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First published online March 9, 2004
Journal of Experimental Biology 207, 1387-1398 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00897
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The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme

Makoto Hirayama, Atsushi Kobiyama*, Shigeharu Kinoshita{dagger} and Shugo Watabe{ddagger}

Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan



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  		Fig. 1. Comparison of the amino acid sequences of two types of medaka Wap65, mWap65-1 and mWap65-2, with those of fish Wap65s and mammalian hemopexins, including goldfish Wap65 (Kikuchi et al., 1995Go), carp Wap65 (Kinoshita et al., 2001), rainbow trout hemopexin-like protein (Miot et al., 1996Go) and hemopexins of rabbit (Morgan et al,. 1993Go), rat (Nikkilä et al., 1991Go) and human (Altruda et al., 1985Go; Takahashi et al., 1985Go). Amino acids identical to those of mWap65-1 are shown as dots; gaps introduced to maximize the alignment are represented by hyphens. Lightly shaded boxes indicate conserved cysteine residues, and heavily shaded boxes indicate conserved histidine residues that are thought to serve as heme axial ligands (Paoli et al., 1999Go). Conserved histidine residues in all Wap65s are shown by blue boxes, and conserved histidine residues in all except for mWap65-2 by green boxes. Black arrowheads in Wap65s and hemopexins (not mWap65-2) indicate cleavage sites for secreted proteins when data are available. The predicted signal peptide regions of mWap65-1 and mWap65-2 were defined using SignalP program and are underlined. White arrowheads in mWap65-1 and mWap65-2 indicate predicted cleavage sites used for the construction of expression vectors for recombinant mWap65-1 and mWap65-2. Potential N-glycosylation sites predicted using PROSITE are shown by red boxes. The numbers in the right margin represent those of amino acids from the N-terminal methionine in premature proteins.

 


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  		Fig. 2. Phylogenetic analysis of fish Wap65s and mammalian hemopexins. The neighbor-joining method with Poisson distance matrix was used to infer the tree topology. Bootstrap values (%) are shown at each branch point of the tree. The scale at the bottom is in units of amino acid substitutions per site. Data are cited for goldfish Wap65 (Kikuchi et al., 1995Go), carp Wap65 (Kinoshita et al., 2001) and rainbow trout hemopexin-like protein (Miot et al., 1996Go), and hemopexins of rabbit (Morgan et al,. 1993Go), rat (Nikkilä et al., 1991Go) and human (Altruda et al., 1985Go; Takahashi et al., 1985Go).

 


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  		Fig. 3. SDS-PAGE of expressed and purified recombinant proteins of the two types of medaka Wap65, mWap65-1 and mWap65-2. (A) Purification of recombinant mWap65-1 (rWap65-1). Lane 1, control total cell lysates of Escherichia coli BL21 used as host cell; lane 2, total cell lysate after induction; lane 3, soluble fraction; lane 4, insoluble fraction; lane 5, the fraction containing solubilized rWap65-1; lane 6, eluate after GSTrap affinity purification. (B) Purification of recombinant mWap65-2 (rWap65-2). Lane 7, control total cell lysates of E. coli BL21 (DE3) used as host cell; lane 8, total cell lysate after induction; lane 9, periplasm fraction; lane 10, soluble fraction; lane 11, insoluble fraction; lane 12, eluate after HiTrap affinity purification. 10–20 µg of protein were loaded in lanes 1–8, 10 and 11, and 5 µg in lanes 9 and 12. M, molecular mass standards (kDa). The positions of rWap65-1 and rWap65-2 in SDS-PAGE gels are marked by black and white arrowheads, respectively.

 


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  		Fig. 4. SDS-PAGE (A) and immunoblotting (B,C) patterns for recombinant proteins mWap65-1 (rWap65-1) and mWap65-2 (rWap65-2), and cytosolic protein fraction (8.0 mg ml–1) and blood solution (2.8 mg ml–1) from adult medaka. 10 µg of proteins were applied per lane and specific anti-rWap65-1 rabbit antiserum (B) and anti-rWap65-2 mouse antiserum (C) were used for immunoblotting. Lanes 1, rWap65-1; lanes 2, rWap65-2; lanes 3, a cytosolic protein fraction extracted from whole individuals of medaka; lanes 4, a blood solution obtained from adult medaka. M, molecular mass standards (kDa). The marks for rWap65-1 and rWap65-2 are as in Fig. 3 and the positions of intact mWap65-1 and mWap65-2 in SDS-PAGE gels are marked by red and blue arrowheads, respectively.

 


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  		Fig. 5. SDS-PAGE (A) and immunoblotting (B,C) patterns of blood solution fractions obtained by hemin–agarose affinity chromatography. Immunoblotting used antisera specific to mWap65-1 (B) and to mWap65-2 (C). Lanes 1, blood solution of 2.8 mg ml–1 obtained from adult medaka; lanes 2, supernatant; lanes 3, eluted fraction; lanes 4, proteins still bound to hemin–agarose after elution; M, molecular mass markers (kDa). 10 µl solution/lane. Arrowheads, see Fig. 4.

 


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  		Fig. 6. RT-PCR analysis of mRNAs encoding mWap65-1 and mWap65-2, and ß-actin, from different tissues of adult medaka. After PCR, amplified products were separated on agarose gels and stained with ethidium bromide.

 


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  		Fig. 7. Quantitative RT-PCR analyses on mRNAs encoding mWap65-1 and mWap65-2, and ß-actin from embryos at various developmental stages (h.p.f.). (A) Agarose gel electrophoresis patterns for amplified products obtained by RT-PCR. Total RNAs were isolated from embryos at different developmental stages (see Table 1). The arrow indicates the stage at which the liver begins to develop. After PCR, amplified products were separated on agarose gels and stained with ethidium bromide. Numbers in the left margin represent base pairs of {phi}X174 DNAs digested with HincII. (B) Changes in relative transcription levels of mWap65-1 and mWap65-2 during ontogeny. The signal intensities of each transcript in agarose gels stained with ethidium bromide were quantified, using Electrophoresis Documentation and Analysis System 120, relative to those of ß-actin at the same stage.

 


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  		Fig. 8. Accumulated mRNA levels of mWap65-1 and mWap65-2 in livers from adult medaka acclimated to 10 and 30°C. (A) Northern blot analysis was performed for four groups each containing three individuals acclimated to either 10 or 30°C. Lanes 1–4 contained 20 µg of total RNAs from the liver of fish acclimated to 10°C, and lanes 5–8 contained those from each group of fish acclimated to 30°C. (B) The transcriptional levels of mWap65-1 and mWap65-2 relative to those of 18S rRNA. RNA blots for mWap65-1 and mWap65-2 were quantified using a Fujix BAS 1000 densitometer, and signal intensities of 18S rRNA in agarose gels stained with ethidium bromide were quantified by using the Electrophoresis Documentation and Analysis System 120. Relative mRNA levels are compared to those of 18S rRNA for the same sample; values are means ± S.D.

 


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  		Fig. 9. Changes in mWap65-1 and mWap65-2 mRNA levels in liver from adult medaka following i.p. injection of LPS. (A) Northern blot analysis for three groups each containing three individuals following injection of either LPS or saline. Lanes 1–3 and 4–6 contained 20 µg of total RNAs from fish on days 2 and 4 after LPS injection, respectively, whereas lanes 7–9 and 10–12 contained those on days 2 and 4 after saline injection, respectively. (B) The transcriptional levels of mWap65-1 and mWap65-2 relative to those of ß-actin. RNA blots were quantified using a Fujix BAS 1000 densitometer. Relative mRNA levels are compared to those of ß-actin for the same sample; values are means ± S.D.

 

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© The Company of Biologists Ltd 2004