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Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus)
Food Chemistry 212 (2016) 341–348
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Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus) Yu-Xin Liu a,b, Da-Yong Zhou a,b,⇑, Dong-Dong Ma a, Yan-Fei Liu a, Dong-Mei Li a,b, Xiu-Ping Dong a,b, Ming-Qian Tan a,b, Ming Du a,b, Bei-Wei Zhu a,b,c,d,⇑ a
School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR China National Engineering Research Center of Seafood, Dalian 116034, PR China The Innovation center of Food Nutrition and Human Health, Beijing 100083, PR China d Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, Tianjin 300457, PR China b c
a r t i c l e
i n f o
Article history: Received 9 January 2016 Received in revised form 16 May 2016 Accepted 27 May 2016 Available online 28 May 2016 Keywords: Sea cucumber (Stichopus japonicus) Autolysis Collagen fibers Cathepsin L Localization Electron histochemistry
a b s t r a c t The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Sea cucumber is a type of echinoderm and one of the important cultured aquatic species in China and many other Asian countries due to its high commercial value (Wu et al., 2009). According to FAO (2013) statistics, the total world production of sea cucumber was about 232 thousand tones (http://www.fao.org/figis/servlet/ TabSelector). Sea cucumber is highly susceptible towards external stimulus. The body wall of sea cucumber undergoes a massive tissue autolysis, also called ‘‘melting” or ‘‘local degeneration”, in response to changes of external environmental factors such as temperature, salt concentration, nutrient deficiency, sunlight exposure, ultraviolet irradiation, and mechanical stimulation (Hill, 2001; Wilkie, 2002; Yang et al., 2015; Zhu et al., 2008a). Autolysis can lead to severe postharvest quality deterioration of sea cucumber, consequently cause heavy economic losses. The body wall of sea cucumber is mainly composed of mutable connective tissue (MCT) with scattered cells (Elphick, 2012; Hill, 2001; Thurmond & Trotter, 1996). In MCT, collagen fibers are ⇑ Corresponding authors at: School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR China. E-mail addresses: [email protected] (D.-Y. Zhou), [email protected] (B.-W. Zhu). http://dx.doi.org/10.1016/j.foodchem.2016.05.173 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
surrounded and separated by a microfibrillar network, which maintain the organization and provide a long-range restoring force (Thurmond and Trotter, 1996; Thurmond, Koob, Bowness, & Trotter, 1997; Wilkie, 2005). Therefore, the dramatic changes in apparent and mechanical properties of body wall of sea cucumber during autolysis must be due to the degradation of MCT. However, the in situ microstructural changes in MCT of sea cucumber during autolysis are less clear. The autolytic degradation of body wall of sea cucumber is supposed to be caused by endogenous enzymolysis of structural elements. To date, cysteine-like proteinase (Qi et al., 2007), cathepsin L-like proteinase (Zhou et al., 2014; Zhu et al., 2008b), cathepsin B-like proteinase (Sun et al., 2011) and gelatinolytic metalloproteinase (GMP) (Wu et al., 2013a) have been purified and characterized from sea cucumber (Stichopus japonicus, S. japonicus). Junqueira et al. (1980) suggested that an endogenous proteinase (maybe Ca2+-activated collagenase) in the body wall of sea cucumber (Isostichopus badionotus) could liberate proteoglycans from interaction with collagens. Hill (2001) further speculated that the autolysis of body wall of sea cucumber might be initiated by the release of proteoglycans by Ca2+-activated collagenase. Wu et al. (2013a) reported that calcium-dependent metalloproteinase from S. japonicus could degrade the collagen isolated from S. japonicus
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body wall in a temperature-dependent mode. In a previous study, we found that endogenous cysteine proteinases were partially involved in the proteolysis of noncollagenous proteins from S. japonicus body wall (Wu et al., 2013b). Autolysis is common for aquatic species. Cathepsin, a type of lysosomal cysteine proteinase, may well be the most active proteinases in the human body (Barrett & Kirschke, 1980). It is widely accepted that cathepsin is one of the most important endogenous proteinase in autolytic degradation of fish (Mukundan, Antony, & Nair, 1986). For example, cathepsin B, D, H and L were involved in the breakdown of muscle structures in several fish species (Bahuaud et al., 2010; Godiksen, Morzel, Hyldig, & Jessen, 2009). Meanwhile, cathepsin L, a representative cathepsin, was thought to be mainly responsible for the modori phenomenon during surimi production (Hu, Morioka, & Itoh, 2007; Zhong et al., 2012). Previous studies indicated that the body wall of sea cucumber is rich in cathepsins (Sun et al., 2011; Zhou et al., 2014; Zhu et al., 2008b). However, lysosomal cathepsin must be released from cells before it makes contact with extracellular substrates and degradation. To our knowledge, there is still no report on the subcellular localization and release of cathepsin in sea cucumber. In the present study, ultraviolet (UV) irradiation was used to induce the autolysis of S. japonicus, and then the changes of microstructures of collagenous tissues as well as tissue and cellular distributions of cathepsin L were investigated by histological and histochemical techniques using light microscope and electron microscopes. Through this research, the in situ changes that occur in collagen fibers, and the mechanism through which lysosomal cysteine proteinases contact with their extracellular substrates were revealed.
with 75 lL of assay buffer containing 340 mM sodium acetate, 60 mM acetic acid, 4 mM disodium EDTA, and 8 mM DTT (pH 5.5, prepared just before use) and pre-incubated at 37 °C for 2 min. Then freshly prepared substrate (75 lL) was added to the mixture to a final concentration of 25 lM. After 10 min of incubation at 37 °C, 300 lL of sodium acetate buffer (pH 4.3) containing 0.1 M chloroacetate was added to the mixture to terminate the reaction. The fluorescence of AMC liberated by hydrolysis was measured by using a spectrofluorometer (F-2700, Hitachi, Tokyo, Japan) with excitation and emission wavelengths of 380 and 460 nm, respectively. AMC was used as a standard reference. A blank sample was prepared by adding the sodium acetate buffer containing chloroacetate before the substrate. One unit of the enzyme activity (U) was defined as the amount of the activity that released 1 nM of AMC per min.
2. Materials and methods
2.6. Histological analysis with scanning electron microscopy
2.1. Chemicals
The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 1.0 cm3 sized pieces, dehydrated through a graded series of 50%, 60%, 70%, 80%, 90%, 100% ethanol, freezedried, mounted on a metal stub, coated with Pd, and imaged by using a JSM-7800F SEM (JEOL Ltd., Tokyo, Japan) which operated at 10 kV. Experiments were repeated at least three times to confirm the results, and the samples were observed carefully without any beam damage. Micrographs were taken from different spots of the same sample at similar magnification and width measurements.
Benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-b-n aphthylamide hydrochloride (Z-Phe-Arg-4MbNAHCl) was purchased from Bachem Inc. (Torrance, CA, USA). Benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-AMC), 7-amino-4-methylcoumarin (AMC), L-cysteine, dithiothreitol (DTT) and cacodylic acid sodium were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All of the other chemicals used were of analytical grade. 2.2. Biological materials Sea cucumbers of the species S. japonica, each 70 ± 10 g, were captured in the Yellow Sea near the coast of Dalian, China, and transported to the laboratory immediately in cold seawater. In laboratory, the sea cucumber was induced to autolysis by 30 min of UV irradiation (15 w, 0.5 m), and left in an air-conditioned room (the temperature was about 20 ± 2 °C) for different intervals before further experiment. 2.3. Preparation of crude cathepsin L The crude cathepsin L was extracted from body wall of S. japonicus according to a procedure described in our previous study (Zhou et al., 2014). 2.4. Assay of enzyme activity toward fluorogenic substrate Cathepsin L activity was determined according to the method of Yamashita and Konagaya (1990), by using Z-Phe-Arg-AMC as a substrate. Briefly, 150 lL of crude cathepsin L solution was mixed
2.5. Histological analysis by using light microscopy The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 0.5 cm3 sized pieces, fixed in 10% paraformaldehyde at 4 °C for 18 h, dehydrated through a graded series of 50%, 60%, 70%, 80%, 90%, 100% ethanol, vitrified in a dimethylbenzene/ethanol (v/v) series of 1:2, 1:1, 2:1, and 1:0, and embedded routinely in paraffin. Tissue sections (5 lm) were prepared by using a Leica EG1150C rotary microtome (Leica Microsystems). Hematoxylin and eosin (H&E) and Van Gieson’s (VG) stain were performed according to the standard techniques (Liu, 2004). The microstructures were observed and photographed by using an Olympus BX51TF optical microscopy & DP70 Digital Camera System (Olympus Co., Tokyo, JP).
2.7. Histochemical localization of cathepsin L by using light microscopy Light microscopic histochemical localization of cathepsin L was carried out according to the process introduced by Smith and Van Frank (1975) with slight modifications. The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 1.0 cm 1.0 cm 0.5 cm sized clumps. The tissue pieces were quick-frozen on a cryostat for 10 min, and then sectioned by using Leica CM1950 freezing microtome (Leica Microsystems) to a thickness of 10 lm. The tissue section was put onto a glass slide, dried naturally, washed twice with double distilled water, and incubated in stain fluid containing substrate. After 60 min of incubation at 37 °C, the stain fluid was discarded and the tissue section was washed three times with double distilled water. The tissue sections were observed and photographed using an Olympus BX51TF optical microscopy & DP70 Digital Camera System (Olympus Co., Tokyo, JP). Control reactions were carried out in medium containing no substrate. Substrate fluid preparation: 1 mg of Z-Phe-Arg-4MbNAHCl was dissolved in 330 lL of dimethyl sulfoxide, and then mixed with 670 lL of Tris-HCl buffer (0.05 M, pH 7.5) containing 10 mM CaCl2; Stain fluid preparation: liquid A: 400 mg of basic fuchsin was mixed with 2 mL of 12 M hydrochloric acid and stirred into a paste. The paste was mixed further with 8 mL of deionized water, heated
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until dissolved, and filtered to recover the liquid A. liquid B: 4% sodium nitrite. The same volumes of liquid A and liquid B were mixed in sealed container and left at 4 °C for 2 min. After the reaction in low temperature, the liquid became yellow, indicating the formation of hexazotized pararosaniline. One milliliter of hexazotized pararosaniline was diluted with 19 mL of Na2HPO4-NaH2PO4 buffer (0.1 M, pH 7.4) to obtain the stain fluid. The same volumes of substrate fluid and stain fluid were mixed to obtain the stain fluid containing substrate. 2.8. Histochemical localization of cathepsin L by using transmission electron microscopy Electronic-histochemical localization of cathepsin L was performed according to the previously reported procedures (Smith and Van Frank, 1975; Ryvnyak, Ryvnyak, & Tudos, 2004), with slight modifications. The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 0.5 cm 0.3 cm 0.3 cm sized pieces, fixed in 1.5% glutaraldehyde in sodium cacodylate buffer (0.05 M, pH 7.4) containing 2% sucrose at 4 °C for 3 h. The materials were washed three times with the same buffer (10 min for each time), and then incubated with the stain liquid containing substrate (same as the fluid used in Section 2.7). After being washed with sodium cacodylate buffer (0.05 M, pH 7.4) for three times, the tissue pieces were post-fixed in 1% osmic acid for 2 h at 4 °C. Osmicated samples were rinsed with 0.1 M phosphate buffer (pH 7.4), and embedded in epon. Sections (70 nm) were obtained by a LKB ultramicrotome and stained with uranyl acetate and lead citrate, and then observed and photographed using JEM-2000EX transmission electron microscopy (JEOL Ltd., Tokyo, JP). Control reactions were carried out in medium containing no substrate. 2.9. Statistical method All the tests were conducted with three replicates. Data were presented as mean ± standard deviation (SD). The statistical analysis was performed by using SPSS 16.0 software (SPSS Inc. Chicago, IL, USA). Differences between means were evaluated by one-way analysis of variance (Post Hoc test: S-N-K). Comparisons that yielded P values < 0.05 were considered significant. 3. Results and discussion 3.1. Changes in apparent properties of S. japonicus in response to UV stimuli The autolysis of sea cucumber was induced by UV irradiation as reported in our previous studies (Yang et al., 2015; Zhu et al., 2008a). After UV irradiation, the body of sea cucumber relaxed, mucoid degeneration took place in body surface with white dermis being exposed, and part of the initially firm body wall even flowed out in a sticky mass. Similar autolysis phenomenon of sea cucumber in response to external stimuli, also called ‘‘melting” or ‘‘local degeneration”, has been reported previously (Hill, 2001; Wilkie, 2002; Yang et al., 2015; Zhu et al., 2008a). Though the autolysis occurred throughout the S. japonicus body wall, the speed and degree were more pronounced in epidermis and outer dermis compared to inner dermis. 3.2. Microstructure of S. japonicus body wall and its change during autolysis In the present study, the microstructural changes involved during autolysis of body wall of S. japonicus were revealed through light microscopic examination of fresh and autolytic tissues (H&E stain). S. japonicus body wall consists of an epidermis which is
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covered by an acellular cuticle, a thick dermis which is composed of an outer pigmented layer and a dense and white inner layer, and an inner circular muscle layer (Byrne, 2001; Koob, KoobEmunds, & Trotter, 1999; Menton & Eisen, 1970). In fresh tissue, the cuticle was an intact, smooth and clear border membrane (Fig. 1a). It became rough in surface, broken, and the boundaries between cuticle and superficial epidermis became blurry along with the progress of autolysis (Fig. 1d). The epidermis of S. japonicus body wall is composed primarily of widely-spaced epidermal cells as well as some granule containing cells (Menton and Eisen, 1970; Koob et al., 1999). Though the outlines of epithelial cells could not be clearly observed by using a light microscopy, the regular distribution of the dark nucleus reflected their regular arrangement (Fig. 1a). In a previous study, Menton and Eisen (1970) also found that the nucleus of the epidermal cells tend to be positioned at the same level in epidermis of sea cucumber. Along with the progress of autolysis, the outlines of nucleus became blurry, and the arrangement of nucleus became disordered with some of them aggregated (Fig. 1d). The changes of nucleus reflect the rupture of epithelial cells during autolysis. The thick S. japonicus dermis is MCT consisting of collagen fibers, microfibrils, ground substances, and scattered cells (Thurmond and Trotter, 1996; Elphick, 2012; Hill, 2001). Collagen fibers are surrounded and separated by a microfibrillar network, which maintain the organization and provide a long-range restoring force (Thurmond and Trotter, 1996; Thurmond et al., 1997; Wilkie, 2005). Under the light microscopic level, the H&E stained fresh dermis showed a fibrous network-like texture (Fig. 1a–b). It is speculated that the fibrous networks are constituted with collagen fibers and microfibrillar networks. In contrast, the outer dermis layer was thinner, which contained less fibrous networks but more ground substances compared to the inner dermis layer. Along with the progress of autolysis, the fibrous networks disappeared (Fig. 1d–e), indicating the degradation of collagen fibers and microfibrillar networks. Moreover, the damage of the fibrous networks in out dermis layer was more pronounced compared to inner dermis layer at 4 h post UV stimuli (Fig. 1d–e). In fresh tissue, the muscle fibers in inner circular muscle layer arranged regularly with clear outline (Fig. 1c). Along with the progress of autolysis, the muscle fibers swelled, mixed, and the arrangement became irregular (Fig. 1f). This means that the inner circular muscle layer is also being degraded during autolysis. VG stain was performed to observe the microstructural changes of collagen fibers in S. japonicus dermis. As Fig. 2a and b show, the collagen fibers in fresh body wall of S. japonicus stained red, whereas the ground substances showed gray. Obviously, the distribution of collagen fibers was denser in inner dermis layer compared to outer dermis layer, which is consistent with the published results (Menton and Eisen, 1970). With the duration of autolysis, the collagen fibers became progressively fainter (Fig. 2c–d), indicating the degradation of collagen fibers. In contrast, the damage of collagen fibers in out dermis layer was more pronounced compared to inner dermis layer. For example, the collagen fibers in outer dermis layer were hard to observe at 4 h post UV stimuli, while they were still cognizable in inner dermis layer (Fig. 2c–d). Generally, the degradation of collagen is very slow due to it is highly resistant to proteolysis (Sanggaard et al., 2012). Therefore, the difference in speed and degree of autolysis of different layers of S. japonicus body wall may partially be due to the difference in density of collagen fibers. 3.3. Ultrastructure of collagen fibers in S. japonicus and its change during autolysis The SEM micrographs of the dermis of sea cucumber are shown in Fig. 3. At lower magnification, the SEM image showed that
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Fig. 1. Light micrographs of fresh and autolytic (4 h post UV stimuli) S. japonicus body wall by H&E staining (magnification 100). a, fresh cuticle (?), epidermis (N) and outer dermis layer (w); b, fresh inner dermis layer; c, fresh inner circular muscle layer (r); d, autolytic cuticle, epidermis and outer dermis layer; e, autolytic inner dermis layer; f, autolytic inner circular muscle layer.
Fig. 2. Light micrographs of fresh and autolytic (4 h post UV stimuli) S. japonicus body wall by V-G staining (magnification 200). a, fresh cuticle, epidermis and outer dermis layer; b, fresh inner dermis layer; c, autolytic cuticle, epidermis and outer dermis layer; d, autolytic inner dermis layer.
rope-like collagen fibers were the main structural units of sea cucumber dermis (Fig. 3a). At higher magnification, the collagen fibers showed a parallel arrangement of collagen fibrils with typical D-banding pattern (Fig. 3b). The parallel organization of the collagen fibrils was so compact that there was no interfibrillar structure could be observed. After autolysis, the collagen fibers disaggregated into collagen fibril bundles and individual fibrils (Fig. 3c–d). Meanwhile, some fragmented interfibrillar bridges on the surface of the disintegrated collagen fibrils were observed (Fig. 3d). Previous studies have suggested that the interfibrillar proteoglycan bridges are mainly responsible for holding collagen fibrils together within a collagen fiber (Liu, Yeh, Lewis, & Luo, 2005; Puxkandl et al., 2002). Our previous study indicated that endogenous cysteine proteinases (mainly cathepsins) could degrade the collagen fibers isolated from sea cucumber dermis. The present study revealed that the degradation of interfibrillar proteoglycan bridges by endogenous proteinases results in the disintegration of collagen fibers.
3.4. Light microscopic histochemical localization of cathepsin L In this study, the distribution of cathepsin L in S. japonicus body wall was investigated through light microscopic examination of fresh and autolytic tissues stained with cathepsin L activity. The substrate for enzyme activity staining is a peptide derivate of 4MbNA. Proteinase hydrolyzes the substrate and liberates the terminal 4 MbNA, which is coupled to diazomium salts such as hexazotized pararosaniline. In this way, the proteinase will be visible in histochemical investigations by localization of an insoluble azo-dye reaction product (Smith and Van Frank, 1975). The light microscopic micrographs of the staining sections showed many point-like orange-yellow reaction products (Fig. 4a–c). This phenomenon accords with the mechanism of the staining method (Smith and Van Frank, 1975). In contrast, the control samples did not show any similar reaction products. The distribution of cathepsin L in S. japonicus body wall was inhomogeneous. Cathepsin L was denser in epidermis and outer
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Fig. 3. Scanning electron micrographs of fresh and autolytic S. japonicus dermis. a, fresh tissue (magnification 1000); b, fresh tissue (magnification 20,000); c, autolytic tissue (magnification 1000); d, autolytic tissue (magnification 20,000). Graph showing fragmented interfibrillar proteoglycan bridges (?).
dermis layer compared to inner dermis layer (Fig. 4a–c), which is consistent with our previous results obtained by using immunohistochemical method (Zhou et al., 2014). To confirm the above results, cathepsin L activity in tissues from different layers was determined. As Fig. 5a shows, the enzyme activity of cathepsin L
in per unit weight of tissue decreased from body surface to body cavity of S. japonicus body wall, which is in good agreement with the results of histochemical method. As a type of lysosomal cysteine enzyme, cathepsin L is supposed to be found inside cells. The epidermis of sea cucumber is composed primarily of epidermal
Fig. 4. Light micrographs of enzyme-histochemical localization of cathepsin L in fresh and autolytic S. japonicus body wall (magnification 200). a, fresh cuticle, epidermis and outer dermis layer; b, fresh outer dermis layer; c, fresh inner dermis layer; d, autolytic cuticle, epidermis and outer dermis layer (1 h post UV stimuli); e, autolytic outer dermis layer (1 h post UV stimuli); f, autolytic inner dermis layer (1 h post UV stimuli); g, autolytic cuticle, epidermis and outer dermis layer (4 h post UV stimuli); h, autolytic outer dermis layer (4 h post UV stimuli); i, autolytic inner dermis layer (4 h post UV stimuli).
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cells as well as some granule containing cells (Koob et al., 1999). Whereas, the dermis of sea cucumber is MCT containing scattered cells (Thurmond and Trotter, 1996; Elphick, 2012; Hill, 2001). Koob et al. (1999) reported that the cell density is notably higher in the outer layer than the inner layer of dermis. Therefore, the density of cathepsin L was positively correlated with the cell density. As described above, the speed and degree of autolysis were more pronounced in epidermis and out dermis layer compared to inner dermis layer. It is speculated that the inhomogeneous distribution of endogenous proteinases may be partially responsible for the phenomenon. With the duration of autolysis, the point-like reaction products of cathepsin L in epidermis and dermis showed a progressive diffusion and weakness (Fig. 4e–i). However, as Fig. 5b shows, the enzyme activity of cathepsin L in per unit weight of tissue in S. japonicus body wall was relatively constant during autolysis. This means that the decrease in staining intensity of cathepsin L cannot be attributed to the deactivation of the enzyme. It is speculated that the release of cathepsin L from cells and its diffusion in tissues result in the decrease in local cathepsin L concentration, which leads to the diffusion and weakness of reaction products of cathepsin L in tissues.
3.5. Electron microscopic histochemical localization of cathepsin L To reveal the subcellular localization of cathepsin L in S. japonicus cells and its release, the transmission electron microscopic examination of fresh and autolytic tissues stained with cathepsin
U/g tissue
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Autolysis time (h) Fig. 5. Enzyme activity of cathepsin L in per unit weight of tissue in different layers of fresh S. japonicus body wall (a) (A, cuticle, epidermis and outer dermis layer; B, inner dermis layer; C, circular muscle layer) and in S. japonicus body wall with different time post of UV stimuli (b). Significant differences between different groups were evaluated by one-way analysis of variance (Post Hoc test: S-N-K). Values of different groups with different up numbers (1 2) are significantly different at P < 0.05.
L activity was carried out. The staining mechanism for electron microscopic enzymorphology is similar with that of light microscopic enzymorphology. However, for electron microscopic staining method, osmium is added after the coupling reaction, it can chelate the azo-dye reaction product (Smith and Van Frank, 1975). The insoluble azo-dye metal chelates will impart enough density to be sufficiently electron opaque for electron microscopic enzymorphology. As Fig. 6a shows, the typical cell in S. japonicus body wall had a multivesicular body and a big oval nuclear. The frequently encountered cells in dermis of sea cucumber include granule containing cell, morula cell and vacuole cell (Menton and Eisen, 1970; Thurmond and Trotter, 1996). However, it was difficult to differentiate them as all the cells had a similar characteristic as the typical cell illustrated in Fig. 6a. As a type of lysosomal enzyme, cathepsin L should be found inside lysosome. However, it was difficult to observe the reaction products of cathepsin L in lysosome due to the very high electron density of the organelle. Interestingly, the reaction products of cathepsin L were present in the inside surface of the membrane of the vacuoles (Fig. 6b). The vacuoles having a very low electron density in cells of sea cucumber have been reported previously, but their definite function is still unknown (Menton and Eisen, 1970; Byrne, 2001). In fresh tissue, the membrane of vacuoles appeared intact (Fig. 6b). After UV stimuli, the membranes of a part of vacuoles and cells fused (Fig. 6c–d), indicating the release of cathepsin L from cells. Our previous study indicated that UV irradiation induced apoptosis of cells in S. japonicus body wall, which may be responsible for the disruption of cells (Yang et al., 2015). The MCT of echinoderms can undergo series of changes including contraction and relaxation as well as degradation (Elphick, 2012; Hill, 2001). Some researchers suggest that the agents affecting MCT change are released from cells through neurosecretorylike processes (Byrne, 2001; Koob et al., 1999). Echinoderm cells generally contain membrane bound vesicles such as vacuoles. It has been speculated that the release of the vesicle contents is partially responsible for the secretion of echinoderm cells (Wilkie, Griffiths, & Glennie, 1990). For example, Matsuno and Motokawa (1992) suggested that the vacuoles in vacuole cells of sea cucumber were calcium-storage, and the release of calcium ions from the vacuole cells was supposed to induce connective tissue catch. The above results showed that cathepsin L was present in the vacuoles of S. japonicus cells in fresh body wall. After UV stimuli, it was liberated from broken vacuoles and cells and diffused into tissues. The body wall of sea cucumber is mainly MCT composed of macromolecules such as collagen fibers and microfibrils, ground substances, as well as scattered cells (Thurmond and Trotter, 1996; Elphick, 2012; Hill, 2001). In this study, the data of light microscopic and scanning electron microscopic histological studies all proved that collagen fibers are the most abundant structural elements in MCT (Figs. 2 and 3). Therefore, it is speculated that the released cathepsin L will contact with the extracellular substrates such as collagen fibers in response to UV stimuli. Meanwhile, as described above, the vacuoles were abundant in the frequently encountered cells in S. japonicus body wall, which could explain the extensive degradation of sea cucumber body wall in response to UV irradiation.
4. Conclusion S. japonicus body relaxed, mucoid degeneration occurred from body surface, white dermis exposed, and part of the initially firm body wall even flowed out in a sticky mass in response to UV stimuli. Though the autolysis occurred throughout the S. japonicus body wall, the speed and degree were more pronounced in epidermis
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Fig. 6. Transmission electron micrographs of cells in fresh (a, b) and autolytic (c, d) S. japonicus body wall. a, a typical cell; b, the reaction products of cathepsin L were present in the inside surface of the membrane of the vacuoles (?); c, membrane of a part of vacuoles fused (?); d, membrane of cell and a part of vacuoles fused (?).
and outer dermis layer compared to inner dermis layer. Histological studies indicated that the density of collagen fibers increased from body surface to body cavity of S. japonicus body wall, which may partially accounts for the difference in speed and degree of autolysis of different layers of body wall. Collagen fibers in fresh S. japonicus dermis were intact and orderly arranged. They disaggregated into collagen fibrils after UV stimuli. Histochemical studies showed that cathepsin L was dense in epidermis and outer dermis layer, but was relatively sparse in inner dermis layer, indicating that the inhomogeneous distribution of endogenous proteinases may be partially responsible for the difference in speed and degree of autolysis of different layers of body wall. In fresh
S. japonicus body wall, cathepsin L was present in the inside surface of the vacuoles of cells. After UV stimuli, cathepsin L was released from cells and diffused into tissues. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. Acknowledgement This work was supported financially by ‘‘The National Natural Science Foundation of China (Grant No. 30901124; 31401520;
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31401519)”, ‘‘Project of Distinguished Professor of Liaoning Province (2015-153)”, ‘‘Organization Project for National Engineering Research Center of Seafood funded by Ministry of Science and Technology of the People’s Republic of China (No. 2012FU125X03)”. References Bahuaud, D., Mørkøre, T., Østbye, T. K., Veiseth-Kent, E., Thomassen, M. S., & Ofstad, R. (2010). Muscle structure responses and lysosomal cathepsins B and L in farmed Atlantic salmon (Salmo salar L.) pre-and post-rigor fillets exposed to short and long-term crowding stress. Food Chemistry, 118, 602–615. Barrett, A. J., & Kirschke, H. (1980). Cathepsin B, Cathepsin H, and cathepsin L. Methods in Enzymology, 80, 535–561. Byrne, M. A. R. I. A. (2001). The morphology of autotomy structures in the sea cucumber Eupentacta quinquesemita before and during evisceration. Journal of Experimental Biology, 204, 849–863. Elphick, M. R. (2012). The protein precursors of peptides that affect the mechanics of connective tissue and/or muscle in the echinoderm Apostichopus japonicus. PLoS ONE, 7, e44492. Godiksen, H., Morzel, M., Hyldig, G., & Jessen, F. (2009). Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss). Food Chemistry, 113, 889–896. Hill, R. B. (2001). Role of Ca(2+) in excitation-contraction coupling in echinoderm muscle: comparison with role in other tissues. Journal of Experimental Biology, 204, 897–908. Hu, Y., Morioka, K., & Itoh, Y. (2007). Hydrolysis of surimi paste from walleye pollock (Theragra chalcogramma) by cysteine proteinase cathepsin L and effect of the proteinase inhibitor (E-64) on gelation. Food Chemistry, 104, 702–708. Junqueira, L. C. U., Montes, G. S., Mourão, P. A. S., Carneiro, J., Salles, L. M. M., & Bonetti, S. S. (1980). Collagen-proteoglycans interactions during autonomy in the sea cucumber, Stichopus badionotus. Revue Canadienne de Biologie, 39, 157–164. Koob, T. J., Koob-Emunds, M. M., & Trotter, J. A. (1999). Cell-derived stiffening and plasticizing factors in sea cucumber (Cucumaria frondosa) dermis. The Journal of Experimental Biology, 202, 2291–2301. Liu, S. X. (2004). Practical techniques of biological histology (1st ed.). Beijing: Science Press (Chapter 6). Liu, X., Yeh, M. L., Lewis, J. L., & Luo, Z. P. (2005). Direct measurement of the rupture force of single pair of decorin interactions. Biochemical and Biophysical Research Communications, 338, 1342–1345. Matsuno, A., & Motokawa, T. (1992). Evidence for calcium translocation in catch connective tissue of the sea cucumber Stichopus chloronotus. Cell and Tissue Research, 267, 307–312. Menton, D. N., & Eisen, A. Z. (1970). The structure of the integument of the sea cucumber, Thyone briareus. Journal of Morphology, 131, 17–35. Mukundan, M. K., Antony, P. D., & Nair, M. R. (1986). A review on autolysis in fish. Fisheries Research, 4, 259–269. Puxkandl, R., Zizak, I., Paris, O., Keckes, J., Tesch, W., Bernstorff, S., Purslow, P., et al. (2002). Viscoelastic properties of collagen: synchrotron radiation investigations and structural model. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 357, 191–197. Qi, H., Dong, X. P., Cong, L. N., Gao, Y., Liu, L., Mikiro, T., & Zhu, B. W. (2007). Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber Stichopus japonicus. Fish Physiology and Biochemistry, 33, 181–188. Ryvnyak, V. V., Ryvnyak, E. I., & Tudos, R. V. (2004). Electron histochemical localization of cathepsin L in the liver. Bulletin of Experimental Biology and Medicine, 137(1), 90–91.
Sanggaard, K. W., Danielsen, C. C., Wogensen, L., Vinding, M. S., Rydtoft, L. M., Mortensen, M. B., Karring, H., et al. (2012). Unique structural features facilitate lizard tail autotomy. PLoS ONE, 7, e51803. Smith, R. E., & Van Frank, R. M. (1975). The use of amino acid derivatives of 4methoxy-beta-naphthylamine for the assay and subcellular localization of tissue proteinases. Frontiers of Biology, 43, 193–249. Sun, L. M., Zhu, B. W., Wu, H. T., Yu, L., Zhou, D. Y., Dong, X. P., Yang, J. F., et al. (2011). Purification and characterization of cathepsin B from the gut of the sea cucumber (Stichopus japonicas). Food Science and Biotechnology, 20, 919–925. Thurmond, F. A., Koob, T. J., Bowness, J. M., & Trotter, J. A. (1997). Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis. Connective Tissue Research, 36, 211–222. Thurmond, F., & Trotter, J. (1996). Morphology and biomechanics of the microfibrillar network of sea cucumber dermis. The Journal of Experimental Biology, 199, 1817–1828. Wilkie, I. C. (2002). Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissue? Journal of Experimental Biology, 205, 159–165. Wilkie, I. C. (2005). Mutable collagenous tissue: overview and biotechnological perspective. Echinodermata (pp. 221–250). Berlin Heidelberg: Springer. Wilkie, I. C., Griffiths, G. V., & Glennie, S. F. (1990). Morphological and physiological aspects of the autotomy plane in the aboral integument of Asterias rubens (Echinodermata). In C. De Ridder, P. Dubois, M. C. Lahaye, & M. Jangoux (Eds.), Echinoderm Research (pp. 301–313). Rotterdam: Balkema. Wu, Z., Chen, H., Wang, W., Jia, B., Yang, T., Zhao, Z., Ding, J., et al. (2009). Differentiation of dried sea cucumber products from different geographical areas by surface desorption atmospheric pressure chemical ionization mass spectrometry. Journal of Agricultural and Food Chemistry, 57, 9356–9364. Wu, H. L., Hu, Y. Q., Shen, J. D., Cai, Q. F., Liu, G. M., Su, W. J., & Cao, M. J. (2013a). Identification of a novel gelatinolytic metalloproteinase (GMP) in the body wall of sea cucumber (Stichopus japonicus) and its involvement in collagen degradation. Process Biochemistry, 48, 871–877. Wu, H. T., Li, D. M., Zhu, B. W., Sun, J. J., Zheng, J., Wang, F. L., Konno, K., et al. (2013b). Proteolysis of noncollagenous proteins in sea cucumber, Stichopus japonicus, body wall: characterisation and the effects of cysteine protease inhibitors. Food Chemistry, 141, 1287–1294. Yamashita, M., & Konagaya, S. (1990). Purification and characterization of cathepsin L from the white muscle of chum salmon, Oncorhynchus keta. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 96, 247–252. Yang, J. F., Gao, R. C., Wu, H. T., Li, P. F., Hu, X. S., Zhou, D. Y., Zhu, B. W., et al. (2015). Analysis of apoptosis in ultraviolet-induced sea cucumber (Stichopus japonicus) melting using terminal eoxynucleotidyl-transferase-mediated dUTP nick endlabeling assay and cleaved caspase-3 immunohistochemistry. Journal of Agricultural and Food Chemistry, 63, 9601–9608. Zhong, C., Cai, Q. F., Liu, G. M., Sun, L. C., Hara, K., Su, W. J., & Cao, M. J. (2012). Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins. Food Chemistry, 133, 1560–1568. Zhou, D. Y., Chang, X. N., Bao, S. S., Song, L., Zhu, B. W., Dong, X. P., Zong, Y., et al. (2014). Purification and partial characterisation of a cathepsin L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue distribution in body wall. Food Chemistry, 158, 192–199. Zhu, B. W., Zhao, L. L., Sun, L. M., Li, D. M., Murata, Y., Yu, L., & Zhan, L. (2008b). Purification and characterization of a cathepsin l-like enzyme from the body wall of the sea cucumber Stichopus japonicus. Bioscience, Biotechnology, and Biochemistry, 72, 1430–1437. Zhu, B. W., Zheng, J., Zhang, Z. S., Dong, X. P., Zhao, L. L., & Tada, M. (2008a). Autophagy plays a potential role in the process of sea cucumber body wall ‘‘melting” induced by UV irradiation. Wuhan University Journal of Natural Sciences, 13, 232–238.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus) Yu-Xin Liu a,b, Da-Yong Zhou a,b,⇑, Dong-Dong Ma a, Yan-Fei Liu a, Dong-Mei Li a,b, Xiu-Ping Dong a,b, Ming-Qian Tan a,b, Ming Du a,b, Bei-Wei Zhu a,b,c,d,⇑ a
School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR China National Engineering Research Center of Seafood, Dalian 116034, PR China The Innovation center of Food Nutrition and Human Health, Beijing 100083, PR China d Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, Tianjin 300457, PR China b c
a r t i c l e
i n f o
Article history: Received 9 January 2016 Received in revised form 16 May 2016 Accepted 27 May 2016 Available online 28 May 2016 Keywords: Sea cucumber (Stichopus japonicus) Autolysis Collagen fibers Cathepsin L Localization Electron histochemistry
a b s t r a c t The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Sea cucumber is a type of echinoderm and one of the important cultured aquatic species in China and many other Asian countries due to its high commercial value (Wu et al., 2009). According to FAO (2013) statistics, the total world production of sea cucumber was about 232 thousand tones (http://www.fao.org/figis/servlet/ TabSelector). Sea cucumber is highly susceptible towards external stimulus. The body wall of sea cucumber undergoes a massive tissue autolysis, also called ‘‘melting” or ‘‘local degeneration”, in response to changes of external environmental factors such as temperature, salt concentration, nutrient deficiency, sunlight exposure, ultraviolet irradiation, and mechanical stimulation (Hill, 2001; Wilkie, 2002; Yang et al., 2015; Zhu et al., 2008a). Autolysis can lead to severe postharvest quality deterioration of sea cucumber, consequently cause heavy economic losses. The body wall of sea cucumber is mainly composed of mutable connective tissue (MCT) with scattered cells (Elphick, 2012; Hill, 2001; Thurmond & Trotter, 1996). In MCT, collagen fibers are ⇑ Corresponding authors at: School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR China. E-mail addresses: [email protected] (D.-Y. Zhou), [email protected] (B.-W. Zhu). http://dx.doi.org/10.1016/j.foodchem.2016.05.173 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
surrounded and separated by a microfibrillar network, which maintain the organization and provide a long-range restoring force (Thurmond and Trotter, 1996; Thurmond, Koob, Bowness, & Trotter, 1997; Wilkie, 2005). Therefore, the dramatic changes in apparent and mechanical properties of body wall of sea cucumber during autolysis must be due to the degradation of MCT. However, the in situ microstructural changes in MCT of sea cucumber during autolysis are less clear. The autolytic degradation of body wall of sea cucumber is supposed to be caused by endogenous enzymolysis of structural elements. To date, cysteine-like proteinase (Qi et al., 2007), cathepsin L-like proteinase (Zhou et al., 2014; Zhu et al., 2008b), cathepsin B-like proteinase (Sun et al., 2011) and gelatinolytic metalloproteinase (GMP) (Wu et al., 2013a) have been purified and characterized from sea cucumber (Stichopus japonicus, S. japonicus). Junqueira et al. (1980) suggested that an endogenous proteinase (maybe Ca2+-activated collagenase) in the body wall of sea cucumber (Isostichopus badionotus) could liberate proteoglycans from interaction with collagens. Hill (2001) further speculated that the autolysis of body wall of sea cucumber might be initiated by the release of proteoglycans by Ca2+-activated collagenase. Wu et al. (2013a) reported that calcium-dependent metalloproteinase from S. japonicus could degrade the collagen isolated from S. japonicus
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body wall in a temperature-dependent mode. In a previous study, we found that endogenous cysteine proteinases were partially involved in the proteolysis of noncollagenous proteins from S. japonicus body wall (Wu et al., 2013b). Autolysis is common for aquatic species. Cathepsin, a type of lysosomal cysteine proteinase, may well be the most active proteinases in the human body (Barrett & Kirschke, 1980). It is widely accepted that cathepsin is one of the most important endogenous proteinase in autolytic degradation of fish (Mukundan, Antony, & Nair, 1986). For example, cathepsin B, D, H and L were involved in the breakdown of muscle structures in several fish species (Bahuaud et al., 2010; Godiksen, Morzel, Hyldig, & Jessen, 2009). Meanwhile, cathepsin L, a representative cathepsin, was thought to be mainly responsible for the modori phenomenon during surimi production (Hu, Morioka, & Itoh, 2007; Zhong et al., 2012). Previous studies indicated that the body wall of sea cucumber is rich in cathepsins (Sun et al., 2011; Zhou et al., 2014; Zhu et al., 2008b). However, lysosomal cathepsin must be released from cells before it makes contact with extracellular substrates and degradation. To our knowledge, there is still no report on the subcellular localization and release of cathepsin in sea cucumber. In the present study, ultraviolet (UV) irradiation was used to induce the autolysis of S. japonicus, and then the changes of microstructures of collagenous tissues as well as tissue and cellular distributions of cathepsin L were investigated by histological and histochemical techniques using light microscope and electron microscopes. Through this research, the in situ changes that occur in collagen fibers, and the mechanism through which lysosomal cysteine proteinases contact with their extracellular substrates were revealed.
with 75 lL of assay buffer containing 340 mM sodium acetate, 60 mM acetic acid, 4 mM disodium EDTA, and 8 mM DTT (pH 5.5, prepared just before use) and pre-incubated at 37 °C for 2 min. Then freshly prepared substrate (75 lL) was added to the mixture to a final concentration of 25 lM. After 10 min of incubation at 37 °C, 300 lL of sodium acetate buffer (pH 4.3) containing 0.1 M chloroacetate was added to the mixture to terminate the reaction. The fluorescence of AMC liberated by hydrolysis was measured by using a spectrofluorometer (F-2700, Hitachi, Tokyo, Japan) with excitation and emission wavelengths of 380 and 460 nm, respectively. AMC was used as a standard reference. A blank sample was prepared by adding the sodium acetate buffer containing chloroacetate before the substrate. One unit of the enzyme activity (U) was defined as the amount of the activity that released 1 nM of AMC per min.
2. Materials and methods
2.6. Histological analysis with scanning electron microscopy
2.1. Chemicals
The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 1.0 cm3 sized pieces, dehydrated through a graded series of 50%, 60%, 70%, 80%, 90%, 100% ethanol, freezedried, mounted on a metal stub, coated with Pd, and imaged by using a JSM-7800F SEM (JEOL Ltd., Tokyo, Japan) which operated at 10 kV. Experiments were repeated at least three times to confirm the results, and the samples were observed carefully without any beam damage. Micrographs were taken from different spots of the same sample at similar magnification and width measurements.
Benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-b-n aphthylamide hydrochloride (Z-Phe-Arg-4MbNAHCl) was purchased from Bachem Inc. (Torrance, CA, USA). Benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-AMC), 7-amino-4-methylcoumarin (AMC), L-cysteine, dithiothreitol (DTT) and cacodylic acid sodium were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All of the other chemicals used were of analytical grade. 2.2. Biological materials Sea cucumbers of the species S. japonica, each 70 ± 10 g, were captured in the Yellow Sea near the coast of Dalian, China, and transported to the laboratory immediately in cold seawater. In laboratory, the sea cucumber was induced to autolysis by 30 min of UV irradiation (15 w, 0.5 m), and left in an air-conditioned room (the temperature was about 20 ± 2 °C) for different intervals before further experiment. 2.3. Preparation of crude cathepsin L The crude cathepsin L was extracted from body wall of S. japonicus according to a procedure described in our previous study (Zhou et al., 2014). 2.4. Assay of enzyme activity toward fluorogenic substrate Cathepsin L activity was determined according to the method of Yamashita and Konagaya (1990), by using Z-Phe-Arg-AMC as a substrate. Briefly, 150 lL of crude cathepsin L solution was mixed
2.5. Histological analysis by using light microscopy The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 0.5 cm3 sized pieces, fixed in 10% paraformaldehyde at 4 °C for 18 h, dehydrated through a graded series of 50%, 60%, 70%, 80%, 90%, 100% ethanol, vitrified in a dimethylbenzene/ethanol (v/v) series of 1:2, 1:1, 2:1, and 1:0, and embedded routinely in paraffin. Tissue sections (5 lm) were prepared by using a Leica EG1150C rotary microtome (Leica Microsystems). Hematoxylin and eosin (H&E) and Van Gieson’s (VG) stain were performed according to the standard techniques (Liu, 2004). The microstructures were observed and photographed by using an Olympus BX51TF optical microscopy & DP70 Digital Camera System (Olympus Co., Tokyo, JP).
2.7. Histochemical localization of cathepsin L by using light microscopy Light microscopic histochemical localization of cathepsin L was carried out according to the process introduced by Smith and Van Frank (1975) with slight modifications. The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 1.0 cm 1.0 cm 0.5 cm sized clumps. The tissue pieces were quick-frozen on a cryostat for 10 min, and then sectioned by using Leica CM1950 freezing microtome (Leica Microsystems) to a thickness of 10 lm. The tissue section was put onto a glass slide, dried naturally, washed twice with double distilled water, and incubated in stain fluid containing substrate. After 60 min of incubation at 37 °C, the stain fluid was discarded and the tissue section was washed three times with double distilled water. The tissue sections were observed and photographed using an Olympus BX51TF optical microscopy & DP70 Digital Camera System (Olympus Co., Tokyo, JP). Control reactions were carried out in medium containing no substrate. Substrate fluid preparation: 1 mg of Z-Phe-Arg-4MbNAHCl was dissolved in 330 lL of dimethyl sulfoxide, and then mixed with 670 lL of Tris-HCl buffer (0.05 M, pH 7.5) containing 10 mM CaCl2; Stain fluid preparation: liquid A: 400 mg of basic fuchsin was mixed with 2 mL of 12 M hydrochloric acid and stirred into a paste. The paste was mixed further with 8 mL of deionized water, heated
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until dissolved, and filtered to recover the liquid A. liquid B: 4% sodium nitrite. The same volumes of liquid A and liquid B were mixed in sealed container and left at 4 °C for 2 min. After the reaction in low temperature, the liquid became yellow, indicating the formation of hexazotized pararosaniline. One milliliter of hexazotized pararosaniline was diluted with 19 mL of Na2HPO4-NaH2PO4 buffer (0.1 M, pH 7.4) to obtain the stain fluid. The same volumes of substrate fluid and stain fluid were mixed to obtain the stain fluid containing substrate. 2.8. Histochemical localization of cathepsin L by using transmission electron microscopy Electronic-histochemical localization of cathepsin L was performed according to the previously reported procedures (Smith and Van Frank, 1975; Ryvnyak, Ryvnyak, & Tudos, 2004), with slight modifications. The body wall tissues sampled from the dorsal surface of S. japonicus were sliced into 0.5 cm 0.3 cm 0.3 cm sized pieces, fixed in 1.5% glutaraldehyde in sodium cacodylate buffer (0.05 M, pH 7.4) containing 2% sucrose at 4 °C for 3 h. The materials were washed three times with the same buffer (10 min for each time), and then incubated with the stain liquid containing substrate (same as the fluid used in Section 2.7). After being washed with sodium cacodylate buffer (0.05 M, pH 7.4) for three times, the tissue pieces were post-fixed in 1% osmic acid for 2 h at 4 °C. Osmicated samples were rinsed with 0.1 M phosphate buffer (pH 7.4), and embedded in epon. Sections (70 nm) were obtained by a LKB ultramicrotome and stained with uranyl acetate and lead citrate, and then observed and photographed using JEM-2000EX transmission electron microscopy (JEOL Ltd., Tokyo, JP). Control reactions were carried out in medium containing no substrate. 2.9. Statistical method All the tests were conducted with three replicates. Data were presented as mean ± standard deviation (SD). The statistical analysis was performed by using SPSS 16.0 software (SPSS Inc. Chicago, IL, USA). Differences between means were evaluated by one-way analysis of variance (Post Hoc test: S-N-K). Comparisons that yielded P values < 0.05 were considered significant. 3. Results and discussion 3.1. Changes in apparent properties of S. japonicus in response to UV stimuli The autolysis of sea cucumber was induced by UV irradiation as reported in our previous studies (Yang et al., 2015; Zhu et al., 2008a). After UV irradiation, the body of sea cucumber relaxed, mucoid degeneration took place in body surface with white dermis being exposed, and part of the initially firm body wall even flowed out in a sticky mass. Similar autolysis phenomenon of sea cucumber in response to external stimuli, also called ‘‘melting” or ‘‘local degeneration”, has been reported previously (Hill, 2001; Wilkie, 2002; Yang et al., 2015; Zhu et al., 2008a). Though the autolysis occurred throughout the S. japonicus body wall, the speed and degree were more pronounced in epidermis and outer dermis compared to inner dermis. 3.2. Microstructure of S. japonicus body wall and its change during autolysis In the present study, the microstructural changes involved during autolysis of body wall of S. japonicus were revealed through light microscopic examination of fresh and autolytic tissues (H&E stain). S. japonicus body wall consists of an epidermis which is
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covered by an acellular cuticle, a thick dermis which is composed of an outer pigmented layer and a dense and white inner layer, and an inner circular muscle layer (Byrne, 2001; Koob, KoobEmunds, & Trotter, 1999; Menton & Eisen, 1970). In fresh tissue, the cuticle was an intact, smooth and clear border membrane (Fig. 1a). It became rough in surface, broken, and the boundaries between cuticle and superficial epidermis became blurry along with the progress of autolysis (Fig. 1d). The epidermis of S. japonicus body wall is composed primarily of widely-spaced epidermal cells as well as some granule containing cells (Menton and Eisen, 1970; Koob et al., 1999). Though the outlines of epithelial cells could not be clearly observed by using a light microscopy, the regular distribution of the dark nucleus reflected their regular arrangement (Fig. 1a). In a previous study, Menton and Eisen (1970) also found that the nucleus of the epidermal cells tend to be positioned at the same level in epidermis of sea cucumber. Along with the progress of autolysis, the outlines of nucleus became blurry, and the arrangement of nucleus became disordered with some of them aggregated (Fig. 1d). The changes of nucleus reflect the rupture of epithelial cells during autolysis. The thick S. japonicus dermis is MCT consisting of collagen fibers, microfibrils, ground substances, and scattered cells (Thurmond and Trotter, 1996; Elphick, 2012; Hill, 2001). Collagen fibers are surrounded and separated by a microfibrillar network, which maintain the organization and provide a long-range restoring force (Thurmond and Trotter, 1996; Thurmond et al., 1997; Wilkie, 2005). Under the light microscopic level, the H&E stained fresh dermis showed a fibrous network-like texture (Fig. 1a–b). It is speculated that the fibrous networks are constituted with collagen fibers and microfibrillar networks. In contrast, the outer dermis layer was thinner, which contained less fibrous networks but more ground substances compared to the inner dermis layer. Along with the progress of autolysis, the fibrous networks disappeared (Fig. 1d–e), indicating the degradation of collagen fibers and microfibrillar networks. Moreover, the damage of the fibrous networks in out dermis layer was more pronounced compared to inner dermis layer at 4 h post UV stimuli (Fig. 1d–e). In fresh tissue, the muscle fibers in inner circular muscle layer arranged regularly with clear outline (Fig. 1c). Along with the progress of autolysis, the muscle fibers swelled, mixed, and the arrangement became irregular (Fig. 1f). This means that the inner circular muscle layer is also being degraded during autolysis. VG stain was performed to observe the microstructural changes of collagen fibers in S. japonicus dermis. As Fig. 2a and b show, the collagen fibers in fresh body wall of S. japonicus stained red, whereas the ground substances showed gray. Obviously, the distribution of collagen fibers was denser in inner dermis layer compared to outer dermis layer, which is consistent with the published results (Menton and Eisen, 1970). With the duration of autolysis, the collagen fibers became progressively fainter (Fig. 2c–d), indicating the degradation of collagen fibers. In contrast, the damage of collagen fibers in out dermis layer was more pronounced compared to inner dermis layer. For example, the collagen fibers in outer dermis layer were hard to observe at 4 h post UV stimuli, while they were still cognizable in inner dermis layer (Fig. 2c–d). Generally, the degradation of collagen is very slow due to it is highly resistant to proteolysis (Sanggaard et al., 2012). Therefore, the difference in speed and degree of autolysis of different layers of S. japonicus body wall may partially be due to the difference in density of collagen fibers. 3.3. Ultrastructure of collagen fibers in S. japonicus and its change during autolysis The SEM micrographs of the dermis of sea cucumber are shown in Fig. 3. At lower magnification, the SEM image showed that
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Fig. 1. Light micrographs of fresh and autolytic (4 h post UV stimuli) S. japonicus body wall by H&E staining (magnification 100). a, fresh cuticle (?), epidermis (N) and outer dermis layer (w); b, fresh inner dermis layer; c, fresh inner circular muscle layer (r); d, autolytic cuticle, epidermis and outer dermis layer; e, autolytic inner dermis layer; f, autolytic inner circular muscle layer.
Fig. 2. Light micrographs of fresh and autolytic (4 h post UV stimuli) S. japonicus body wall by V-G staining (magnification 200). a, fresh cuticle, epidermis and outer dermis layer; b, fresh inner dermis layer; c, autolytic cuticle, epidermis and outer dermis layer; d, autolytic inner dermis layer.
rope-like collagen fibers were the main structural units of sea cucumber dermis (Fig. 3a). At higher magnification, the collagen fibers showed a parallel arrangement of collagen fibrils with typical D-banding pattern (Fig. 3b). The parallel organization of the collagen fibrils was so compact that there was no interfibrillar structure could be observed. After autolysis, the collagen fibers disaggregated into collagen fibril bundles and individual fibrils (Fig. 3c–d). Meanwhile, some fragmented interfibrillar bridges on the surface of the disintegrated collagen fibrils were observed (Fig. 3d). Previous studies have suggested that the interfibrillar proteoglycan bridges are mainly responsible for holding collagen fibrils together within a collagen fiber (Liu, Yeh, Lewis, & Luo, 2005; Puxkandl et al., 2002). Our previous study indicated that endogenous cysteine proteinases (mainly cathepsins) could degrade the collagen fibers isolated from sea cucumber dermis. The present study revealed that the degradation of interfibrillar proteoglycan bridges by endogenous proteinases results in the disintegration of collagen fibers.
3.4. Light microscopic histochemical localization of cathepsin L In this study, the distribution of cathepsin L in S. japonicus body wall was investigated through light microscopic examination of fresh and autolytic tissues stained with cathepsin L activity. The substrate for enzyme activity staining is a peptide derivate of 4MbNA. Proteinase hydrolyzes the substrate and liberates the terminal 4 MbNA, which is coupled to diazomium salts such as hexazotized pararosaniline. In this way, the proteinase will be visible in histochemical investigations by localization of an insoluble azo-dye reaction product (Smith and Van Frank, 1975). The light microscopic micrographs of the staining sections showed many point-like orange-yellow reaction products (Fig. 4a–c). This phenomenon accords with the mechanism of the staining method (Smith and Van Frank, 1975). In contrast, the control samples did not show any similar reaction products. The distribution of cathepsin L in S. japonicus body wall was inhomogeneous. Cathepsin L was denser in epidermis and outer
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Fig. 3. Scanning electron micrographs of fresh and autolytic S. japonicus dermis. a, fresh tissue (magnification 1000); b, fresh tissue (magnification 20,000); c, autolytic tissue (magnification 1000); d, autolytic tissue (magnification 20,000). Graph showing fragmented interfibrillar proteoglycan bridges (?).
dermis layer compared to inner dermis layer (Fig. 4a–c), which is consistent with our previous results obtained by using immunohistochemical method (Zhou et al., 2014). To confirm the above results, cathepsin L activity in tissues from different layers was determined. As Fig. 5a shows, the enzyme activity of cathepsin L
in per unit weight of tissue decreased from body surface to body cavity of S. japonicus body wall, which is in good agreement with the results of histochemical method. As a type of lysosomal cysteine enzyme, cathepsin L is supposed to be found inside cells. The epidermis of sea cucumber is composed primarily of epidermal
Fig. 4. Light micrographs of enzyme-histochemical localization of cathepsin L in fresh and autolytic S. japonicus body wall (magnification 200). a, fresh cuticle, epidermis and outer dermis layer; b, fresh outer dermis layer; c, fresh inner dermis layer; d, autolytic cuticle, epidermis and outer dermis layer (1 h post UV stimuli); e, autolytic outer dermis layer (1 h post UV stimuli); f, autolytic inner dermis layer (1 h post UV stimuli); g, autolytic cuticle, epidermis and outer dermis layer (4 h post UV stimuli); h, autolytic outer dermis layer (4 h post UV stimuli); i, autolytic inner dermis layer (4 h post UV stimuli).
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cells as well as some granule containing cells (Koob et al., 1999). Whereas, the dermis of sea cucumber is MCT containing scattered cells (Thurmond and Trotter, 1996; Elphick, 2012; Hill, 2001). Koob et al. (1999) reported that the cell density is notably higher in the outer layer than the inner layer of dermis. Therefore, the density of cathepsin L was positively correlated with the cell density. As described above, the speed and degree of autolysis were more pronounced in epidermis and out dermis layer compared to inner dermis layer. It is speculated that the inhomogeneous distribution of endogenous proteinases may be partially responsible for the phenomenon. With the duration of autolysis, the point-like reaction products of cathepsin L in epidermis and dermis showed a progressive diffusion and weakness (Fig. 4e–i). However, as Fig. 5b shows, the enzyme activity of cathepsin L in per unit weight of tissue in S. japonicus body wall was relatively constant during autolysis. This means that the decrease in staining intensity of cathepsin L cannot be attributed to the deactivation of the enzyme. It is speculated that the release of cathepsin L from cells and its diffusion in tissues result in the decrease in local cathepsin L concentration, which leads to the diffusion and weakness of reaction products of cathepsin L in tissues.
3.5. Electron microscopic histochemical localization of cathepsin L To reveal the subcellular localization of cathepsin L in S. japonicus cells and its release, the transmission electron microscopic examination of fresh and autolytic tissues stained with cathepsin
U/g tissue
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80 60 40 20 0 0
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Autolysis time (h) Fig. 5. Enzyme activity of cathepsin L in per unit weight of tissue in different layers of fresh S. japonicus body wall (a) (A, cuticle, epidermis and outer dermis layer; B, inner dermis layer; C, circular muscle layer) and in S. japonicus body wall with different time post of UV stimuli (b). Significant differences between different groups were evaluated by one-way analysis of variance (Post Hoc test: S-N-K). Values of different groups with different up numbers (1 2) are significantly different at P < 0.05.
L activity was carried out. The staining mechanism for electron microscopic enzymorphology is similar with that of light microscopic enzymorphology. However, for electron microscopic staining method, osmium is added after the coupling reaction, it can chelate the azo-dye reaction product (Smith and Van Frank, 1975). The insoluble azo-dye metal chelates will impart enough density to be sufficiently electron opaque for electron microscopic enzymorphology. As Fig. 6a shows, the typical cell in S. japonicus body wall had a multivesicular body and a big oval nuclear. The frequently encountered cells in dermis of sea cucumber include granule containing cell, morula cell and vacuole cell (Menton and Eisen, 1970; Thurmond and Trotter, 1996). However, it was difficult to differentiate them as all the cells had a similar characteristic as the typical cell illustrated in Fig. 6a. As a type of lysosomal enzyme, cathepsin L should be found inside lysosome. However, it was difficult to observe the reaction products of cathepsin L in lysosome due to the very high electron density of the organelle. Interestingly, the reaction products of cathepsin L were present in the inside surface of the membrane of the vacuoles (Fig. 6b). The vacuoles having a very low electron density in cells of sea cucumber have been reported previously, but their definite function is still unknown (Menton and Eisen, 1970; Byrne, 2001). In fresh tissue, the membrane of vacuoles appeared intact (Fig. 6b). After UV stimuli, the membranes of a part of vacuoles and cells fused (Fig. 6c–d), indicating the release of cathepsin L from cells. Our previous study indicated that UV irradiation induced apoptosis of cells in S. japonicus body wall, which may be responsible for the disruption of cells (Yang et al., 2015). The MCT of echinoderms can undergo series of changes including contraction and relaxation as well as degradation (Elphick, 2012; Hill, 2001). Some researchers suggest that the agents affecting MCT change are released from cells through neurosecretorylike processes (Byrne, 2001; Koob et al., 1999). Echinoderm cells generally contain membrane bound vesicles such as vacuoles. It has been speculated that the release of the vesicle contents is partially responsible for the secretion of echinoderm cells (Wilkie, Griffiths, & Glennie, 1990). For example, Matsuno and Motokawa (1992) suggested that the vacuoles in vacuole cells of sea cucumber were calcium-storage, and the release of calcium ions from the vacuole cells was supposed to induce connective tissue catch. The above results showed that cathepsin L was present in the vacuoles of S. japonicus cells in fresh body wall. After UV stimuli, it was liberated from broken vacuoles and cells and diffused into tissues. The body wall of sea cucumber is mainly MCT composed of macromolecules such as collagen fibers and microfibrils, ground substances, as well as scattered cells (Thurmond and Trotter, 1996; Elphick, 2012; Hill, 2001). In this study, the data of light microscopic and scanning electron microscopic histological studies all proved that collagen fibers are the most abundant structural elements in MCT (Figs. 2 and 3). Therefore, it is speculated that the released cathepsin L will contact with the extracellular substrates such as collagen fibers in response to UV stimuli. Meanwhile, as described above, the vacuoles were abundant in the frequently encountered cells in S. japonicus body wall, which could explain the extensive degradation of sea cucumber body wall in response to UV irradiation.
4. Conclusion S. japonicus body relaxed, mucoid degeneration occurred from body surface, white dermis exposed, and part of the initially firm body wall even flowed out in a sticky mass in response to UV stimuli. Though the autolysis occurred throughout the S. japonicus body wall, the speed and degree were more pronounced in epidermis
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Fig. 6. Transmission electron micrographs of cells in fresh (a, b) and autolytic (c, d) S. japonicus body wall. a, a typical cell; b, the reaction products of cathepsin L were present in the inside surface of the membrane of the vacuoles (?); c, membrane of a part of vacuoles fused (?); d, membrane of cell and a part of vacuoles fused (?).
and outer dermis layer compared to inner dermis layer. Histological studies indicated that the density of collagen fibers increased from body surface to body cavity of S. japonicus body wall, which may partially accounts for the difference in speed and degree of autolysis of different layers of body wall. Collagen fibers in fresh S. japonicus dermis were intact and orderly arranged. They disaggregated into collagen fibrils after UV stimuli. Histochemical studies showed that cathepsin L was dense in epidermis and outer dermis layer, but was relatively sparse in inner dermis layer, indicating that the inhomogeneous distribution of endogenous proteinases may be partially responsible for the difference in speed and degree of autolysis of different layers of body wall. In fresh
S. japonicus body wall, cathepsin L was present in the inside surface of the vacuoles of cells. After UV stimuli, cathepsin L was released from cells and diffused into tissues. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. Acknowledgement This work was supported financially by ‘‘The National Natural Science Foundation of China (Grant No. 30901124; 31401520;
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31401519)”, ‘‘Project of Distinguished Professor of Liaoning Province (2015-153)”, ‘‘Organization Project for National Engineering Research Center of Seafood funded by Ministry of Science and Technology of the People’s Republic of China (No. 2012FU125X03)”. References Bahuaud, D., Mørkøre, T., Østbye, T. K., Veiseth-Kent, E., Thomassen, M. S., & Ofstad, R. (2010). Muscle structure responses and lysosomal cathepsins B and L in farmed Atlantic salmon (Salmo salar L.) pre-and post-rigor fillets exposed to short and long-term crowding stress. Food Chemistry, 118, 602–615. Barrett, A. J., & Kirschke, H. (1980). Cathepsin B, Cathepsin H, and cathepsin L. Methods in Enzymology, 80, 535–561. Byrne, M. A. R. I. A. (2001). The morphology of autotomy structures in the sea cucumber Eupentacta quinquesemita before and during evisceration. Journal of Experimental Biology, 204, 849–863. Elphick, M. R. (2012). The protein precursors of peptides that affect the mechanics of connective tissue and/or muscle in the echinoderm Apostichopus japonicus. PLoS ONE, 7, e44492. Godiksen, H., Morzel, M., Hyldig, G., & Jessen, F. (2009). Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss). Food Chemistry, 113, 889–896. Hill, R. B. (2001). Role of Ca(2+) in excitation-contraction coupling in echinoderm muscle: comparison with role in other tissues. Journal of Experimental Biology, 204, 897–908. Hu, Y., Morioka, K., & Itoh, Y. (2007). Hydrolysis of surimi paste from walleye pollock (Theragra chalcogramma) by cysteine proteinase cathepsin L and effect of the proteinase inhibitor (E-64) on gelation. Food Chemistry, 104, 702–708. Junqueira, L. C. U., Montes, G. S., Mourão, P. A. S., Carneiro, J., Salles, L. M. M., & Bonetti, S. S. (1980). Collagen-proteoglycans interactions during autonomy in the sea cucumber, Stichopus badionotus. Revue Canadienne de Biologie, 39, 157–164. Koob, T. J., Koob-Emunds, M. M., & Trotter, J. A. (1999). Cell-derived stiffening and plasticizing factors in sea cucumber (Cucumaria frondosa) dermis. The Journal of Experimental Biology, 202, 2291–2301. Liu, S. X. (2004). Practical techniques of biological histology (1st ed.). Beijing: Science Press (Chapter 6). Liu, X., Yeh, M. L., Lewis, J. L., & Luo, Z. P. (2005). Direct measurement of the rupture force of single pair of decorin interactions. Biochemical and Biophysical Research Communications, 338, 1342–1345. Matsuno, A., & Motokawa, T. (1992). Evidence for calcium translocation in catch connective tissue of the sea cucumber Stichopus chloronotus. Cell and Tissue Research, 267, 307–312. Menton, D. N., & Eisen, A. Z. (1970). The structure of the integument of the sea cucumber, Thyone briareus. Journal of Morphology, 131, 17–35. Mukundan, M. K., Antony, P. D., & Nair, M. R. (1986). A review on autolysis in fish. Fisheries Research, 4, 259–269. Puxkandl, R., Zizak, I., Paris, O., Keckes, J., Tesch, W., Bernstorff, S., Purslow, P., et al. (2002). Viscoelastic properties of collagen: synchrotron radiation investigations and structural model. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 357, 191–197. Qi, H., Dong, X. P., Cong, L. N., Gao, Y., Liu, L., Mikiro, T., & Zhu, B. W. (2007). Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber Stichopus japonicus. Fish Physiology and Biochemistry, 33, 181–188. Ryvnyak, V. V., Ryvnyak, E. I., & Tudos, R. V. (2004). Electron histochemical localization of cathepsin L in the liver. Bulletin of Experimental Biology and Medicine, 137(1), 90–91.
Sanggaard, K. W., Danielsen, C. C., Wogensen, L., Vinding, M. S., Rydtoft, L. M., Mortensen, M. B., Karring, H., et al. (2012). Unique structural features facilitate lizard tail autotomy. PLoS ONE, 7, e51803. Smith, R. E., & Van Frank, R. M. (1975). The use of amino acid derivatives of 4methoxy-beta-naphthylamine for the assay and subcellular localization of tissue proteinases. Frontiers of Biology, 43, 193–249. Sun, L. M., Zhu, B. W., Wu, H. T., Yu, L., Zhou, D. Y., Dong, X. P., Yang, J. F., et al. (2011). Purification and characterization of cathepsin B from the gut of the sea cucumber (Stichopus japonicas). Food Science and Biotechnology, 20, 919–925. Thurmond, F. A., Koob, T. J., Bowness, J. M., & Trotter, J. A. (1997). Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis. Connective Tissue Research, 36, 211–222. Thurmond, F., & Trotter, J. (1996). Morphology and biomechanics of the microfibrillar network of sea cucumber dermis. The Journal of Experimental Biology, 199, 1817–1828. Wilkie, I. C. (2002). Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissue? Journal of Experimental Biology, 205, 159–165. Wilkie, I. C. (2005). Mutable collagenous tissue: overview and biotechnological perspective. Echinodermata (pp. 221–250). Berlin Heidelberg: Springer. Wilkie, I. C., Griffiths, G. V., & Glennie, S. F. (1990). Morphological and physiological aspects of the autotomy plane in the aboral integument of Asterias rubens (Echinodermata). In C. De Ridder, P. Dubois, M. C. Lahaye, & M. Jangoux (Eds.), Echinoderm Research (pp. 301–313). Rotterdam: Balkema. Wu, Z., Chen, H., Wang, W., Jia, B., Yang, T., Zhao, Z., Ding, J., et al. (2009). Differentiation of dried sea cucumber products from different geographical areas by surface desorption atmospheric pressure chemical ionization mass spectrometry. Journal of Agricultural and Food Chemistry, 57, 9356–9364. Wu, H. L., Hu, Y. Q., Shen, J. D., Cai, Q. F., Liu, G. M., Su, W. J., & Cao, M. J. (2013a). Identification of a novel gelatinolytic metalloproteinase (GMP) in the body wall of sea cucumber (Stichopus japonicus) and its involvement in collagen degradation. Process Biochemistry, 48, 871–877. Wu, H. T., Li, D. M., Zhu, B. W., Sun, J. J., Zheng, J., Wang, F. L., Konno, K., et al. (2013b). Proteolysis of noncollagenous proteins in sea cucumber, Stichopus japonicus, body wall: characterisation and the effects of cysteine protease inhibitors. Food Chemistry, 141, 1287–1294. Yamashita, M., & Konagaya, S. (1990). Purification and characterization of cathepsin L from the white muscle of chum salmon, Oncorhynchus keta. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 96, 247–252. Yang, J. F., Gao, R. C., Wu, H. T., Li, P. F., Hu, X. S., Zhou, D. Y., Zhu, B. W., et al. (2015). Analysis of apoptosis in ultraviolet-induced sea cucumber (Stichopus japonicus) melting using terminal eoxynucleotidyl-transferase-mediated dUTP nick endlabeling assay and cleaved caspase-3 immunohistochemistry. Journal of Agricultural and Food Chemistry, 63, 9601–9608. Zhong, C., Cai, Q. F., Liu, G. M., Sun, L. C., Hara, K., Su, W. J., & Cao, M. J. (2012). Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins. Food Chemistry, 133, 1560–1568. Zhou, D. Y., Chang, X. N., Bao, S. S., Song, L., Zhu, B. W., Dong, X. P., Zong, Y., et al. (2014). Purification and partial characterisation of a cathepsin L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue distribution in body wall. Food Chemistry, 158, 192–199. Zhu, B. W., Zhao, L. L., Sun, L. M., Li, D. M., Murata, Y., Yu, L., & Zhan, L. (2008b). Purification and characterization of a cathepsin l-like enzyme from the body wall of the sea cucumber Stichopus japonicus. Bioscience, Biotechnology, and Biochemistry, 72, 1430–1437. Zhu, B. W., Zheng, J., Zhang, Z. S., Dong, X. P., Zhao, L. L., & Tada, M. (2008a). Autophagy plays a potential role in the process of sea cucumber body wall ‘‘melting” induced by UV irradiation. Wuhan University Journal of Natural Sciences, 13, 232–238.