Expression And Functional Identification Of TRP14 And YcaCR Gene From Amphioxus Branchiostoma Belcheri

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. It is well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis. Sequencing of the whole genome of Florida amphioxus offers a new opportunity to the research of molecular and evolutionary biology of amphioxus. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus TRP14 gene, AmphiTRP14 and amphioxus ycaC-related gene, AmphiycaCR from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtaoensis.AmphiTRP14 obtained from the gut cDNA library of amphioxus B. belcheri is 775 bp long and its longest open-reading frame (ORF) codes for a protein of 123 amino acids with a predicted molecular mass of approximately 14 kDa and an isoelectric point of 4.8. The deduced amino acid sequence shares 56% identity to human TRP14, and had a Trx-like motif Cys-Pro-Asp-Cys (CPDC). It has fiveβ-sheets and fiveα-helixes, characteristic of human TRP14. There are a number of putative TRP14 sequences from a variety of organisms including animals, plants and bacteria. The phylogenetic tree constructed by neighbor-joining method using the sequences of TRP14 proteins, putative TRP14 proteins and representative Trx proteins shows that both AmhiTRP14 and human TRP14 as well as putative TRP14 proteins are clustered together, separating from Trx proteins. These imply that TRP14 is a highly conserved ubiquitous protein with the active site CPDC. A search of the recently completed draft assembly and automated annotation of B. floridae genome reveals the presence of a Florida amphioxus TRP14 cDNA and its genomic DNA sequence. Sequence comparison demonstrates that AmphiTRP14 shares 94% identity to the deduced protein encoded by Florida amphioxus gene at the amino acid level. Analysis of the genomic structure exhibites that Florida amphioxus TRP14 gene consists of four exons and three introns, which is in accord with the genomic structures from deuterostomes such as human, bovine, mouse, rat and sea urchin, while putative TRP14 genes in prostostomes like fruit fly and nematode comprise three exons and two introns. These demonstrate the evolutionary emergence of the four-exon-three-intron organization of TRP14 genes after the split of protostome/deuterostome, which is highly conserved since then. An expression vector including the entire open reading frame of AmphiTRP14 and a 5'additional tag of pET28a is constructed and transformed into E. coli. Recombinant protein is expressed and purified. AmphiTRP14, which contains W40, W75 and W99, yielded a 2.7-fold increase in tryptophan fluorencence when reduced, suggesting changes in the microenvironment around the Trp (W) residues juxtaposing the CXXC active site. AmphiTRP14 exhibits a concentration-dependent disulfide reductase activity toward insulin, which contains one intramolecular and two intermolecular disulfides. It shows that AmphiTRP14 is a disulfide reductase. In situ hybridization histochemistry reveals that AmphiTRP14 transcript is abundant in the hepatic caecum and ovary, and at a lower level present in the hind-gut, endostyle, epipharyngeal groove, gill and testis, while it is absent in the neural tube, notochord and muscle. It is clear that TRP14 gene is expressed in amphioxus in a tissue-specific manner. This is further corroborated by immunohistochemical staining using the rabbit antiserum against the purified recombinant AmphiTRP14, which showed that AmphiTRP14 was predominantly localized in the hepatic caecum, ovary and hind-gut, and weakly in the testis and gill. This suggests that AmphiTRP14 may play a fundamental but tissue-specific role in food digestion for example, or alternatively reflect differences in the tissue susceptibility to oxidative damage.This paper also reports the characterization and expression of AmphiycaCR. The cDNA is 891 bp long and the ORF codes for a protein of 201 amino acids with a predicted molecular mass of approximately 22 kDa and an isoelectric point of 7.2. The deduced protein has a ycaC related domain, which belongs to the the isochorismatase superfamily. Like ycaC, AmphiycaCR had the putative catalytic triad: D19, R(K)84 and C118 (numbering as in E.coli) and the predicted structure has three-layerα-β-αsandwich topology structure, characteristic of the ycaC family. The phylogenetic tree constructed by neighbor-joining method using the sequences of putative ycaCR proteins and representative members of the isochorismatse superfamily shows that AmphiycaCR and other putative ycaCR proteins are clustered together, separating from other subfamilies of the isochorismatase superfamily. These imply that ycaCR is a highly conserved protein with the active sites of ycaC subfamily. An expression vector including the entire ORF of AmphiycaCR and a 5'additional tag of pET28a is constructed and transformed into E. coli. Recombinant protein is expressed and purified. Assays for isochorismatase, nicotinamidase and N-carbamoylsarcosine amidohydrolase demonstrate that AmphiycaCR doesn't show the activity of the three enzymes. Pull down assay shows that there are two proteins interacting with AmphiycaCR. One is amphioxus native ycaCR protein and the other is creatine kinase. These showed that AmphiycaCR may be form polymers and indirectly involved in energy transduction. In situ hybridization histochemistry reveals that AmphiycaCR transcript is abundant in the hepatic caecum, gut, endostyle and ovary, and at a lower level present in the epidermis, epipharyngeal groove, gill, testis, muscle, neural tube and notochord. It is clear that AmphiycaCR gene is expressed in a ubiquitous expression pattern. This is further corroborated by immunohistochemical staining using the rabbit antiserum against the purified AmphiycaCR, which shows that AmphiycaCR is predominantly localized in the hepatic caecum, endostyle, ovary and hind-gut, and weakly in the testis and gill. This suggests that AmphiycaCR may be a novel member of isochoristamase superfamily, which is mainly localized in the mitochondrial fraction, and is capable of interacting with the creatine kinase. The ubiquitous expression pattern suggests that Amphi plays a conserved fundamental cellular role in amphioxus, lending additional support to the proposal that Amphi is involved in energy transduction. The predominant expression in the gill, hepatic caecum, hind-gut, endostyle, and ovary may be in line with a rapid energy turnover in these tissues.