Change Of Glycoproteins In Autolytic Sea Cucumber(Stichopus Japonicus) Body Wall And Its Mechanism

Sea cucumber has high capacity in autolysis.In response to external stimuli,the body of sea cucumber relaxed,mucoid degeneration took place in body surface with white dermis being exposed.The massive tissue autolysis can lead to severe postharvest quality deterioration of sea cucumber,consequently cause heavy economic losses.The body wall of sea cucumber is a type of mutable connective tissue(MCT),which is mainly composed of collagen,glycoprotein and glycosaminoglycan(GAG).To date,studies on autolysis of sea cucumber are mainly focused on collagen.Nevertheless,research on glycoprotein and GAG in autolytic sea cucumber is very scarce.In this study,the sequential changes of microfibrils and collagen fibers in autolytic sea cucumber was observed and analyzed.Meanwhile,the microfibril networks were prepared from sea cucumber body wall,and typsin(a representative serine protease),endogenous cysteine protease and endogenous matrix metalloproteinase were used to hydrolyze the network to reveal the relationship between proteases and degradation of the networks.Furthermore,crude collagen fibers(CCF)and pepsin solubilized collagen(PSC)were prepared form the autolytic sea cucumber body wall and the structure of them were analyzed to reveal the structural changes in collagen components during autolysis.The present study was aimed to reveal the sequential changes of microfibrils and collagen fibers in autolytic sea cucumber and demonstrate the relationship between endogenous proteases and degradation of the microfibril networks.Light microscopic examination showed that the MCT of sea cucumber body wall is composed of the networks of collagen fibers and microfibrils.Along with the progression of autolysis,collagen fibers became sparse,and the density between collagen fibers decreased.However,the structures of collagen fibers were still integrated after 24 h of autolysis.By contrast,the damage on microfibril networks was more significant.After 24 h of autolysis,the microfibril networks between collagen fibers were disappeared.This indicated that there was a sequential change in microstructures of autolytic sea cucumber,namely,the damage of microfibrils occurred early,and then damage of collagen fibers took place.Microfibril networks were prepared from sea cucumber body wall,and various types of proteases were used to hydrolyze the networks.Transmission electron microscopy(TEM)observation showed that the microfibril had characteristic bead-like structure.Monosaccharide analysis indicated that the microfibrils had similar monosaccharide composition with those reported in literature.Trypsin could significantly degrade the microfibril networks and release soluble GAG and protein(peptides).However,treatment with endogenous cysteine protease could only release soluble proteins,while the degrading effect of endogenous matrix metalloproteinase was relatively slight.The above results indicated that the degrading effects on microfibrils of various proteases were different.To reveal the structural changes of collagen components in autolytic sea cucumber,CCF and PSC were prepared from sea cucumber body wall with autolysis time within 24 h,and the structures of them were analyzed.Results indicated that the CCF was mainly composed of collagen fibrils.After 24 h of autolysis,the characteristic D-banding pattern of CCK was still complete and clear,and the infrared spectrum of CCF also retained.At the same time,the infrared spectrum,ultraviolet spectrum and SDS-PAGE pattern of PSC were maintained during 24 h of autolysis.The above results indicated that there was no significant change in collagen components in sea cucumber with autolysis time less than 24 h.