acclimation associated 65-kDa protein (Wap65), in the liver of fresh water and seawater milkfish, Chanos chanos.

The milkfish (Chanos chanos), an important aquaculture species, is intolerant to cold environments. Temperature fluctuations in the environment affect the physiological response, behavior, and survival rate of the fish. The warm-temperature-acclimation associated 65-kDa protein (Wap65) of teleosts was identified after heat shock treatment and has two isoforms. Both the isoforms were involved in the induction of immune responses in fish. They showed high degree of sequence conservation with the mammalian hemopexin and had high affinity for heme, which helped in the neutralization of free-heme and its transport to the liver. In this study, we isolated and characterized the two isoforms of wap65 genes (Ccwap65-1 and Ccwap65-2) from the liver of milkfish. The Ccwap65-1 and Ccwap65-2 are mainly expressed in livers of milkfish. In hypothermal treatment, the expression levels of Ccwap65-2 in the livers of SW and FW milkfish were up-regulated after exposure to low temperature (18 °C) for 12 h and 96 h compared to those in the normal temperature (28 °C) group, respectively. After intraperitoneal injection of lipopolysaccharide (LPS), the expression of Ccwap65-2 was elevated in both SW and FW milkfish, whereas that of Ccwap65-1 was not affected in both the groups. Thus, Ccwap65-2 expressed in the milkfish liver under hypothermal stress was identified as a novel immune biomarker. In addition, according to the transcriptome database, up-regulation of the other immune-response genes indicated increased pathogen infection status under hypothermal stress. Acute increase in the expression of hepatic Ccwap65-2 in response to pathogen infection might lead to better cold tolerance of SW milkfish compared to that of the FW individuals upon cold challenge.

The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

Molecular cloning, characterization and expression analysis of a novel wap65-1 gene from Plecoglossus altivelis.

Warm temperature acclimation associated 65-kDa protein 1 (WAP65-1) is a specific fish plasma glycoprotein that is possibly involved in various physiological or pathological processes. In this study, we obtained the cDNA and genomic DNA sequences of the Plecoglossus altivelis wap65-1 (Pawap65-1) gene. Multiple sequence alignment showed that Pawap65-1 is similar in structure to wap65-1 in fish. Phylogenetic analysis revealed that Pawap65-1 is most closely related to that of a rainbow trout. Pawap65-1 transcripts are present in various tissues and are most abundant in the liver. We expressed recombinant PaWAP65-1 in Escherichia coli and raised antiserum against it in mouse. Western blot analysis revealed that the higher molecular mass of PaWAP65-1 in blood plasma was caused by post-translational N-glycosylation. Quantitative real-time quantitative PCR (qPCR) and Western blot analysis data showed that the hepatic mRNA and blood plasma levels of PaWAP65-1 were both influenced by warm temperature acclimation and cadmium exposure, but not by Listonella anguillarum infection, hypo-osmotic, or cold temperature acclimation. In conclusion, our data reveals that PaWAP65-1 is a stress-related protein, and may play a role in fish acclimation to warm temperature and cadmium exposure.

Modulation of warm-temperature-acclimation-associated 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments.

Two paralogous isoform cDNAs of warm-temperature-acclimation-associated 65-kDa protein (Wap65-1 and Wap65-2) were isolated from the cypriniform species, mud loach (Misgurnus mizolepis), and characterized. The deduced amino acid sequences of the two mud loach Wap65 isoforms (mlWap65-1 and mlWap65-2) share moderate levels of sequence homology with their corresponding orthologues from teleosts and with human hemopexin, a possible mammalian homologue. Both isoforms display conserved features, including essential motifs and/or residues that are important for the protein structure of hemopexin. In overall, mlWap65-2 is more homologous to human hemopexin than is mlWap65-1. Both mud loach Wap65 transcripts are predominantly expressed in liver, although the transcripts are ubiquitously detectable in most tissues with variable basal expression. Both mlWap65 isoforms are differentially regulated during embryonic development, and the changes in transcript levels during embryogenesis are greater for mlWap65-2 than for mlWap65-1. The transcription of the mlWap65 genes is differentially modulated by various stimuli, including thermal changes, immune challenge (lipopolysaccharide injection or bacterial infection), and heavy metal exposure (cadmium, copper, or nickel). The isoform mlWap65-1 is more responsive to warm temperature treatments than mlWap65-2, whereas mlWap65-2 is much more strongly stimulated by immune and heavy metal challenges than is mlWap65-1. Taken together, the results of this study suggest that mud loach Wap65 isoforms are potentially involved in multiple cellular pathways and that the two mud loach Wap65 isoforms undergo functional partitioning or subfunctionalization.

Evolution of a multifunctional gene: The warm temperature acclimation protein Wap65 in the European seabass Dicentrarchus labrax.

The warm temperature acclimation protein Wap65 has been shown to be involved in temperature acclimation, in immune response as well as in development. In teleosts, two types of Wap65 proteins, Wap65-1 and Wap65-2 are found, both acting as a multifunctional agent in several biological processes. In the present study we identified both transcripts Wap65-1 and Wap65-2 for the European seabass (Dicentrarchus labrax), examined their evolutionary rate and performed selection tests. The two paralogues were shown to be under moderate positive selection indicating their evolutionary adaptation. This functional diversification was further explored through expression studies. Both transcripts were differentially expressed during development as well as in various tissues and pathogen challenges, showing that Wap65-1 and Wap65-2 have evolved diverse functions. These results direct to the hypothesis that Wap65 proteins may, similarly to heat-shock proteins, have a general role in cell physiology.

The warm temperature acclimation protein Wap65 as an immune response gene: its duplicates are differentially regulated by temperature and bacterial infections.

The warm temperature acclimation related 65kDa protein (Wap65) in teleost fish shares high structural similarities with mammalian hemopexins. Recent studies using microarray analysis indicated that this temperature acclimation protein may also be involved in immune responses. To provide evidence of its potential involvement in immune responses after bacterial infections, we have identified and characterized two types of Wap65 genes in channel catfish, referred to as Wap65-1 and Wap65-2, respectively. While Wap65-1 and Wap65-2 are both structurally similar to the mammalian hemopexins, they exhibit highly differential patterns of spatial expression. Wap65-1 was expressed in a wide range of tissues, whereas Wap65-2 was only expressed in the liver. Their regulation with warm temperature and bacterial infections was also highly different: Wap65-1 was constitutively expressed, whereas Wap65-2 was highly regulated by both warm temperature and bacterial infections, and warm temperature and bacterial infections appeared to synergistically induce the expression of Wap65-2. The great contrast of expression patterns and regulation of the two catfish Wap65 genes suggested both neofunctionalization and partitioning of their functions. Phylogenetic analysis indicated that the duplicated catfish Wap65 genes were evolved not only from whole genome duplication, but also from tandem, intrachromosomal gene duplications. Taken together, the results of this study suggest that Wap65 genes are not only important for its classical role as a warm temperature acclimation protein, but more importantly, may also function as an immune response protein.

Is 100KF an isoform of hemopexin? Immunochemical characterization of the vasoactive plasma factor 100KF.

The human vasoactive plasma factor 100KF has been proposed to play a role in minimal change disease in relapse. Since preliminary data suggested similarity between 100KF and the human plasma glycoprotein hemopexin (Hx), this study was conducted to compare 100KF with purified Hx for sequence homology, immunostaining properties in Western and dot-blot assays, ability to affect glomerular ecto-ATPase and glomerular polyanions in vitro, as well as their glomerular permeability increasing effect following alternate perfusion into the rat kidney ex vivo. 100KF was purified from normal pooled plasma according to standard chromatographic techniques, and from the same batch Hx was prepared using affinity chromatography. A second batch of Hx was prepared directly from human serum according to a standard protocol. (For comparison, additional Hx samples obtained from other centers were also included in the study.) The results show: (1) 100% homology of 100KF with plasma Hx after internal sequence analysis; (2) positive staining of the eluate with both monoclonal and polyclonal anti-Hx IgG as well as anti-100KF IgG in dot-blot assays, and similar bands on Western blotting using the same antibodies; (3) affection of glomerular polyanions and glomerular ecto-ATPase after incubation of kidney tissue with either 100KF or Hx (1.5 respectively 1.0 mg/ml; 1.0 h, 37 degrees C), as detected by computerized histochemical quantification; and (4) significant enhancement of urinary protein leakage after Hx perfusion followed by diluted rat serum into the rat kidney ex vivo (Hx: 210.65+/-49.79 microg protein leakage per min versus heat-inactivated Hx control: 112.2+/-49.18 microg per min [both n = 6]). From these data and from the observation that both Hx and 100KF activity can be inhibited by serine protease inhibitors but not by broad spectrum collagenase inhibitors, it is concluded that Hx may be closely related or identical to the active moiety of 100KF.

The warm temperature acclimation-related 65-kDa protein, Wap65, in goldfish and its gene expression.

cDNAs encoding a warm temperature acclimation-related protein (Wap65) were cloned from the muscle and hepatopancreas cDNA libraries of the warm temperature-acclimated goldfish Carassius auratus, and their nucleotide sequences containing 5'- and 3'-noncoding regions together with their polyadenylation signal were determined. The deduced amino acid sequence of Wap65 was 31% homologous to rat hemopexin. However, goldfish Wap65 lacked a few possible glycosylation sites and presumed functional histidine residues, implying that it may have different functions from hemopexin. Wap65 contained a leader peptide of 30 amino acids and a mature protein region of 415 amino acids. Southern blot analysis demonstrated that the protein is expressed by a single copy gene in the goldfish haploid genome. In RNA blot analysis using isolated cDNA clones, a single transcript of about 2.0 kilobases was detected in the hepatopancreas but not in brain, muscle, or hemocytes. The abundancy of this transcript markedly increased in the hepatopancreas as a result of warm temperature acclimation. Electrophoretic analysis of plasma proteins revealed a good correlation of plasma Wap65 levels to those of the corresponding transcript in the hepatopancreas, suggesting that serum Wap65 concentrations are regulated mainly by transcript levels in the hepatopancreas via the secretion process.

Intracellular distribution of haem after uptake by different receptors. Haem-haemopexin and haem-asialo-haemopexin.

How the interaction of haemopexin with two different receptors affects its subsequent metabolism and 'intracellular' haem transport was examined by using mesohaem-haemopexin and mesohaem-asialo-haemopexin. The physical properties of the two haem proteins, including their absorption and c.d. spectra, are similar. Binding studies in vitro showed that haem-asialo-haemopexin interacts with both the haemopexin-specific and galactose-specific receptors on liver plasma membranes, but that haem-haemopexin interacts only with the haemopexin receptor. In vivo haem-asialo-haemopexin rapidly interacts with the liver via the galactose-specific receptor, since the protein is extensively catabolized and uptake is blocked by asialofetuin. Haem iron from haem-asialo-haemopexin is not accumulated in the liver to the same extent as from intact haem-haemopexin, and the native sialylated protein is not proteolysed. Moreover, after fractionation of homogenized liver by using colloidal-silica gradients, liver-associated haem-haemopexin and haem-asialo-haemopexin produced distinctly different patterns for both protein and ligand, consistent with their uptake by two distinct receptors. These results demonstrate that the interaction of haemopexin with different receptors influences its subsequent metabolic fate and that haem iron from haem-haemopexin is efficiently conserved only if it enters the liver cell via the specific haemopexin receptor.