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Isolation and characterization of acid-soluble bluefin tuna ( Thunnus orientalis ) skin collagen

Isolation and characterization of acid-soluble bluefin tuna ( Thunnus orientalis ) skin collagen In this study, we isolated and characterized the acid-soluble skin collagen of Pacific bluefin tuna (PBT, Thunnus orientalis). The PBT skin collagen was composed of two α chains (α1 and α2) and one β chain. The denaturation temperature of PBT collagen was low although it was rich in proline and hydroxyproline. The primary structure of PBT skin collagen was almost identical to that of calf and salmon skin collagen; however, it differed with respect to the epitope recognition of the antibody against salmon type I collagen. These results suggest that the primary structure of skin collagen was highly conserved among animal species, although partial sequences that included the epitope structure differed among collagens. Keywords: Pacific bluefin tuna, Thunnus orientails, Collagen, Epitope, Proline, Hydroxyproline Background temperature than collagen of terrestrial animals (Nagai Collagen is a major structural protein that is widely et al., 1999; Nagai et al., 2010; Senaratne et al., 2006), distributed in animal connective tissues. The primary which is conducive for assimilation by the human digest- structure of collagen is unique as it contains a glycine- ive system. rich repeat sequence (Gly-X-Y), in which prolyl and In 2002, the first full-cycle aquaculture of Pacific bluefin hydroxyprolyl residues at the X and Y positions deter- tuna (PBT) was successfully performed at Kindai mine the triple helical secondary structure (Gordon and University, Japan (Sawada et al., 2005). Currently, more Hahn, 2010; Ramshaw et al., 1998). Collagen is widely than 40,000 cultured juveniles are available from the used in the food, cosmetic, biomedical, and pharmaceut- bioventure company, A-Marine Kindai (Wakayama, ical industries. Commercial sources of collagen are Japan). This increased supply of bluefin tuna has triggered mainly derived from mammals such as cows and pigs. research into the effective use of the unused parts of the Marine collagen is advantageous over mammalian tuna, such as its skin and organs, to avoid environmental collagen because (i) marine animals are not affected by pollution and to promote economic sufficiency. Therefore, infectious diseases such as avian influenza, bovine we have focused on PBT skin as a collagen-rich underused spongiform encephalopathy (BSE), transmissible spongi- resource for functional food. Previously, we reported that form encephalopathy (TSE), and foot and mouth disease dietary PBT skin protein and collagen hydrolysis exerts (FMD) observed in pigs and cattle, (ii) the consumption hepato-protective action in CCl -intoxicated mice (Tanaka of marine collagen is acceptable to people with religious et al., 2012). In addition, the collagen from PBT, but not restrictions, and (iii) it has a lower thermal denaturation from salmon, mackerel, and carp, also reduced HepG2 and HeLa cell growth in a dose-dependent manner, * Correspondence: kawamury@kyoto-wu.ac.jp suggesting the existence of a PBT skin collagen-specific Department of Applied Biological Chemistry, Graduate School of Agriculture, primary structure and/or higher-order structural conform- Kindai University, 3327-204 Naka-machi, Nara 631-8505, Japan ation (Han et al., 2011). However, little is known about Present address: Department of Food Nutrition and Biochemistry, Kyoto Women’s University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto characteristic feature and structural information of iso- 605-8501, Japan lated PBT skin collagen. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 2 of 8 Studies on the early life history of PBT, which The collagen sample was stored at − 20 °C until further addressed the morphological changes (Kaji et al. 1996; analysis. Miyashita et al. 2001), chemical content, enzyme activ- ities (Takii et al. 1997), and the development of the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis digestive system during PBT embryogenesis (Miyashita (SDS-PAGE) and peptide mapping et al. 1998), have provided information required for the SDS-PAGE was performed using the Tris-HCl/glycine development of mass seeding techniques. However, buffer system and 7.5% polyacrylamide gel described by laboratory-reared PBT suffer high mortality during the Laemmli (1970) using the Tris-HCl/glycine buffer rapid somatic growth stage in their early life (Sawada et system with a 7.5% resolving gel and 4% stacking gel. The al. 2005; Tanaka et al. 2007). For example, PBT collagen sample was dissolved in sample buffer (0.5 M possesses very sensitive skin, which renders its handling Tris-HCl, pH 6.8, containing 8% SDS, 30% glycerol, 0.2% difficult during rearing of this species. Over 40% PBT ju- bromophenolblue) containing 5% β-mercaptoethanol and veniles die of skin injuries that are incurred during then boiled for 5 min. Collagen samples (50 μg/well) were transportation with hand nets in the first week of trans- applied to sample wells and electrophoresed. The sepa- fer of these land-based farmed juveniles to open net rated proteins were stained with Coomassie Brilliant Blue cages (Ishibashi et al., 2009). Therefore, it is important R-250. Peptide mapping was performed as described by to understand the property of type I collagen, which is a Yata et al. (2001). The isolated collagens were digested major component of PBT skin. with lysyl endopeptidase (Wako Pure Chemicals, Japan) at In this study, we isolated skin collagen from PBT and an enzyme/substrate ratio of 1:100 (w/w). Peptides characterized certain properties. generated by the protease digestion were separated by SDS-PAGE using 7.5% gel. The separated proteins and Methods peptide were stained with Coomassie Brilliant Blue R-250. Materials Calf and salmon skin type I collagens were purchased Amino acid composition from Wako Pure Chemicals (Osaka, Japan). All chemi- The collagen sample was hydrolyzed in 6 N HC1 at cals used in this study were of the highest purity 110 °C for 24 h. The hydrolysates were analyzed using available. an L-8800 automated amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan). Isolation of PBT skin collagen PBT (24–32 days after hatching) was obtained in an un- Denaturation temperature frozen state at 4 °C within 24 h after catching the tuna As previously reported by Nomura et al. (1996), the from culture fields of Aquaculture Research Institute, denaturation temperature of PBT skin collagen in 0.5 M Uragami Station, Kindai University, Japan. The skin was acetic acid was measured using an Autopol III automatic dissected from the body and stored at − 20 °C. Bluefin polarimeter (Rudolph Research Co. Flanders, N J) at tuna skin collagen was isolated using a previously 589 nm. reported procedure (Han et al., 2011)with slight modifica- tions. All steps of the extraction was performed at 4 °C. UV-Vis spectra The skin of PBT without the muscles and scales was cut The ultraviolet absorption spectra of collagen were re- into small pieces. The pieces were soaked in 0.1 M NaOH corded using a spectrophotometer (U-0080D, HITACHI, for 24 h with stirring. The NaOH solution was changed Japan) from 190 to 400 nm. The isolated collagen was every 8 h to remove non-collagenous proteins and pig- dissolved in 0.5 M acetic acid to obtain a concentration ments. The pieces were washed with distilled water until of 0.05% (m/v). neutral pH was obtained. The pieces were then defatted with methanol/chloroform (2:3) and washed with metha- Fourier transform infrared spectroscopy (FTIR) nol and distilled water. For extracting collagen, the defat- Attenuated total reflection (ATR)-FTIR spectra of collagen ted pieces were stirred in 10 volumes (w/v) of 0.5 M acetic was obtained using a Nicolet 6700 FTIR Spectrometer acid for 24 h. Pepsin (3130 U/mg solid; Nacalai Tesque (Thermo Fisher Scientific, USA) equipped with ATR − 1 Inc. Kyoto, Japan) was then added to the supernatant accessory. Spectra were recorded from 4000 to 500 cm at − 1 (7 μg/L), and the mixture was gently stirred for 48 h. Col- adataacquisition rate of 0.5cm per point. lagen was precipitated by salting out with 25% (w/v)NaCl and centrifuged at 5000×g for 30 min. The precipitate was Cross-reactivity of PBT type I skin collagen with salmon dissolved in 0.5 M acetic acid and centrifuged (15,000×g, collagen antibody 60 min). The supernatant was dialyzed with stirring for The cross-reactivity of PBT type I skin collagen with 24 h against five changes of distilled water and lyophilized. IgG-purified guinea pig antibody against salmon type I Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 3 of 8 collagen was examined using three enzyme-linked im- munosorbent assays (ELISA). Direct ELISA ELISA plates were coated with serial dilutions of collagen in phosphate-buffered saline (PBS) to determine the linear range of the sigmoid curve. Next, the plates were blocked with blocking buffer (200 μL) (Blocking One-P, Nacalai Tesque Inc. Kyoto, Japan) for 1 h at room temperature. Horseradish peroxidase (HRP)-labeled antibody against salmon collagen (100 μL) was added at 1/1000 dilution in PBS and incubated for 1 h at room temperature. The specificity for binding with immunoglobulins of salmon collagen antibodies was previously tested using western blot (data not shown). After incubation with 3, 3′,5, 5′- tetramethylbenzidine (TMB) substrate buffer, absorbance was measured using a plate spectrophotometer at 405 nm. Sandwich ELISA ELISA plates were coated with 100 μLof10 μg/mL Fig. 1 SDS polyacrylamide gel (7.5%) electrophoretic pattern for salmon collagen antibody for 13 h at 4 °C. After block- acid-soluble collagen. M.W. Marker: molecular weight marker ing, the plates were incubated with serial dilutions of collagen (100 μL) in PBS for 1 h at room temperature. Next, HRP-labeled salmon collagen antibody (100 μL) acid in the PBT skin collagen with a content of 27.58%. was added at 1/1000 dilution in PBS and incubated for This is similar to the glycine content of calf (Giraud-Guille 1 h at room temperature. The colorimetric method was et al., 2000) and salmon skin gelatin (Arnesen and performed as mentioned above using the TMB substrate. Gildberg, 2007). In addition, PBT skin collagen had high content of proline, alanine, and arginine; however, cysteine Inhibition ELISA and phenylalanine were not detected. Serial dilutions of collagen were coated on ELISA plates. After coating, the plate was incubated for 1 h at room Table 1 Amino acid compositions of bluefin tuna skin type I temperature (200 μL). Pre-incubated (1 h at room collagen temperature) dilutions of a salmon collagen and HRP- Amino acid % labeled salmon collagen antibody were added and incubated Hydroxyproline 6.41 for 1 h at room temperature. The colorimetric method was performed as mentioned above using the TMB substrate. Aspartic acid 3.95 Threonine 2.49 Results Serine 3.41 Isolation of PBT skin collagen Glutamic acid 6.81 In this study, the acid-soluble skin collagen of PBT was Proline 10.45 isolated. The final protein recovery rate of the PBT skin Glycine 27.58 collagen was 2.1 g/100 g and the dry yield was 5.4%. The isolated PBT skin collagen was analyzed using SDS- Alanine 9.68 PAGE. The separation pattern shows that PBT skin Valine 2.11 collagen was composed of two α chains (α1 and α2) and Methionine 1.29 one β chain similar to calf and salmon collagen (Fig. 1). Isoleucine 1.15 The estimated molecular weights for the α1 and α2 Leucine 2.14 chains were approximately 120 and 112 kDa, respectively, Tyrosine 0.35 which is similar to previous observations (Nalinanon et al., 2007). Hydroxylysine 1.16 Lysine 2.63 Amino acid composition of PBT skin collagen Histidine 0.79 Table 1 shows the amino acid composition of the PBT Arginine 9.90 skin collagen. Glycine was the most abundant amino Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 4 of 8 UV-Vis spectra of PBT skin collagen Figure 2 shows the UV-Vis spectra of PBT skin collagens scanned at 190–590 nm. The major peak was observed at 238 nm. There was also a swell distribution between 250 and 280 nm. Denaturation temperature of PBT skin collagen As shown in Fig. 3, the change in optical rotation of PBT skin collagen in solution started at 20 °C and finished at 29 °C. Thus the denaturation temperature (mid-point, Tm) of the PBT skin collagen was estimated as 24.5 °C. Peptide mapping Peptide mapping was performed to compare the primary Fig. 3 Denaturation curve of PBT skin collagen estimated from structure of PBT skin collagen with calf and salmon skin col- specific rotation lagen. The electrophoretic patterns of lysyl endopeptidase- digested PBT, calf, and salmon skin collagen were observed on a 7.5% denaturing polyacrylamide gel. As shown in Fig. 4, sandwich ELISA, suggesting that its epitope structure the electrophoretic pattern of PBT skin collagen was similar differed from that of salmon collagen (Fig. 5b). The PBT to those of calf and salmon skin collagen, indicating that the collagen recognized the antibody in inhibition ELISA, al- cleavage site of PBT skin collagen by lysyl endopeptidase though the reactivity to the antibody was appreciably was almost identical to those of calf and salmon skin weaker compared to that of salmon collagen (Fig. 5c). collagen. The difference in cross-reactivity of PBT and salmon col- lagen might reflect the variations in epitope recognition Cross-reactivity of PBT type I skin collagen with salmon of the antibody. collagen antibody To compare the partial sequences and higher-order FTIR spectra of PBT skin collagen structure of PBT skin collagen with calf, and salmon skin Figure 6 shows the FTIR spectra of PBT and calf skin collagen, direct, sandwich, and inhibition ELISA were collagen. The spectra of PBT skin collagen were roughly established. We examined cross-reactivity between the similar to those of calf collagen. The spectra of PBT dis- isolated PBT skin collagen and IgG-purified guinea pig persions demonstrated a characteristic pattern reflecting antibody against salmon type I collagen. As shown in Fig. 4, the calibration ranges established using direct, sandwich, and inhibition ELISA were 10–1000, 10– 10,000, and 10–100,000 ng/mL, respectively. In the direct ELISA, the PBT skin collagen reacted with the antibody against salmon type I collagen, but the reactiv- ity was almost similar to that of calf collagen (Fig. 5a). In addition, the PBT skin collagen was not detected in Fig. 4 Peptide maps of lysyl endopeptidase digests of PBT, calf, and Fig. 2 Ultraviolet spectra of PBT skin collagen salmon skin collagens. M.W. Marker: molecular weight marker Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 5 of 8 Fig. 5 Determination of the antibody reactivities of PBT, calf, and salmon collagen. a Direct ELISA, b sandwich ELISA, and c inhibition ELISA. An IgG-purified guinea pig antibody against salmon type I collagen was used. All ELISA were performed in triplicate and the data were expressed as the mean value − 1 the amide I band at 1657 cm , the amide II band at structure of the protein, and the amide III band − 1 − 1 1553 cm , and amide III band at 1241 cm , derived demonstrated the existence of a helical structure from C=O stretching, N–H bending vibrations, and C– (Muyonga et al., 2004, 2004). These results suggest H stretching (Payne and Veis, 1988), respectively. The the existence of helical arrangements in the extracted amide I band, which is associated with the secondary PBT collagen. Fig. 6 FTIR spectra of PBT skin collagen Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 6 of 8 Discussion Tryptophan and phenylalanine are not present in the In this study, we isolated and characterized certain prop- PBT collagen and the tyrosine content was 0.35%. Be- erties of PBT skin collagen. The PBT skin collagen was cause, it is generally considered that most proteins that composed of two α chains (α1 and α2) and one β chain. absorb at 280 nm of the UV-Vis spectra contain tyro- This result is similar to previous reports on collagen sine, tryptophan, and phenylalanine, the absorption peak characteristics of other fish species (Tan and Chang at 280 nm was weak. The major peak at 238 nm was 2018; Muyonga et al., 2004; Yata et al., 2001). UV-vis slightly different from the skin collagen of largefin long- and FTIR spectra of PBT skin collagen resembled that of barbel (Zhang et al., 2009) at 232 nm and collagen of type I collagen reported previously. All these data sug- abalone gastropod muscle (Dong et al., 2012) at 233 nm. gest that the isolated collagen is a typical type I collagen. These differences might be due to differences in amino In the present study, we did not perform proximate ana- acid content between PBT collagen and other collagen. lysis of fish skin during the isolation process. The pos- The electrophoretic patterns of lysyl endopeptidase- sible differences in the yield obtained during the digested PBT was similar to those of calf and salmon isolation process between these species are a limitation skin collagen as well as the electrophoretic pattern for of this study. acid-soluble collagen. Therefore, the primary structure The denaturation temperature of the PBT skin colla- of PBT skin collagen, including the cleavage site by lysyl gen was lower than that of other fish collagen. The endopeptidase, was almost identical to that of calf and thermal denaturation temperature of collagen is related salmon skin collagen. However, the cross-reactivity of to the proline and hydroxyproline content (Wong, PBT type I skin collagen with salmon collagen antibody 1989). The Pro and Hyp content in PBT skin type I was weak. The difference in cross-reactivity of PBT and collagen were 10.5 and 6.4%, respectively; the ratio of Pro salmon collagen might reflect the variations in epitope to Hyp in PBT is higher than that in salmon (Arnesen and recognition of the antibody. These results suggest that Gildberg, 2007), big eye snapper (Kittiphattanabawon et although the primary structure of collagen type I is al., 2005), and skate (Hwang et al., 2007). However, the highly conserved in animal species, the partial sequences thermal denaturation temperature of PBT skin collagen that include the epitope structure differ significantly. An was lower than that of salmon (28.7 °C), torafugu, and antibody against PBT collagen is required for more skate (28.8 °C). accurate characterization of tuna collagen. Previous studies have revealed the primary structure of type I and II procollagen α1 chain in some fishes (Saito Further studies et al., 2001; Hwang et al., 2006; Zhang et al., 2016). We Most fish collagens are composed of two α1 and one α2 cloned the cDNA for PBT procollagen α1 (I) (Tanaka et chains (Gómez-Guillén et al., 2002; Muyonga et al., al., 2014) and predicted that the PBT procollagen α1 (I) 2004). Piez (1965) reported that cod skin collagen has might contain high numbers of Gly-Gly sequences (Gly- three variants of α chains (α1, α2, and α3) that differ in Gly and Gly-Gly-Gly) in the triple-helical region. The amino acid composition. Subsequently, the α3 chain was number of Gly-Gly sequences in PBT procollagen α1(I) identified in collagen of other fish skin. Although the was 14, whereas the number in zebrafish, rainbow trout, PBT skin collagen may contain the α3 chain, its presence and torafugu were 4, 22, and 11, respectively. Since Gly is was not determined using ion exchange chromatography the smallest amino acid, the Gly-Gly sequence likely con- in this study. Therefore, further studies are required to tributes to the partial skew in the triple helix structure elucidate this point. and the decrease in thermal stability. While the PBT pro- In the present study, we did not calculate the extrac- collagen α1 (I) contains a high number of Gly-Gly se- tion efficiency of skin collagen halfway during the quence, it is not the highest among fish procollagen α1(I) extraction process. However, this efficiency will be calcu- reported previously. Thus, further rationalization for the lated by determining the hydroxyproline content in the low thermal stability of PBT skin collagen is required. In sample in our next study. In addition, the proximate addition, two Ser residues (1253 and 1270) that play a analysis of fish skin and the yield during the isolation crucial role in the interactions of the procollagen α chains process was not performed. The differences in the yield (Dion and Myers, 1987) were not found in the C-terminal obtained during the isolation process between these region of the PBT procollagen α1 (I) chain. This indicated animal species are a limitation of this study. that PBT collagen might easily accrue distortion in its In addition, type I collagen has been identified as cross- protein structure, which might contribute to its low de- reactive allergen for fish allergies (Hamada et al., 2001). naturation temperature. PBT possesses delicate skin, Although the difference in cross-reactivity of PBT and which renders handling difficult during rearing this salmon collagen was showed in this study, Kobayashi et al. species. The primary structure of the PBT skin collagen (2016) clarified that pooled serum obtained from patients could possibly explain the sensitive nature of its skin. with fish collagen-specific allergies exhibited IgE reactivity Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 7 of 8 to extracts from Atlantic salmon (Salmo salar)and School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan. ADEKA Corporation, 7-2-35 Higashi-ogu, Arakawa-ku, Tokyo 116-8554, Japan. yellowfin tuna (Thunnus albacares) by direct and inhib- Aquaculture Research Institute, Uragami Station, Kindai University, 468-3 ition ELISA. The cross-reactivity of bluefin tuna collagen 5 Uragami, Nachikatsuura, Higashimuro, Wakayama 649-5145, Japan. Present with salmon collagen antibody provided information rele- address: Department of Food Nutrition and Biochemistry, Kyoto Women’s University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, vant for structural studies. 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Isolation and characterization of acid-soluble bluefin tuna ( Thunnus orientalis ) skin collagen

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2018 The Author(s)
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2234-1757
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10.1186/s41240-018-0084-1
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Abstract

In this study, we isolated and characterized the acid-soluble skin collagen of Pacific bluefin tuna (PBT, Thunnus orientalis). The PBT skin collagen was composed of two α chains (α1 and α2) and one β chain. The denaturation temperature of PBT collagen was low although it was rich in proline and hydroxyproline. The primary structure of PBT skin collagen was almost identical to that of calf and salmon skin collagen; however, it differed with respect to the epitope recognition of the antibody against salmon type I collagen. These results suggest that the primary structure of skin collagen was highly conserved among animal species, although partial sequences that included the epitope structure differed among collagens. Keywords: Pacific bluefin tuna, Thunnus orientails, Collagen, Epitope, Proline, Hydroxyproline Background temperature than collagen of terrestrial animals (Nagai Collagen is a major structural protein that is widely et al., 1999; Nagai et al., 2010; Senaratne et al., 2006), distributed in animal connective tissues. The primary which is conducive for assimilation by the human digest- structure of collagen is unique as it contains a glycine- ive system. rich repeat sequence (Gly-X-Y), in which prolyl and In 2002, the first full-cycle aquaculture of Pacific bluefin hydroxyprolyl residues at the X and Y positions deter- tuna (PBT) was successfully performed at Kindai mine the triple helical secondary structure (Gordon and University, Japan (Sawada et al., 2005). Currently, more Hahn, 2010; Ramshaw et al., 1998). Collagen is widely than 40,000 cultured juveniles are available from the used in the food, cosmetic, biomedical, and pharmaceut- bioventure company, A-Marine Kindai (Wakayama, ical industries. Commercial sources of collagen are Japan). This increased supply of bluefin tuna has triggered mainly derived from mammals such as cows and pigs. research into the effective use of the unused parts of the Marine collagen is advantageous over mammalian tuna, such as its skin and organs, to avoid environmental collagen because (i) marine animals are not affected by pollution and to promote economic sufficiency. Therefore, infectious diseases such as avian influenza, bovine we have focused on PBT skin as a collagen-rich underused spongiform encephalopathy (BSE), transmissible spongi- resource for functional food. Previously, we reported that form encephalopathy (TSE), and foot and mouth disease dietary PBT skin protein and collagen hydrolysis exerts (FMD) observed in pigs and cattle, (ii) the consumption hepato-protective action in CCl -intoxicated mice (Tanaka of marine collagen is acceptable to people with religious et al., 2012). In addition, the collagen from PBT, but not restrictions, and (iii) it has a lower thermal denaturation from salmon, mackerel, and carp, also reduced HepG2 and HeLa cell growth in a dose-dependent manner, * Correspondence: kawamury@kyoto-wu.ac.jp suggesting the existence of a PBT skin collagen-specific Department of Applied Biological Chemistry, Graduate School of Agriculture, primary structure and/or higher-order structural conform- Kindai University, 3327-204 Naka-machi, Nara 631-8505, Japan ation (Han et al., 2011). However, little is known about Present address: Department of Food Nutrition and Biochemistry, Kyoto Women’s University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto characteristic feature and structural information of iso- 605-8501, Japan lated PBT skin collagen. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 2 of 8 Studies on the early life history of PBT, which The collagen sample was stored at − 20 °C until further addressed the morphological changes (Kaji et al. 1996; analysis. Miyashita et al. 2001), chemical content, enzyme activ- ities (Takii et al. 1997), and the development of the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis digestive system during PBT embryogenesis (Miyashita (SDS-PAGE) and peptide mapping et al. 1998), have provided information required for the SDS-PAGE was performed using the Tris-HCl/glycine development of mass seeding techniques. However, buffer system and 7.5% polyacrylamide gel described by laboratory-reared PBT suffer high mortality during the Laemmli (1970) using the Tris-HCl/glycine buffer rapid somatic growth stage in their early life (Sawada et system with a 7.5% resolving gel and 4% stacking gel. The al. 2005; Tanaka et al. 2007). For example, PBT collagen sample was dissolved in sample buffer (0.5 M possesses very sensitive skin, which renders its handling Tris-HCl, pH 6.8, containing 8% SDS, 30% glycerol, 0.2% difficult during rearing of this species. Over 40% PBT ju- bromophenolblue) containing 5% β-mercaptoethanol and veniles die of skin injuries that are incurred during then boiled for 5 min. Collagen samples (50 μg/well) were transportation with hand nets in the first week of trans- applied to sample wells and electrophoresed. The sepa- fer of these land-based farmed juveniles to open net rated proteins were stained with Coomassie Brilliant Blue cages (Ishibashi et al., 2009). Therefore, it is important R-250. Peptide mapping was performed as described by to understand the property of type I collagen, which is a Yata et al. (2001). The isolated collagens were digested major component of PBT skin. with lysyl endopeptidase (Wako Pure Chemicals, Japan) at In this study, we isolated skin collagen from PBT and an enzyme/substrate ratio of 1:100 (w/w). Peptides characterized certain properties. generated by the protease digestion were separated by SDS-PAGE using 7.5% gel. The separated proteins and Methods peptide were stained with Coomassie Brilliant Blue R-250. Materials Calf and salmon skin type I collagens were purchased Amino acid composition from Wako Pure Chemicals (Osaka, Japan). All chemi- The collagen sample was hydrolyzed in 6 N HC1 at cals used in this study were of the highest purity 110 °C for 24 h. The hydrolysates were analyzed using available. an L-8800 automated amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan). Isolation of PBT skin collagen PBT (24–32 days after hatching) was obtained in an un- Denaturation temperature frozen state at 4 °C within 24 h after catching the tuna As previously reported by Nomura et al. (1996), the from culture fields of Aquaculture Research Institute, denaturation temperature of PBT skin collagen in 0.5 M Uragami Station, Kindai University, Japan. The skin was acetic acid was measured using an Autopol III automatic dissected from the body and stored at − 20 °C. Bluefin polarimeter (Rudolph Research Co. Flanders, N J) at tuna skin collagen was isolated using a previously 589 nm. reported procedure (Han et al., 2011)with slight modifica- tions. All steps of the extraction was performed at 4 °C. UV-Vis spectra The skin of PBT without the muscles and scales was cut The ultraviolet absorption spectra of collagen were re- into small pieces. The pieces were soaked in 0.1 M NaOH corded using a spectrophotometer (U-0080D, HITACHI, for 24 h with stirring. The NaOH solution was changed Japan) from 190 to 400 nm. The isolated collagen was every 8 h to remove non-collagenous proteins and pig- dissolved in 0.5 M acetic acid to obtain a concentration ments. The pieces were washed with distilled water until of 0.05% (m/v). neutral pH was obtained. The pieces were then defatted with methanol/chloroform (2:3) and washed with metha- Fourier transform infrared spectroscopy (FTIR) nol and distilled water. For extracting collagen, the defat- Attenuated total reflection (ATR)-FTIR spectra of collagen ted pieces were stirred in 10 volumes (w/v) of 0.5 M acetic was obtained using a Nicolet 6700 FTIR Spectrometer acid for 24 h. Pepsin (3130 U/mg solid; Nacalai Tesque (Thermo Fisher Scientific, USA) equipped with ATR − 1 Inc. Kyoto, Japan) was then added to the supernatant accessory. Spectra were recorded from 4000 to 500 cm at − 1 (7 μg/L), and the mixture was gently stirred for 48 h. Col- adataacquisition rate of 0.5cm per point. lagen was precipitated by salting out with 25% (w/v)NaCl and centrifuged at 5000×g for 30 min. The precipitate was Cross-reactivity of PBT type I skin collagen with salmon dissolved in 0.5 M acetic acid and centrifuged (15,000×g, collagen antibody 60 min). The supernatant was dialyzed with stirring for The cross-reactivity of PBT type I skin collagen with 24 h against five changes of distilled water and lyophilized. IgG-purified guinea pig antibody against salmon type I Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 3 of 8 collagen was examined using three enzyme-linked im- munosorbent assays (ELISA). Direct ELISA ELISA plates were coated with serial dilutions of collagen in phosphate-buffered saline (PBS) to determine the linear range of the sigmoid curve. Next, the plates were blocked with blocking buffer (200 μL) (Blocking One-P, Nacalai Tesque Inc. Kyoto, Japan) for 1 h at room temperature. Horseradish peroxidase (HRP)-labeled antibody against salmon collagen (100 μL) was added at 1/1000 dilution in PBS and incubated for 1 h at room temperature. The specificity for binding with immunoglobulins of salmon collagen antibodies was previously tested using western blot (data not shown). After incubation with 3, 3′,5, 5′- tetramethylbenzidine (TMB) substrate buffer, absorbance was measured using a plate spectrophotometer at 405 nm. Sandwich ELISA ELISA plates were coated with 100 μLof10 μg/mL Fig. 1 SDS polyacrylamide gel (7.5%) electrophoretic pattern for salmon collagen antibody for 13 h at 4 °C. After block- acid-soluble collagen. M.W. Marker: molecular weight marker ing, the plates were incubated with serial dilutions of collagen (100 μL) in PBS for 1 h at room temperature. Next, HRP-labeled salmon collagen antibody (100 μL) acid in the PBT skin collagen with a content of 27.58%. was added at 1/1000 dilution in PBS and incubated for This is similar to the glycine content of calf (Giraud-Guille 1 h at room temperature. The colorimetric method was et al., 2000) and salmon skin gelatin (Arnesen and performed as mentioned above using the TMB substrate. Gildberg, 2007). In addition, PBT skin collagen had high content of proline, alanine, and arginine; however, cysteine Inhibition ELISA and phenylalanine were not detected. Serial dilutions of collagen were coated on ELISA plates. After coating, the plate was incubated for 1 h at room Table 1 Amino acid compositions of bluefin tuna skin type I temperature (200 μL). Pre-incubated (1 h at room collagen temperature) dilutions of a salmon collagen and HRP- Amino acid % labeled salmon collagen antibody were added and incubated Hydroxyproline 6.41 for 1 h at room temperature. The colorimetric method was performed as mentioned above using the TMB substrate. Aspartic acid 3.95 Threonine 2.49 Results Serine 3.41 Isolation of PBT skin collagen Glutamic acid 6.81 In this study, the acid-soluble skin collagen of PBT was Proline 10.45 isolated. The final protein recovery rate of the PBT skin Glycine 27.58 collagen was 2.1 g/100 g and the dry yield was 5.4%. The isolated PBT skin collagen was analyzed using SDS- Alanine 9.68 PAGE. The separation pattern shows that PBT skin Valine 2.11 collagen was composed of two α chains (α1 and α2) and Methionine 1.29 one β chain similar to calf and salmon collagen (Fig. 1). Isoleucine 1.15 The estimated molecular weights for the α1 and α2 Leucine 2.14 chains were approximately 120 and 112 kDa, respectively, Tyrosine 0.35 which is similar to previous observations (Nalinanon et al., 2007). Hydroxylysine 1.16 Lysine 2.63 Amino acid composition of PBT skin collagen Histidine 0.79 Table 1 shows the amino acid composition of the PBT Arginine 9.90 skin collagen. Glycine was the most abundant amino Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 4 of 8 UV-Vis spectra of PBT skin collagen Figure 2 shows the UV-Vis spectra of PBT skin collagens scanned at 190–590 nm. The major peak was observed at 238 nm. There was also a swell distribution between 250 and 280 nm. Denaturation temperature of PBT skin collagen As shown in Fig. 3, the change in optical rotation of PBT skin collagen in solution started at 20 °C and finished at 29 °C. Thus the denaturation temperature (mid-point, Tm) of the PBT skin collagen was estimated as 24.5 °C. Peptide mapping Peptide mapping was performed to compare the primary Fig. 3 Denaturation curve of PBT skin collagen estimated from structure of PBT skin collagen with calf and salmon skin col- specific rotation lagen. The electrophoretic patterns of lysyl endopeptidase- digested PBT, calf, and salmon skin collagen were observed on a 7.5% denaturing polyacrylamide gel. As shown in Fig. 4, sandwich ELISA, suggesting that its epitope structure the electrophoretic pattern of PBT skin collagen was similar differed from that of salmon collagen (Fig. 5b). The PBT to those of calf and salmon skin collagen, indicating that the collagen recognized the antibody in inhibition ELISA, al- cleavage site of PBT skin collagen by lysyl endopeptidase though the reactivity to the antibody was appreciably was almost identical to those of calf and salmon skin weaker compared to that of salmon collagen (Fig. 5c). collagen. The difference in cross-reactivity of PBT and salmon col- lagen might reflect the variations in epitope recognition Cross-reactivity of PBT type I skin collagen with salmon of the antibody. collagen antibody To compare the partial sequences and higher-order FTIR spectra of PBT skin collagen structure of PBT skin collagen with calf, and salmon skin Figure 6 shows the FTIR spectra of PBT and calf skin collagen, direct, sandwich, and inhibition ELISA were collagen. The spectra of PBT skin collagen were roughly established. We examined cross-reactivity between the similar to those of calf collagen. The spectra of PBT dis- isolated PBT skin collagen and IgG-purified guinea pig persions demonstrated a characteristic pattern reflecting antibody against salmon type I collagen. As shown in Fig. 4, the calibration ranges established using direct, sandwich, and inhibition ELISA were 10–1000, 10– 10,000, and 10–100,000 ng/mL, respectively. In the direct ELISA, the PBT skin collagen reacted with the antibody against salmon type I collagen, but the reactiv- ity was almost similar to that of calf collagen (Fig. 5a). In addition, the PBT skin collagen was not detected in Fig. 4 Peptide maps of lysyl endopeptidase digests of PBT, calf, and Fig. 2 Ultraviolet spectra of PBT skin collagen salmon skin collagens. M.W. Marker: molecular weight marker Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 5 of 8 Fig. 5 Determination of the antibody reactivities of PBT, calf, and salmon collagen. a Direct ELISA, b sandwich ELISA, and c inhibition ELISA. An IgG-purified guinea pig antibody against salmon type I collagen was used. All ELISA were performed in triplicate and the data were expressed as the mean value − 1 the amide I band at 1657 cm , the amide II band at structure of the protein, and the amide III band − 1 − 1 1553 cm , and amide III band at 1241 cm , derived demonstrated the existence of a helical structure from C=O stretching, N–H bending vibrations, and C– (Muyonga et al., 2004, 2004). These results suggest H stretching (Payne and Veis, 1988), respectively. The the existence of helical arrangements in the extracted amide I band, which is associated with the secondary PBT collagen. Fig. 6 FTIR spectra of PBT skin collagen Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 6 of 8 Discussion Tryptophan and phenylalanine are not present in the In this study, we isolated and characterized certain prop- PBT collagen and the tyrosine content was 0.35%. Be- erties of PBT skin collagen. The PBT skin collagen was cause, it is generally considered that most proteins that composed of two α chains (α1 and α2) and one β chain. absorb at 280 nm of the UV-Vis spectra contain tyro- This result is similar to previous reports on collagen sine, tryptophan, and phenylalanine, the absorption peak characteristics of other fish species (Tan and Chang at 280 nm was weak. The major peak at 238 nm was 2018; Muyonga et al., 2004; Yata et al., 2001). UV-vis slightly different from the skin collagen of largefin long- and FTIR spectra of PBT skin collagen resembled that of barbel (Zhang et al., 2009) at 232 nm and collagen of type I collagen reported previously. All these data sug- abalone gastropod muscle (Dong et al., 2012) at 233 nm. gest that the isolated collagen is a typical type I collagen. These differences might be due to differences in amino In the present study, we did not perform proximate ana- acid content between PBT collagen and other collagen. lysis of fish skin during the isolation process. The pos- The electrophoretic patterns of lysyl endopeptidase- sible differences in the yield obtained during the digested PBT was similar to those of calf and salmon isolation process between these species are a limitation skin collagen as well as the electrophoretic pattern for of this study. acid-soluble collagen. Therefore, the primary structure The denaturation temperature of the PBT skin colla- of PBT skin collagen, including the cleavage site by lysyl gen was lower than that of other fish collagen. The endopeptidase, was almost identical to that of calf and thermal denaturation temperature of collagen is related salmon skin collagen. However, the cross-reactivity of to the proline and hydroxyproline content (Wong, PBT type I skin collagen with salmon collagen antibody 1989). The Pro and Hyp content in PBT skin type I was weak. The difference in cross-reactivity of PBT and collagen were 10.5 and 6.4%, respectively; the ratio of Pro salmon collagen might reflect the variations in epitope to Hyp in PBT is higher than that in salmon (Arnesen and recognition of the antibody. These results suggest that Gildberg, 2007), big eye snapper (Kittiphattanabawon et although the primary structure of collagen type I is al., 2005), and skate (Hwang et al., 2007). However, the highly conserved in animal species, the partial sequences thermal denaturation temperature of PBT skin collagen that include the epitope structure differ significantly. An was lower than that of salmon (28.7 °C), torafugu, and antibody against PBT collagen is required for more skate (28.8 °C). accurate characterization of tuna collagen. Previous studies have revealed the primary structure of type I and II procollagen α1 chain in some fishes (Saito Further studies et al., 2001; Hwang et al., 2006; Zhang et al., 2016). We Most fish collagens are composed of two α1 and one α2 cloned the cDNA for PBT procollagen α1 (I) (Tanaka et chains (Gómez-Guillén et al., 2002; Muyonga et al., al., 2014) and predicted that the PBT procollagen α1 (I) 2004). Piez (1965) reported that cod skin collagen has might contain high numbers of Gly-Gly sequences (Gly- three variants of α chains (α1, α2, and α3) that differ in Gly and Gly-Gly-Gly) in the triple-helical region. The amino acid composition. Subsequently, the α3 chain was number of Gly-Gly sequences in PBT procollagen α1(I) identified in collagen of other fish skin. Although the was 14, whereas the number in zebrafish, rainbow trout, PBT skin collagen may contain the α3 chain, its presence and torafugu were 4, 22, and 11, respectively. Since Gly is was not determined using ion exchange chromatography the smallest amino acid, the Gly-Gly sequence likely con- in this study. Therefore, further studies are required to tributes to the partial skew in the triple helix structure elucidate this point. and the decrease in thermal stability. While the PBT pro- In the present study, we did not calculate the extrac- collagen α1 (I) contains a high number of Gly-Gly se- tion efficiency of skin collagen halfway during the quence, it is not the highest among fish procollagen α1(I) extraction process. However, this efficiency will be calcu- reported previously. Thus, further rationalization for the lated by determining the hydroxyproline content in the low thermal stability of PBT skin collagen is required. In sample in our next study. In addition, the proximate addition, two Ser residues (1253 and 1270) that play a analysis of fish skin and the yield during the isolation crucial role in the interactions of the procollagen α chains process was not performed. The differences in the yield (Dion and Myers, 1987) were not found in the C-terminal obtained during the isolation process between these region of the PBT procollagen α1 (I) chain. This indicated animal species are a limitation of this study. that PBT collagen might easily accrue distortion in its In addition, type I collagen has been identified as cross- protein structure, which might contribute to its low de- reactive allergen for fish allergies (Hamada et al., 2001). naturation temperature. PBT possesses delicate skin, Although the difference in cross-reactivity of PBT and which renders handling difficult during rearing this salmon collagen was showed in this study, Kobayashi et al. species. The primary structure of the PBT skin collagen (2016) clarified that pooled serum obtained from patients could possibly explain the sensitive nature of its skin. with fish collagen-specific allergies exhibited IgE reactivity Tanaka et al. Fisheries and Aquatic Sciences (2018) 21:7 Page 7 of 8 to extracts from Atlantic salmon (Salmo salar)and School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan. ADEKA Corporation, 7-2-35 Higashi-ogu, Arakawa-ku, Tokyo 116-8554, Japan. yellowfin tuna (Thunnus albacares) by direct and inhib- Aquaculture Research Institute, Uragami Station, Kindai University, 468-3 ition ELISA. The cross-reactivity of bluefin tuna collagen 5 Uragami, Nachikatsuura, Higashimuro, Wakayama 649-5145, Japan. Present with salmon collagen antibody provided information rele- address: Department of Food Nutrition and Biochemistry, Kyoto Women’s University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, vant for structural studies. Therefore, epitope recognition Japan. by anti-collagen antibody might differ among tuna species. However, further studies are required to understand its Received: 7 November 2017 Accepted: 17 January 2018 structural integrity. References Conclusion Arnesen JA, Gildberg A. Extraction and characterisation of gelatine from Atlantic In summary, the PBT skin collagen is composed of two salmon (Salmo salar) skin. Bioresour Technol. 2007;98:53–7. Dion AS, Myers JC. COOH-terminal propeptides of the major human procollagens: α chains (α1 and α2) and one β chain. The PBT collagen structural, functional and genetic comparisons. J Mol Biol. 1987;193:127–43. has low denaturation temperature, although it is rich in Dong X, Yuan Q, Qi H, Yang J, Zhu B, Zhou D, Murata Y, Ye W. Isolation and proline and hydroxyproline. The primary structure of characterization of pepsin-soluble collagen from abalone (Haliotis discus hannai) gastropod muscle part II. Food Sci Technol Res. 2012;18:271–8. PBT skin collagen was approximately identical to that of Giraud-Guille MM, Besseau L, Chopin C, Durand P, Herbage D. Structural aspects calf and salmon skin collagen; however, it varied from of fish skin collagen which forms ordered arrays via liquid crystalline states. the others with respect to epitope recognition of the Biomaterials. 2000;21:899–906. Gómez-Guillén MC, Turnay J, Fernández-Dıaz MD, Ulmo N, Lizarbe MA, Montero antibody against salmon type I collagen. Further studies P. Structural and physical properties of gelatin extracted from different are required for understanding the specific primary or marine species: a comparative study. Food Hydrocoll. 2002;16:25–34. higher-order structure of PBT collagen. Gordon MK, Hahn RA. Collagens. Cell Tissue Res. 2010;339:247–57. Hamada Y, Nagashima Y, Shiomi K. Identification of collagen as a new fish Abbreviations allergen. Biosci Biotechnol Biochem. 2001;65:285–91. BSE: Bovine spongiform encephalopathy; ELISA: Enzyme-linked immunosorbent Han SH, Uzawa Y, Moriyama T, Kawamura Y. Effect of collagen and collagen peptides assays; FMD: Foot and mouth disease; PBS: Phosphate-buffered saline; from bluefin tuna abdominal skin on cancer cells. Health. 2011;3:129–34. PBT: Pacific bluefin tuna; SDS-PAGE: Sulfate-polyacrylamide gel electrophoresis; Hwang JH, Mizuta S, Yokoyama Y, Yoshinaka R. Purification and characterization TSE: Transmissible spongiform encephalopathy of molecular species of collagen in the skin of skate (Raja kenojei). Food Chem. 2007;100:921–5. Acknowledgements Hwang JH, Yokoyama Y, Mizuta S, Yoshinaka R. cDNA cloning and The authors would like to thank the hatchery staff of the Fish Nursery characterization of type I procollagen α1 chain in the skate Raja Kenojei. Centers, Uragami Station, Kindai University for their assistance in this study. Comp Biochem Physiol B: Biochem Mol Biol. 2006;144:1–10. IshibashiY,Honryo T,Saida K, Hagiwara A, MiyashitaS,SawadaY, Okada T, Kurata M. Funding Artificial lighting prevents high night-time mortality of juvenile Pacific bluefin tuna, This work was supported in part by a Grant-in-Aid for the Global COE Program Thunnus orientalis, caused by poor scotopic vision. Aquaculture. 2009;293:157–63. from the Ministry of Education, Culture, Sports, Science, and Technology of Kaji T, Tanaka M, Takahashi Y, Oka M, Ishibashi N. Preliminary observations on Japan and the Sapporo Bioscience Foundation. development of Pacific bluefin tuna Thunnus thynnus (Scombridae) larvae reared in the laboratory, with special reference to the digestive system. Mar Availability of data and materials Freshw Res. 1996;47:261–9. All datasets generated during and/or analyzed during the current study are Kittiphattanabawon P, Benjakul S, Visessanguan W, Nagai T, Tanaka M. available from the corresponding author on reasonable request. 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Journal

Fisheries and Aquatic Sciences – Springer Journals

Published: Dec 1, 2018

Keywords: Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology

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