Comparative Analysis Of Prolyl Endopeptidases In Fish And Shellfish

Prolyl endopeptidase(PEP,EC3.4.21.26)is a special kind of protease in the serine protease family,which involved in many biological processes,such as learning and memory,cell proliferation and differentiation,and glucose metabolism by cleaving oligopeptides with length less than 33 amino acids and containing proline residues However,compared with PEPs in mammalian,Few studies on PEP in aquatic animals have been investigated.The functions of PEP in aquatic animals and whether there are differences between PEPs in different aquatic animals remains to be studied.In this thesis,using Lateolabrax japonicas,Hypophthalmichthys molitrix and Haliotis discus hannai as main research objects,a comparative analysis on the differences of properties and structures of PEPs in fish and shellfish was performed.Previous studies confirmed the existence of PEP in the muscle of sea bass and silver carp,while no work on PEP from shellfish has been reported.We identified that the enzyme activity of PEP in the gonad of abalone was the highest.PEP was then purified from muscle of sea bass,silver carp and abalone gonad by ammonium sulfate fractionation and a series of column chromatographies.The molecular weights of these PEPs were 84 k Da,80 k Da and 82 k Da,respectively indicating their difference among species.Enzymatic analysis revealed that the optimum temperatures of PEPs from sea bass and silver carp were both 35?,while that of abalone PEP was 25?,the optimum p Hs of the three PEPs were all 6.0,suggesting PEPs are active at low temperature and weak acidic conditions.Analysis of substrate specificity and protease inhibitor effect on PEPs further confirmed that PEPs belong to a special class of serine protease family.Real-time fluorescence quantitative PCR results showed that PEP had the highest expression level in brain of fish and gonad of abalone.The expression level of PEP in the process of male and female gonad development revealed that the expression level of PEP was the highest in the late stage of female gonad maturation and maximum level was in the middle stage of male gonad development.Different expression levels of PEP in stages of gonad development indicated that PEP may play critical role in the development of gonad physilogically.In order to compare the amino acid sequences of the three PEPs,in this thesis,the open reading frame of PEP from silver carp was cloned,which contained 2223 base pairs(bp)in length encoding 741 amino acid residues.Comparing with PEP sequences in sea bass and abalone,PEP sequences in sea bass and silver carp encoded 35 amino acid residues more than that in abalone at the N-terminal.The homology of PEPs between sea bass and abalone was58.7%,silver carp and abalone was 58.97%,sea bass and silver carp was 98.65%.It was predicted that the theoretical molecular weight of PEP from sea bass was 84.1 k Da,isoelectric point was 5.91,including 91 negatively charged amino acids and 76 positively charged amino acids.The theoretical molecular weight of PEP from silver carp was 84.2 k Da,isoelectric point was 5.93,containeing 99 negatively charged amino acids and 77 positively charged amino acids.The theoretical molecular weight of PEP from abalone was 80.3 k Da,isoelectric point was 5.55,containing 95 negatively charged amino acids and 75 positively charged amino acids.The structures of PEPs from sea bass and silver carp were similar to abalone PEP(PDB: 6JYM)by homology modeling,including a typical catalytic triad structure with typical characteristics of serine protease family.It was of interest to notice that the structures of PEPs contain a typical ?/?protease characteristic withcatalytic domain and ?-propeller.In order to investigate the effect of PEP endogenous inhibitor(PEPI)on the properties and structure of PEP,PEPI was purified from abalone by heat treatment,ammonium sulfate precipitation and a series of column chromatographies.SDS-PAGE and PAS staining showed that PEPI was a glycoprotein with molecular weight of 12 k Da.PEPI was a reversible competitive inhibitor,having high thermal and p H stabilities,which can maintain 80% inhibitory activity in the range of 40-90? and p H 5-12.In the presence of PEPI,the content of ?+?structure decreased by 2.7% compared with PEP alone,and the thermal denaturation temperature of PEP increased from 52? to 65? indicating the formation of PEP-PEPI complex and the increase of thermal stability.In this thesis,enzymatic analysis of PEPs from sea bass,silver carp and abalone was performed and their characteristics were compared in detail.The ORF sequence of PEP from silver carp was cloned.Differences of amino acid sequences,secondary and tertiary structures of the three PEPs were analyzed.The differences of PEP expression level in different tissues of sea bass and abalone were further analyzed,with a purpose to establish a theoretical basis for further exploring the function of PEP in aquatic animals.Study on the interaction mechanism of PEP and PEPI provided an important reference for exploring the regulation of PEPI on PEP in aquatic animals...