Structural, Expression And Functional Analysis Of Chitotriosidase-like And Lysozyme Genes From Amphioxus Branchiostoma Japonicum

Amphioxus is a transitional species between invertebrates and vertebrates, whichis used as an important model organism for studying the origin and evolution ofvertebrates. The hepatic diverticulum of amphioxus has long been regarded as theprecursor of vertebrate liver, which plays a role in immune defense. We report herethe cloning, expression and functional analysis of the genes BjChTl (Branchiostomajaponicum chitotriosidase-like) and Lys (lysozyme), which both are involved inimmune defense in amphioxus. At the same time to demonstrate the two genes areassociated with the innate immune of amphioxus, the results of this research alsoprovide more experimental basis for the origin of the vertebrate liver.The mammalian chitinase family18consists of two memmbers，chitotriosidase(ChT) and acidic chitinase (AMCase). Despite the enormous progress on mammalianChT study, little information regarding ChT is available to date in lower animals. ByPCR and RACE reaction we obtained the full length of BjChTl (Branchiostomajaponicum chitotriosidase-like). The full length of the cDNA is1805bp and thelength of ORF is1503bp. Its5-untranslated region (UTR) is93bp and its3-UTR is209bp. The ORF of the cDNA encods a deduced protein of501amino acids with acalculated molecular weight of about56.9kDa and an isoelectric point (pI) of5.97.The protein has a putative N-terminal signal peptide of17amino acids, the29-399amino acid residues is a catalytic domain, the406-423acid residues is the hingeregion and the448-500acid residues is chitin binding region. BjChTl has the typicalstructure of family18chitinases. Sequence analysis revealed that BjChTl sharedapproximately33–43%and30–34%sequence identity to vertebrate and invertebratechitinases, respectively. Additionally, BjChTl was closely identical to mammalianChT (~46%) and AMCase (~41%) at amino acid level. The single B. floridaechitotriosidase-like cDNA encoded a protein with~87.6%identity to BjChTl atamino acid level, suggesting that BjChTl is highly conserved in interspecies. Alignment of BjChTl with ChT and AMCase revealed the core of BjChTl to be wellconserved. The phylogenetic analysis showed that BjChTl appears the commonancestor of ChT and AMCases. As ChT-like gene is highly conserved in both B.japonicum and B.floridae, the genomic structure of B. floridae ChTl gene thus allowsus to compare the amphioxus gene structure with that of vertebrate ChT genes. Allthe vertebrate ChT genes contained11–12exons. Although amphioxus ChTl gene has10exons and9introns, but its exon2and exon4have the intron insert. Additionally,each exon of amphioxus ChTl gene possessed an identical counterpart in thevertebrate ChT genes. These demonstrate that the general genomic organization ofChT genes is highly conserved through-out chordate evolution in terms of bothexon–intron structure and sequence homology, hinting at the clue that ChT genetranscription is regulated similarly in cephalochordate and vertebrates. To researchthe function of different domains，we expression the full length and the truncatedBjChTl. Like human ChT, recombinant BjChTl was able to bind to chitin particlesderived from crab shells, to hydrolyze artificial chitin substrate4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside and to inhibit the growth ofthe fungus C. albicans. These indicate that BjChTl also acts as a chiti-nolytic enzymeas well as a fungistatic molecule. Interestingly, BjChTl is mainly expressed in thehepatic caecum and hind gut. This suggests that BjChTl in vivo may be involved infood digestion via degrading chitin as well as in immune responsevia inhibitingfungal growth. However, BjChTl-CD’s enzymatic activity was almost completely lostand its antifungal activity significantly reduced. It shows that the chitin-bindingdomain is necessary for the immunocompetence of BjChTl.Lysozymes include six types: chicken-type (c-type), goose-type (g-type),invertebrate-type (i-type), T4phage (phage-type), bacterial and plant. Both c-typeand g-type as well as i-type lysozymes belong to animal lysozymes. Bioinformaticsanalyses revealed that amphioxus contains all the three types of animal lysozymes,which is the only species that possesses the three types of lysozymes among all theextant animals. Therefore, it will be of vital significance to investigate the structure,expression and function of the lysozymes from amphioxus. The full length of amphioxus g-type lysozyme is1014bp，the open reading frame is801bp，5′-UTR is83bp，3′-UTR is130bp，with a signal peptide of15amino acids, encoding267amino acids, and the protein’s molecular weight is30.03kDa, as well as theisoelectric point is6.3. The full length of amphioxus c-type lysozyme is651bp，theopen reading frame is423bp，5′-UTR is60bp，3′-UTR is168bp，with a signalpeptide of17amino acids, encoding141amino acids, and the protein’s molecularweight is17.69kDa, as well as the isoelectric point is6.64. The full length ofamphioxus i-type lysozyme is1053bp，the open reading frame is501bp，5′-UTR is56bp，3′-UTR is496bp，with a signal peptide of21amino acids, encoding167amino acids, and the protein’s molecular weight is20.02kDa, as well as theisoelectric point is7.84. The result of Multiple sequence alignment shows that thethree types of lysozymes are all have the catalytic active sites, g-type lysozyme ofamphioxus has SLT domain and substrate binding site, c-type lysozyme ofamphioxus has8conserved cysteine residues, but i-type lysozyme of amphioxus hasonly one isomerase active site. The identity of g-type lysozyme between amphioxusand vertebrate is32%-50%, between amphioxus and invertebrate is about40%; theidentity of c-type lysozyme between amphioxus and other species is between31%-44%; the identity of i-type lysozyme between amphioxus and other species isassociated with the genetic relationship，that means the genetic relationship is closer,the consistency is higher. The change of genomic structure of g-type lysozyme islittle；the exon3of c-type lysozyme has intron insert in the evolutionary process，butthe intron insertion region is not in the catalytic active site region；i-type lysozymeshows degradation trend during species evolution. G-type and c-type lysozymes weremainly expersse in the hepatic caecum and midgut, i-type lysozyme was mainlyexpersses in the gill and the notochord. The recombinate protiens all have lysozymeactivity, it shows that the three types of lysozymes have biological function andmaybe involved in the innate immunity of amphioxus.