Identification, Expression And Functional Analysis Of Calreticulin In Amphioxus Branchiostoma Japonicum

Calreticulin（CRT） is a46-kDa Ca2+binding protein which resides in the lumen of theER. It consists of three distinct structural and functional domains: a globular N-domain, anextended P-domain and an acidic C-domain. The N-domain contains an N-terminal cleavablesignal sequence that directs it to the ER, and the double cysteine residues that are involved inthe disulfide bond formation. The N-terminal domain is highly conserved among species. TheC-domain is the least conserved among CRTs, but includes a highly conserved motif KDEL(Lys-Asp-Glu-Leu) involved in ER retention/retrieval. Between the N-and C-domains ofCRT is the proline-rich P-domain.CRTs have been identified in a wide range of species including plants, invertebrates andvertebrates, but no information is available about CRT to date in the primitive chordateamphioxus, an evolutionarily important organisms. In this study, we reported for the first timethe cloning and identification of CRT in the amphioxus Branchiostoma japonicum, namedBjcrt, which consisted by5’-UTR87bp, an ORF1275bp, and3’-UTR1486bp, overalllength is2848bp. Predict amphioxus CRT is composed of424amino acids, included an16amino acids singnal peptide, and molecular size is about49.5kDa. The deduced424-amino-acid-long protein of amphioxus CRT consisted of a signal peptide, three domains(N-, P-, C-domains) and an ER retrieval signal sequence (KDEL). It contains all the featurestypical of CRTs, including the N-domain with the signature motifs KHEQKIDCGGGYI andIMFGPDICG, the P domain with two sets of triplle repeats A (PXXIXDPDAXKPEDWDE)and B (GXWXPPXIXNPXYX), and the conserved endoplasmic reticulum retention sequence(KDEL).The exon-intron structure analysis showed that both amphioxus and vertebrate CRT geneshad9coding exons interspaced by8introns, and the length of each coding exon ofamphioxus CRT gene closely resembled the corresponding coding exon of vertebrate CRT genes. These demonstrate that CRT is highly conserved throughout chordate evolution,hinting at the clue that CRT gene transcription is regulated similarly in primitive chordate(amphioxus) and vertebrates. The phylogenetic analysis based on and CRT sequencesrevealed that Bjcrt positioned at the base of vertebrate CRTs, suggesting that Bjcrt is theancient form of vertebrate CRTs.CRT is expressed in the various tissues of amphioxus, with the most abundant expressionin the notochordate. CRT is a multifunctional protein and its broad tissue distribution may berelated to its functions in the different tissues, which demands further study.rBjCRT was expressed in E. coli and purified by affinity chromatography on a Ni-NTAresin column. We demonstrated for the first time that the recombinant BjCRT (rBjCRT) wasable to bind the Gram-negative bacterium Escherichia coli and the Gram-positive bacteriumStaphylococcus aureus, and BjCRT was able to promote the phagocytosis of E. coli and S.aureus by sea bass macrophages. It is clear that amphioxus CRT has an opsonic activity,capable of promoting the killing of invading bacteria by the phagocytotic cells of the host.In summary, this study reports the cloning, identification and expression pattern of theCRT gene in amphioxus. It also shows that amphioxus CRT was able to bind to bothGram-negative and positive bacteria such as E. coli and S. aureus, and able to promote thephagocytosis of E. coli and S. aureus by macrophages. These highlights that CRT canfunction as an opsonin， a novel function in immunity, supporting that CRT is animmune-relevant molecule associated with host immune responses.