Study On The Genetic Information Of Complement Components Of The Whitespotted Bamboo Shark(Chiloscyllium Plagiosum) And Their Hepatic Expression Patterns

The complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions.As a pivotal effector arm of innate immunity,the complement system destroys and removes foreign invaders or infectious agents rapidly and potently,and bridging to adaptive immune response,hence it significantly contributes to the health status of the host.On the evolutionary perspective,the complement system is so ancient that its origin could be traced back as far as protostomes,since the central complement component,C3,and its homologues have been reported from some ancient invertebrates.However,it is generally accepted that an integrated modern complement system was established after the emergence of jawed vertebrates.Therefore,elasmobranchs,especially sharks could serve as excellent animal models to study ancestral complement components due to the unique phylogenetic position.Previously,several nucleic acid sequences had been identified in the cDNA library of regenerated liver of shark,Chiloscyllium plagiosum,suggesting a possible role of complement during liver regeneration.In this study,cloning and structural characterization of the full-length cDNA of C8a,C9 and factor I,as well as partial sequence of C3 and C4 of Chiloscyllium plagiosum are addressed,moreover,the hepatic expression of those complement components has been investigated.Specificly,the main works are included as follows:Molecular cloning of the alpha subunit of complement component C8(CpC8a)of Chiloscyllium plagiosumAmong the process of MAC assembly,a subunit of C8(C8a)is the first protein that traverses the lipid bilayer,and then initiates the recruitment of C9 molecules to form pore on target membranes.In this part,a full-length cDNA of C8a(CpC8a)is identified from Chiloscyllium plagiosum(whitespotted bamboo shark)by RACE.The CpC8a cDNA is 2183 bp in length,encoding a protein of 591 amino acids.The deduced CpC8α exhibits 89%,69%and 44%identity with nurse shark,frog and human orthologs,respectively.Sequence alignment indicates that the C8a is well conserved during the evolution process from sharks to mammals,with the same modular architecture as well as the identical cysteine composition in the mature protein.Phylogenetic analysis places CpC8a and nurse shark C8a in cartilaginous fish clade,in parallel with the teleost taxa,to form the C8a cluster with higher vertebrates.Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8a to mammals.Finally,expression analysis revealed CpC8a transcripts were constitutively highly expressed in shark liver,with much less expression in other tissues.The well conserved structure and properties suggests an analogous function of CpC8a to mammalian C8a,though it remains to be confirmed by further study.Molecular cloning of complement component C9(CpC9)of Chiloscyllium plagiosumAs the last component in the complement cascade,C9 is the key molecule in MAC assembly:the polymerization of C9 constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions,providing the cytotoxic effect of complement system.A full-length cDNA of C9(CpC9)is identified from Chiloscyllium plagiosum by RACE.The CpC9 cDNA is 2263 bp in length,encoding a protein of 603 amino acids,which shares 42%and 43%identity with human and Xenopus C9 respectively.Through sequence alignment and comparative analysis,the CpC9 protein was found well conserved,with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal.Phylogenetic analysis places it in a clade with C9 orthologs in higher veterbrate and as a sister taxa to the Xenopus.Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver,with much less or even undetectable expression in other tissues;demonstrating liver is the primary tissue for C9 synthesis.To sum up,the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response.Molecular cloning of the complement factor I(CpFI)of Chiloscyllium plagiosum Complement factor I(FI)is a plasma serine proteinase that plays an essential role in the modulation of the complement cascade.In the presence of substrate modulating cofactors(Factor H,C4bp,CR1,etc),FI cleaves the activation products of C3(i.e.C3b)and C4(i.e.C4b)to limit complement activity.In this part,the full length cDNA of factor I(CpFI)is isolated from the liver of the whitespotted bamboo shark(Chiloscyllium plagiosum).The CpFI cDNA is 2326 bp in length,encoding a protein of 671 amino acids,which shares 72-80%identity with FI of other sharks,higher than the teleosts(37-40%)and mammals(44-47%).The sequence alignment and comparative analysis indicates the FI proteins are well conserved,with the typical modular architecture and identical active sites throughout vertebrate evolution,suggesting the conservation in function.However,the additional sequence present between the leader peptide(LP)and the factor I membrane attack complex(FIMAC)domain in other fishes is also found in CpFI,which consists of two kind of tandem repeats.Phylogenetic analysis suggests that factor I of cartilaginous fish diverged prior to the emergence of mammals,as a sister taxa to the teleosts.On the other hand,expression analysis revealed that CpFI is ubiquitously distributed in several tissues with the constructive expression in liver,which might reflect the species and tissues specificity of FI expression pattern.Together with the earlier reports,the presense ofFI in differerent sharks suggests the existence of a well developed complement regulation mechanism in cartilaginous fish.Molecular characterization of partial cDNA sequence of complement C3(CpC3)and C4(CpC3)in Chiloscyllium plagiosumInteractions of complement factors C3,C4 and C5 are paramount for complement activation cascade.Actually,all three proteins are structural homologues arosed from a common ancestral molecule by gene duplications occurred in the early stage of vertebrate evolution.In this part,partial cDNA sequences of C3 and C4(CpC3 and CpC4)are cloned from the liver of the whitespotted bamboo shark(Chiloscyllium plagiosum).The sequence alignment and comparative analysis based on the partial sequences indicates a well conserved structure of C3 family,with the characteristic modular architecture and considerable identity.The evolutionary conservation is further confirmed by phylogenetic analysis.Expression analysis find that CpC3 is expressed in a wide assortment of organs with the constructive expression in liver,in contrast,CpC4 appears to be primarily expressed in liver.While it is possible that in Chiloscyllium plagiosum,the expression pattern of complement is component-specific.The hepatic expression patterns of complement components of Chiloscyllium plagiosumThe liver,which plays a crucial role in maintaining metabolic homeostasis,is a major site that biosynthesizes complement components found in plasma.However,indications of an association between liver and the complement system rely not only on that fact but also on growing evidence suggesting the importance of the liver as a target organ for effector complement compounds.It is more apparent that complement can act on the liver in a direct or indirect manner through receptors or cytokines.As mentioned above,several complement components of Chiloscyllium plagiosum were cloned and characterized,hereon,their hepatic expression patterns are studied through a partial hepatectomy model.It turns out that the basal expression of CpC3 in the normal liver outclass that of other components.As is the case for the pathological condition:induced by the injury,a rapid expression increase is commonly visible for all of the shark complement components,however,the most dramatic change occurs in CpC3.The unique hepatic expression profiles might shed light on the study of the relationship between the shark complement system and liver,which contributes to the comprehensive knowledge of the ancient complement system.