Autolysis Of Sea Cucumber Research Articles

An analysis combining proteomics and transcriptomics revealed a regulation target of sea cucumber autolysis

Sea cucumber is a valuable seafood product and autolysis is the main concern for the aquaculture industry. This study employed proteomics and transcriptomics to investigate the autolysis mechanism of sea cucumbers. The fresh sea cucumber was exposed to UV light to induce autolysis. The body wall samples were cut off to analyze by proteomics and transcriptomics. The angiotensin-converting enzyme (ACE) inhibitor of teprotide and the activator of imatinib were gastric gavage to live sea cucumbers, respectively, to identify the regulation target. Autolysis occurrence was evaluated by appearance, soluble peptide, and hydroxyproline content. Four gene-protein pairs were ACE, AJAP10923, Heme-binding protein 2-like, and Ficolin-2-like. Only the ACE protein and gene changed synchronously and a significant down-regulation of ACE occurred in the autolysis sea cucumbers. Teprotide led to a 1.58-fold increase in the TCA-soluble protein content and a 1.57-fold increase in hydroxyproline content. No significant differences were observed between imatinib-treated sea cucumbers and fresh ones regarding TCA-soluble protein content or hydroxyproline levels (P > 0.05). ACE inhibitor accelerated the autolysis of sea cucumber, but ACE activator inhibited the autolysis. Therefore, ACE can serve as a regulatory target for autolysis in sea cucumbers.

The exogenous autophagy inducement alleviated the sea cucumber (Stichopus japonicus) autolysis with exposure to stress stimuli of ultraviolet light.

Autolysis is the most important restrictive factor for the live sea cucumber trade and commercial transportation. Thus, it is essential to investigate the mechanism of autolysis activation or deactivation in the sea cucumber. In this study, monodansylcadaverine staining and Western blotting experiment methods indicated the implication of autophagy in the ultraviolet (UV) exposed sea cucumbers. The health condition was observed after the sea cucumbers (Stichopus japonicus) were gastric perfusion with autophagic inhibitor (3-methyladenine) or inducer (rapamycin) and exposure to UV light for half an hour. The protein expressions of LC3-II and Atg5 appeared immediately after UV exposure and then vanished 1h later. The autophagosome formation in coelomic fluid cells confirmed the autophagy appearance pattern of LC3-II and Atg5. The sea cucumber individuals maintained the health condition during the entire event of autophagy. The autophagic inhibitor along with UV exposure contributed to sea cucumber's swollen intestinal tissues, but the autophagic inducer functioned to alleviate and neutralize the UV effect. The autophagy procedure analysis demonstrated that autophagy plays a role to maintain the health condition of sea cucumber during autolysis inducement. The autolysis of sea cucumber can be alleviated or postponed by the exogenous autophagy inducer and this finding would benefit the live sea cucumber transportation. © 2021 Society of Chemical Industry.

Inhibition of ultraviolet-induced sea cucumber (Stichopus japonicus) autolysis by maintaining coelomocyte intracellular calcium homeostasis

Apoptosis plays a critical role in sea cucumber autolysis. To investigate the ultraviolet (UV)-induced apoptosis, sea cucumbers with and without injection of BAPTA-AM (cytosolic calcium chelator) were exposed to UV (15 W/m2) for 30 min. The results showed that UV irradiation caused several changes in sea cucumber coelomocytes, including calcium imbalance, abnormal morphology of endoplasmic reticulum, upregulation of pro-apoptotic proteins CRT, CHOP, and caspases 9 and 3, and downregulation of anti-apoptotic protein Bcl-2. A comparison between the two groups showed that injection of the calcium chelator into sea cucumbers helped maintain coelomocyte intracellular calcium homeostasis and suppressed other abnormal changes caused by ER stress, indicating apoptosis in sea cucumbers is mediated by calcium imbalance and follows the activation of the ER stress pathway. Therefore, this study broadens understanding of the apoptotic mechanism involved in sea cucumber autolysis, which is helpful in developing preservative agents for sea cucumbers.

Type I collagen from sea cucumber (Stichopus japonicus) and the role of matrix metalloproteinase-2 in autolysis

Autolysis is a major issue affecting the quality of sea cucumber during transportation and processing. To further clarify the mechanism, characteristics of type I collagen from sea cucumber Stichopus japonicas and its degradation by recombinant matrix metalloproteinase-2 (rMMP-2) were investigated. Scanning electron microscopic (SEM) images showed that the body wall of fresh sea cucumber with intact collagen fibrils was disaggregated after autolysis. Pepsin-soluble collagen of Stichopus japonicas (SjPSC) was prepared and its filamentous feature was observed using SEM. Circular dichroism spectrum analysis showed that SjPSC had native triple helix structure and its denaturation temperature was 34.0 °C using differential scanning calorimeter. The α chain gene of SjPSC encoding 1400 amino acid residues was cloned. A polyclonal antibody against SjPSC was prepared and was used to detect collagen degradation during autolysis. The effect of rMMP-2 on the body wall strongly suggested that MMP-2 had an important role in sea cucumber autolysis by disaggregating collagen fibres into fibrils.

Inhibitory effect of natural metal ion chelators on the autolysis of sea cucumber (Stichopus japonicus) and its mechanism

Live sea cucumbers (Stichopus japonicus) were stored in a solution containing oxalic acid and tea polyphenols as natural metal ion chelators. The inhibitory effects of these chelators on the autolysis phenomenon and the underlying mechanism of action were investigated for the first time by using scanning electron microscopy, differential scanning calorimetry, low-field nuclear magnetic resonance and confocal laser scanning microscopy. External stimuli cause autolysis through the release of calcium ions (Ca2+) from cells into the extracellular connective tissue, initiating activity of the matrix metalloprotease (MMP) in the sea cucumber body wall (SCBW). MMP subsequently degrades the microfibrillar networks, that support the interconnecting collagen fibres and the interfibrillar proteoglycan bridges linking the collagen fibrils, to release the water restricted within the interspaces between collagen fibres and collagen fibrils, ultimately causing mucoid degeneration of SCBW. The natural metal ion chelators significantly inhibited the activation of MMP by chelating Ca2+, consequently effectively preventing the autolysis of SCBW.

In vivo mechanism of action of matrix metalloprotease (MMP) in the autolysis of sea cucumber (Stichopus japonicus)

Matrix metalloprotease (MMP) is the key endogenous protease that boosts the autolysis of sea cucumber, while its action and mechanism in vivo are still unclear. MMP inhibitor (1,10-phenanthroline) was injected into coelomic cavity of sea cucumber (Stichopus japonicus) and its effects on autolysis and mechanism of action were investigated for the first time by using scanning electron microscopy, multi-functional in vivo imaging system, and confocal laser scanning microscopy. In response to external stimuli, the level of Ca2+ in live sea cucumber dermis increased, which may activate MMP activity. MMP inhibitor could diffuse from coelomic cavity into body wall and significantly prevented the activation of MMP by chelating Ca2+, and thus, retarded the autolysis of fresh sea cucumber by preventing degradation of interfibrillar proteoglycan bridges between adjacent collagen fibrils. This work provides information toward understanding the mechanism behind the sea cucumber autolysis and demonstrate a potential approach to prevent the autolysis. Practical applications Autolysis occurs in sea cucumber in response to external stimulation. Matrix metalloproteinase (MMP) has been found to be the key endogenous protease that boosts the autolysis of sea cucumber body wall (SCBW). However, the role of MMP in the autolysis of SCBW in vivo and the mechanisms involved remain unclear. In this study, MMP inhibitor (1,10-phenanthroline) was injected into the body cavity of live sea cucumber, and its effects on in situ changes in SCBW were studied. Our results indicated that the activity of endogenous MMP in live SCBW was activated in response to external stimulation, which may be responsible for the autolysis. After injection into coelomic cavity, MMP inhibitor could diffuse into SCBW and significantly reduced the progress of typical morphological and microstructural changes of autolytic sea cucumber. Through chelating Ca2+, the MMP inhibitor could prevent the activation of MMP, and thus reduced the autolysis of fresh sea cucumber.

Effects of collagenase type I on the structural features of collagen fibres from sea cucumber (Stichopus japonicus) body wall

The autolysis of sea cucumber is caused by depolymerisation of collagen fibres and unfolding of fibrils. In order to highlight the role of collagenase in sea cucumber autolysis, collagen fibres from sea cucumber were hydrolysed with collagenase type I. Electron microscopy (EM) results indicated the collagenase caused partial depolymerisation of collagen fibres into fibrils due to the fracture of proteoglycan interfibrillar bridges, as well as uncoiling of collagen fibrils. Chemical analysis and SDS-PAGE both indicated collagenase induced a time-dependent release of glycosaminoglycans (GAGs) and soluble proteins, which further demonstrated the degradation of proteoglycan interfibrillar bridges. Collagenase also degraded collagens by releasing soluble hydroxyproline (Hpy), with the dissolution rate of Hyp reaching 11.11% after 72 h. Fourier transform infrared analysis showed that collagenase caused the reduction of intermolecular interactions and structural order of collagen. Hence, collagenase participated in the autolysis of sea cucumber by deteriorating both macromolecular and monomeric collagens.

The role of matrix metalloprotease (MMP) to the autolysis of sea cucumber (Stichopus japonicus).

Sea cucumber (Stichopus japonicus) is easy to autolysis in response to a variety of environmental and mechanical factors. In the current study, collagen fibres were extracted from fresh sea cucumber body wall and then incubated with endogenous matrix metalloprotease (MMP) of sea cucumber. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC), chemical analysis and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis were utilized to demonstrate the changes in collagen fibres, collagen fibrils and collagen proteins. Moreover, a verification experiment was also carried out to confirm the contribution of MMP to the autolysis of sea cucumber. Endogenous MMP caused complete depolymerization of collagen fibres into smaller collagen fibril bundles and collagen fibrils due to the fracture of proteoglycan interfibrillar bridges. Meanwhile, endogenous MMP also caused partial degradation of collagen fibrils by releasing soluble hydroxyproline and pyridinium cross-links. Furthermore, the treatment with MMP inhibitor (1,10-phenanthroline) prevented the autolysis of tissue blocks from S. japonicus dermis. Endogenous MMP was the key enzyme in the autolysis of sea cucumber, while its action still focused on high-level structures of collagens especially collagen fibres. © 2019 Society of Chemical Industry.

Contribution of Cathepsin L to Autolysis of Sea Cucumber Stichopus japonicus Intestines

ABSTRACTIn order to provide the fundamental basis for control and application of sea cucumber autolysis, the contribution of cathepsin L to intestinal proteolysis in sea cucumber (Stichopus japonicus) was investigated. Cathepsin L mRNA expression was significantly up-regulated by 1.80 ± 0.44-fold with ultraviolet irradiation (p< 0.05). According to the protein pattern identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and determination of the release of trichloroacetic acid (TCA)-soluble oligopeptide, high intestinal protein degradation was observed at pH 4.4 and 48°C. Intestinal protein proteolysis was strongly inhibited by cathepsin L inhibitors, including trans-epoxysuccinyl-L-leucyl-amido (4-guani-dino)butane (E-64), iodoacetic acid (IA), and antipain (AP). These results imply that cathepsin L is involved in intestinal autolysis in the sea cucumber S. japonicus.Abbreviations: TCA: trichloroacetic acid; Z-Phe-Arg-AMC: Z-Phe-Arg-7-amido-4-methylcou-marin; IA: iodoacetic acid; E-64: trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane; AP: antipain; SBTI: soybean trypsin inhibitor; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; RT-PCR: reverse transcription-polymerase chain reaction

Action of trypsin on structural changes of collagen fibres from sea cucumber (Stichopus japonicus)

Trypsin, a representative serine proteinase, was used to hydrolyse the collagen fibres from sea cucumber (Stichopus japonicus) to highlight the role of serine proteinase in the autolysis of sea cucumber. Partial disaggregation of collagen fibres into collagen fibrils upon trypsin treatment occurred. The trypsin treatment also caused a time-dependent release of water-soluble glycosaminoglycans and proteins. Therefore, the degradation of the proteoglycan bridges between collagen fibrils might account for the disaggregation of collagen fibrils. For trypsin-treated collagen fibres (72 h), the collagen fibrils still kept their structural integrity and showed characteristic D-banding pattern, and the dissolution rate of hydroxyproline was just 0.21%. Meanwhile, Fourier transform infrared analysis showed the collagen within trypsin-treated collagen fibres (72 h) still retaining their triple-helical conformation. These results suggested that serine proteinase participated in the autolysis of S. japonicus body wall by damaging the proteoglycan bridges between collagen fibrils and disintegrating the latter.

Molecular cloning and functional characterization of cathepsin D from sea cucumber Apostichopus japonicus

Cathepsin D (CTSD, EC 3.4.23.5) belongs to aspartic protease family, which is located in lysosomes and is distributed in diverse tissues and cells. CTSD has a wide variety of physiological functions, owing to its proteolytic activity in degradating proteins and peptides. In the current study, the full length cDNA of sea cucumber (Apostichopus japonicus) cathepsin D (AjCTSD) was firstly cloned, then the association between AjCTSD and sea cucumber autolysis was investigated. The full length cDNA of AjCTSD was 2896 bp, with an open reading frame (ORF) for 391 amino acids. AjCTSD was widely expressed in body wall, muscle and intestine; the expression level was the highest in intestine, followed by muscle and body wall. Compared to fresh tissues, AjCTSD expression levels were significantly increased in all examined autolytic tissues. The purified recombinant AjCTSD promoted the degradation of sea cucumber muscle. In conclusion, AjCTSD contributed to sea cucumber muscle autolysis.

Structural and biochemical changes in dermis of sea cucumber (Stichopus japonicus) during autolysis in response to cutting the body wall

The autolysis of sea cucumber body wall is caused by endogenous proteolysis of its structural elements. However, changes in collagen fibrils, collagen fibres and microfibrils, the major structural elements in sea cucumber body wall during autolysis are less clear. Autolysis of sea cucumber (S. japonicus) was induced by cutting the body wall, and the structural and biochemical changes in its dermis were investigated using electron microscopy, differential scanning calorimetry, infrared spectroscopy, electrophoresis, and chemical analysis. During autolysis, both collagen fibres and microfibrils gradually degraded. In contrast, damage to microfibrils was more pronounced. Upon massive autolysis, collagen fibres disaggregated into collagen fibril bundles and individual fibrils due to the fracture of interfibrillar bridges. Meanwhile, excessive unfolding of collagen fibrils occurred. However, there was only slight damage to collagen monomers. Therefore, structural damage in collagen fibres, collagen fibrils and microfibrils rather than monomeric collagen accounts for autolysis of S. japonicus dermis.

Effects of endogenous cysteine proteinases on structures of collagen fibres from dermis of sea cucumber (Stichopus japonicus)

Autolysis of sea cucumber, caused by endogenous enzymes, leads to postharvest quality deterioration of sea cucumber. However, the effects of endogenous proteinases on structures of collagen fibres, the major biologically relevant substrates in the body wall of sea cucumber, are less clear. Collagen fibres were prepared from the dermis of sea cucumber (Stichopus japonicus), and the structural consequences of degradation of the collagen fibres caused by endogenous cysteine proteinases (ECP) from Stichopus japonicus were examined. Scanning electron microscopic images showed that ECP caused partial disaggregation of collagen fibres into collagen fibrils by disrupting interfibrillar proteoglycan bridges. Differential scanning calorimetry and Fourier transform infrared analysis revealed increased structural disorder of fibrillar collagen caused by ECP. SDS-PAGE and chemical analysis indicated that ECP can liberate glycosaminoglycan, hydroxyproline and collagen fragments from collagen fibres. Thus ECP can cause disintegration of collagen fibres by degrading interfibrillar proteoglycan bridges.

Characterization of proteolysis in muscle tissues of sea cucumber Stichopus japonicus.

The proteolysis in muscle tissues of sea cucumber Stichopus japonicus (sjMTs) was characterized. The proteins from sjMTs were primarily myosin heavy chains (MHCs), paramyosin (Pm), and actin (Ac) having a molecular mass of approximately 200, 98, and 42 kDa, respectively. Based on SDS-PAGE analysis and quantification of trichloroacetic acid (TCA)-soluble peptides released, degradation of muscle proteins from sjMTs was favorable at pH 5 and 50°C. Proteolysis of MHCs was mostly inhibited by cysteine protease inhibitors, including trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane (E-64) and antipain (AP). E-64 and AP completely inhibited the degradation of Pm and Ac, while iodoacetic acid showed a partially inhibitory effect. These results indicated that the proteolysis of sjMTs was mainly attributed to cysteine proteases. Avoidance of setting the tissues at 40-50°C and slightly acidic condition and inhibition of cysteine proteases are helpful for decreasing sea cucumber autolysis.

Characterization of a Main Extracellular Matrix Autoenzyme from the Dermis of Sea Cucumber Stichopus monotuberculatus: Collagenase

Sea cucumber is easy to undergo autolysis, causing serious economic loss in the aquaculture, fishery, and food processing. However, research on the autolysis of sea cucumber is limited. In the present study, the autoenzyme that could hydrolyze the structural protein of body wall was identified and characterized in sea cucumber Stichopus monotuberculatus. Although the main edible part of sea cucumber was the body wall, its microstructure was shown to be almost composed of collagen fibers belonging to extracellular matrix, indicating that the body wall of S. monotuberculatus belonged to dermis tissue. Starting from this perspective, crude collagen fibrils from dermis of S. monotuberculatus was isolated, and pepsin solubilized collagen was also obtained by hydrolyzing collagen telopeptides from crude collagen fibrils. Crude collagen fibrils and pepsin solubilized collagen were then acting as substrates, respectively, to identify dermis autoenzyme which had hydrolytic activity on dermis collagen. After that, a collagenase of 45 kDa was identified with the method of collagen zymograph, and the content of soluble protein was suppressed by collagenase inhibitor significantly in the autolysis of dermis. Finally, the effect of environmental conditions on collagenase activity was studied, results showed its best activity was at 40°C and pH 8, and divalent metal ion Mn2+ was essential for its activity. As a whole, our results showed that the dermis of sea cucumber was composed of collagen fibers, and collagenase was the main enzyme resulting in autolysis of sea cucumber.

Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus)

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.

Apoptosis induction is involved in UVA-induced autolysis in sea cucumber Stichopus japonicus

Autolysis easily happens to sea cucumber (Stichopus japonicus, S. japonicus) for external stimulus like UV exposure causing heavy economic losses. Therefore, it is meaningful to reveal the mechanism of S. japonicas autolysis. In the present study, to examine the involvement of apoptosis induction in UVA-induced autolysis of S. japonicas, we investigated the biochemical events including the DNA fragmentation, caspase-3 activation, mitogen-activated protein kinases (MAPKs) phosphorylation and free radical formation. Substantial morphological changes such as intestine vomiting and dermatolysis were observed in S. japonicus during the incubation after 1-h UVA irradiation (10W/m2). The degradation of the structural proteins and enhancement of cathepsin L activity were also detected, suggesting the profound impact of proteolysis caused by the UVA irradiation even for 1h. Furthermore, the DNA fragmentation and specific activity of caspase-3 was increased up to 12h after UVA irradiation. The levels of phosphorylated p38 mitogen activated protein kinase (MAPK) and phosphorylated c-Jun.-N-terminal kinase (JNK) were significantly increased by the UVA irradiation for 1h. An electron spin resonance (ESR) analysis revealed that UVA enhanced the free radical formation in S. japonicas, even through we could not identify the attributed species. These results suggest that UVA-induced autolysis in S. japonicas at least partially involves the oxidative stress-sensitive apoptosis induction pathway. These data present a novel insight into the mechanisms of sea cucumber autolysis induced by external stress.

ROS production in homogenate from the body wall of sea cucumber Stichopus japonicus under UVA irradiation: ESR spin-trapping study

Sea cucumber Stichopus japonicus (S. japonicus) shows a strong ability of autolysis, which leads to severe deterioration in sea cucumber quality during processing and storage. In this study, to further characterize the mechanism of sea cucumber autolysis, hydroxyl radical production induced by ultraviolet A (UVA) irradiation was investigated. Homogenate from the body wall of S. japonicas was prepared and subjected to UVA irradiation at room temperature. Electron Spin Resonance (ESR) spectra of the treated samples were subsequently recorded. The results showed that hydroxyl radicals (OH) became more abundant while the time of UVA treatment and the homogenate concentration were increased. Addition of superoxide dismutase (SOD), catalase, EDTA, desferal, NaN3 and D2O to the homogenate samples led to different degrees of inhibition on OH production. Metal cations and pH also showed different effects on OH production. These results indicated that OH was produced in the homogenate with a possible pathway as follows: O2−→H2O2→OH, suggesting that OH might be a critical factor in UVA-induced S. japonicus autolysis.

Effect of matrix metalloproteinase on autolysis of sea cucumber Stichopus japonicus

To investigate the relationship between matrix metalloproteinase (MMPs) and autolysis of sea cucumber Stichopus japonicus, the dermis homogenate was incubated at 25°C to induce autolysis. EDTA Na2 and 1,10-phenanthroline were used to verify the effect of MMPs on autolysis, which was measured by soluble protein and protein pattern. Soluble protein level increased during a 6-h autolysis process. SDS-PAGE demonstrated obvious protein degradation with the concomitant occurrence of degradation products. The above two indicators could be inhibited significantly by EDTA Na2 and 1,10-phenanthroline, indicating that MMPs might play a significant role in autolysis of sea cucumber.

Identification of a novel gelatinolytic metalloproteinase (GMP) in the body wall of sea cucumber (Stichopus japonicus) and its involvement in collagen degradation

Body wall that mainly consists of collagen and polysaccharides is the edible part of sea cucumber and is easy to go autolysis, while the proteinase(s) responsible for autolysis remains unclear. In the present study, a gelatinolytic metalloproteinase (GMP) from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatographic steps including DEAE-Sephacel, Sephacryl S-200, Q-Sepharose and Phenyl-Sepharose. The molecular mass of GMP as estimated by SDS-PAGE and gelatin zymography was 45kDa. The enzyme revealed high activity at a slightly alkaline pH range (8.0–9.0) and the optimal temperature was at 40–45°C. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the activity, whereas other proteinase inhibitors did not show any effect. Peptide mass fingerprinting of the enzyme obtained 3 peptide fragments with a total of 58 amino acid residues, which was 91.4% identical to an alkaline metalloprotease from Pseudomonas fluorescens, strongly suggesting it is a metalloproteinase. Divalent metal ion Ca2+ is essential for its activity, indicating it is a calcium-dependent metalloproteinase. Furthermore, GMP hydrolyzed collagen effectively at 37°C and gradually even at 4°C, suggesting its involvement in the autolysis of sea cucumber.