Collagen Of Sea Cucumber(Stichopus Japonicus)and Proteinases Responsible For Autolysis

Sea cucumbers are unique inverterbarates with special texture.They are widely consumed as traditional food in China,Japan,Korea and other Asian countries.In China,the production of sea cucumber is increasing annually to meet consumers'demand.However,sea cucumbers are easily subject to autolysis,which is a major problem in transportation and processing.The degradation of structural proteins by endogenous proteinases was proposed as the major cause for autolysis.Collagen is a significant nutritional element of sea cucumber,accounting for approximately 70%of total protein in sea cucumber.It is also the major component in the extracellular matrix,and contributes toughness and strength.The degradation of collagen not only leads to nutrient loss,but also seriously affects stability of body wall,it is thus considered as the major reason attributing to sea cucumber autolysis.In order to illuminate the metabolism of collagen and deliver important information of the mechanism of autolysis,the present study investigated the characteristics of sea cucumber collagen and the degradation of collagen by endogenous proteinases.Pepsin-soluble collagen was purified from the body wall of sea cucumber Stichopus japonicus?Sj PSC?.SjPSC was a homotrimer consisting of three?chains and the molecular weight of?chain was approximately 130 kDa on SDS-PAGE.Fourier transform infrared and circular dichroism?CD?spectrum analysis demonstrated that it possesses native triple helix structure.Differential scanning calorimeter revealed that the denaturation temperature of SjPSC was 34.0±0.12?.A polyclonal antibody was prepared against SjPSC and it specifically reacted with collagens from two kinds of sea cucumber?Stichopus japonicus and Acaudina leucoprocta?in Western blot.The full-length sequence of?chain gene of SjPSC?SjCOL??was for the first time cloned,revealing an ORF of 4203 bp encoding a putative protein of 1400 amino acid residues.The deduced amino acid sequence of SjPSC included a signal peptide,an N-propeptide,an N-terminal non-triple helix domain?N-telopeptide?,a C-terminal non-triple helix domain?C-telopeptide?,a C-propeptide and a central triple helix domain.BLAST analysis showed that SjCOL?share less than 50%identities to?chains of type I collagens from sea urchin?Strongylocentrotus purpuratus?,abalone?Haliotis discus hannai?,tilapia?Oreochromis niloticus?,and pig?Sus scrofa domesticus?.Analysis of the sequence in the triple helix domain of SjCOL?revealed that the number of Gly-Gly-Y?9?was higher than that from abalone,tilapia and pig while the number of Gly-Pro-Pro?21?was lower.The physicochemical properties and amino acid sequences of collagen provide a theoretical basis for studying the changes of collagen and mechanism of endogenous proteinase in sea cucumber autolysis.Matrix metalloproteinases?MMPs?are critical proteinases responsible for degradation of collagens.Because of its low content,it is difficult to obtain natural MMP from sea cucumber,although its activity was high.Therefore,preparation of recombination MMP with enzymatic activity is a more practical way for studying its role during sea cucumber autolysis.A complete coding region of MMP-2 gene was cloned from the body wall and the ORF was 1977 bp encoding658 amino acid residues.The deduced amino acid sequence of MMP-2 contains a signal peptide,a propeptide domain,a catalytic domain with three repeats of fibronectin-type II region and a C-terminal hemopexin-like domain.The catalytic domain of MMP,encoding 351 amino acid residues(N₁₁₁ to T₄₆₁),was successfully expressed in Escherichia coli and the recombinant MMP-2?rMMP-2?was purified to homogeneity.The molecular weight of rMMP-2 was approximately 40kDa.CD specturum revealed that the transition temperature of rMMP-2 was 56.80±0.25°C.Enzymatic characterization of rMMP-2 showed that it hydrolyzed gelatin most effectively at pH8.5 and 40°C,suggesting it is a slight alkaline proteinase.In addition,Ca²⁺was required for optimal gelatinolytic activity.The rMMP-2 degraded collagen effectively and the hydrolysate revealed scavenging activities on both 2,2'-diphenyl-1-picrylhydrazyl free radical and hydrogen peroxide.These results suggested that MMP-2 is involved in collagen degradation during sea cucumber autolysis.A proteinase with collagenolytic activity was isolated to high purity from sea cucumber viscera in our previous research,and peptide mass fingerprinting?PMF?revealed that it belongs to the serine proteinase?SP?family.Similar to MMP,the low content of SP suggest it is necessary to obtain enzymatic active recombinant SP?rSP?for further study of the relationship between SP and collagen degradation.A complete coding region of SP gene was cloned and the ORF was 1134bp in length with 377 amino acid residues.The recombinant SP?rSP?with enzyme activity was successfully expressed in Escherichia coli and purified to homogeneity.It is of interest to notice that the rSP was similar to native SP exhibiting highest enzymatic activity at pH 7.5 and 35°C.The rSP could degrade collagen to peptides effectively and approximately 85%of hydrolysate was less than 5 kDa as analyzed by gel permeation chromatography.Furthermore,the cleavage sites of rSP on SjPSC were determined by LC-MS/MS.rSP cleavage preferentially occurred at sites of Arg at P₁ while less occasion was detected with Lys and even Gly at P₁.SP was thus proposed to play a key role in the degradation of collagen,contributing to the autolysis of sea cucumber.In summary,in the present study,the gene of SjPSC was cloned from sea cucumber?Stichopus japonicas?.The biochemical and physicochemical properties of collagen was studied comprehensively and systematically.The full length sequences of MMP-2 and SP were cloned,recombination MMP-2 and SP were expressed individually and their enzymatic properties were investigated in detail.Degradation of collagen by enzymatic active rMMP and rSP strongly suggested the involvement of their native forms in sea cucumber autolysis.rMMP-2 with high purity and activity was applied in the preparation of bioactive collagen peptides.The cleavage sites of SP on collagen were determined.The present study laid a foundation for futher investigation of the metabolic mechanism of collagen and revealed critical proteinases responsible for the autolysis of sea cucumber.