The Study On Generation Mechanism Of AMP And The Activity Of Gonad Polysaccharides And Polypeptides Of Apostichopus Japonicus Based On Proteomics And Other Techniques

Apostichopus japonicus?AJ?is mainly distributed in the cold waters around northeast Asia because of its lack of adaptive immune system,which can only rely on innate immune system to protect against external adverse stimuli.Antimicrobial peptides?AMP?are important components of the innate immune system and play an important role in aquatic animals' response to external environmental stress.Compared with the research on its functional mechanism,the research on its generation mechanism is less.The research on the AMP generation mechanism will be helpful to the prevention and control of the disease in AJ culture.Meanwhile,the enzymatic hydrolysis technology of Apostichopus japonicus gonad?AJG?was optimized,and a variety of active peptides and polysaccharides were prepared.Then by means of proteomics,cell biology and animal models,their activities were studied in anti-tumor,anti-oxidation and immunity enhancement.This paper mainly consists of the following parts:1.AJ antibacterial test and determination of immune-related enzyme activitiesIn order to explore the AMP generation mechanism of AJ,the representative pathogenic bacteria Vibro Splendidus and ecological control bacteria Halomonas were selected to stimulate AJ,and then detect the dynamic changes of the key enzymes related to immunity.It was found that in a short period?0?24 h?,enzyme activities in AJ in all experimental groups were significantly decreased or increased.Through the analysis of bacterial community,in the experiment,with the addition of 2 exogenous dominant bacteria,Alteromonadales can multiply,which is represented by Pseudoalteromonas.However,with a long stage,the bacterial community diversity decreased gradually.2.The proteomics study of AMP generation mechanismThe changes of proteome in AJ were detected by using the iTRAQ labeled quantitative technique.The results showed that the variety and quantity of differentially expressed proteins increased with time,and the different degree was also increasing.The common biological process?BP?of Tr1₁2 h group and Tr2₁2 h group was S-adenosylhomocysteine catabolic process.Cellular component?CC?analysis showed that there was continuity in each experimental group.Molecular function?MF?analysis found that the common MF in the Tr1₁2 h group and Tr2₁2 h group was adenosylhomocysteinase activity which was important to the methylation function.At earlier stage the numbers of signalling pathways were small,mainly including phagosome pathway and so on.In the later stage,a series of signalling pathways related to epithelial tissue defense were initiated,such as adherens junction?ADJ?pathway,cytoskeleton regulation,and so on.The protein-protein interaction?PPI?analysis showed that proteins in the ribosome pathway were up-regulated at 12 h,while the epithelial tissue defense related proteins,such as actin and alpha-catenin,were up-regulated at 24 h.3.The peptidomics study on AMP generation mechanismThe changement of peptidome was detected by peptidomics technique.The results showed that most of the peptides were up-regulated in each experimental group,and the proportion of peptides with high content?>30%?of hydrophobic AA was very high.The main BPs of trace proteins in control group and Tr1 group were glycerol metabolism and glucose metabolism.The BPs in Tr2 group mainly included depolymerization of actin and protein targeting to plasma membrane.The CC of the control group was mostly concentrated in transport vesicle membrane,while Tr1 and Tr2 groups were concentrated in the extracellular region and peroxisomes.The MF analysis showed that,in addition to the activity of isomerase activity and dehydrogenase activity shared with the control group,the Tr1 group also participated in the binding of actin monomers.The MF of Tr2 group is concentrated in actin monomer binding and cytoskeletal protein binding.The results of signalling pathway analysis showed that the KEGG signalling pathways of the control group and the Tr1 group were the same,focusing on glucose metabolism and fatty acid metabolism.Tr2 groups focused on AA metabolism and fatty acid metabolism.The results of PPI analysis showed that proteins participated in multiple signalling pathways in each experimental group were belonged to a kind of peroxidase bifunctional enzyme.In addition,three proteins including actin were up-regulated in Tr2 groups.The isolation and antibacterial experiments of AMP extracts showed that,except for H1,all extracts had significant inhibitory effect?P<0.05?,and V1?cutting-off Mr<1 ku?had the best effect.Through the prediction and analysis of AMP,we screened a polypeptide sequence AJAMP with 18 amino acids and the hydrophobicity AA content of 61.11%,and the sequence was strictly conservative.After comparison and analysis of relevant AMPs in the general AMP database,it is speculated that this AMP may be produced by degrading actin.4.Enzymatic hydrolysis process optimization and activity of PolypeptidesWith AJS as the material,the optimized enzymatic hydrolysis parameters of papain?AP?,protamex?CP?and flavourzyme?FP?were determined,and the hydrolysis degree under optimum condition was 32.25%?31.20%and 12.00%respectively.The results of separation and purification and antioxidant activity of Apostichopus japonicus spermary peptide?AJSP?and Apostichopus japonicus egg peptide?AJEP?showed that AJSP with cutting-off Mr<1 ku had a strong scavenging ability to ·OH in vitro.AA analysis results showed that it was rich in content of acid A A and hydrophobicity AA.The mouse antioxidant model showed that AJSP was able to protect mice against oxidative damage.Three AJEPs with cutting-off Mr<1 ku,1?5 ku and 5?10 ku respectively,all showed strong abilities of scavenging free radicals.Continue to use the IEC and HSCCC for purification,a kind of pure chromatographic AJEP was obtained.Then,the chemical composition and Mr distribution of AJEP and the AJBP were detected,and their Mr was found between 130?1600 u and 130?2500 u.AJEP1 and AJBP1 SLP experimental results confirmed that they were beneficial to the growth of mouse lymphocytes in vitro.Mice models experiments further confirmed that they can enhance the immunity of mice.5.Research on the anti-tumor mechanism of AJS polysaccharide based on iTRAQ technologyThree kinds of polysaccharides?SP,SPS1 and SPSS1?prepared in the process of purification were selected to carry out the experiment of anti-tumor cell HepG2 in vitro.Physical and chemical analysis confirmed that they had typical polysaccharide characteristics and high sulfate content.The dominant monosaccharides in SPSS 1 were Gal and Lac,while in SPSS2,they were Fuc and Lac.SPSS1 was entered by endocytosis,promoting the release of CTS and cholesterol by reducing the stability of lysosomal membrane,and activating the ROS pathway.By affecting APC/C and Mad2,tumor cells were inhibited in the metaphase of division.By inhibiting the key enzyme activity in the metabolic pathway,the growth of tumor cells and the generation of key molecules needed for infection were reduced.Meanwhile,the activity of some enzymes is enhanced to promote the decomposition of key substances such as LPA.Through the above comprehensive action,SPSS1 plays an anti-tumor activity.