Study On The Biological Function Of C-end Cysteine Of Turbot Translationally Controlled Tumor Protein

The translationally controlled tumor protein (TCTP) is a highly conservative, ubiquitously expressed and fairly abundant protein. It can be widely found in animals, plants and fungi and has attracted more and more attentions due to its potential roles in various biological and medical processes. It involves in cell growth, binding calcium ion, histamine release, anti-malarial, antioxidant, etc. and plays key role in these biological processes. The expression of TCTP is highly regulated in transcripition and translation level. Thus studying the relationship between its structure and function is of great theoretical and practical significance. It was found that the amino acis sequence of known TCTPs are highly conserved in the last position of C-end with a cysteine (Cys) shared by all animals from fungal to mammalian. Of interest, turbot (Scophthamus maximus) translationally controlled tumor protein (SmTCTP) contains only one Cys (aa170), and this Cys is located in the C terminal of the protein. Therefore, to study the contributaion of the C-end Cys of SmTCTP to its biological function is of scientific importance to understand the relationship between its structure and function.It was reported that Brugia malayi (Brugia malayi) TCTP has antioxidant activity and this antioxidant function is due to the ajacent three Cys (aa63, aa81 and aa92) in the mid of the protein. In this paper we first examined the role of the C-end Cys of SmTCTP in its antioxidant activity by Cysâ†'Ala mutant. We successfully constructed 170Cysâ†'Ala mutant recombinant plasmid (pET30a-Mutant-SmTCTP) by PCR amplification using designed mutant primers based on the known sequence of SmTCTP. After the empty vector plasmid(pET30a), the wild SmTCTP expression plasmid (pET30a-wild-SmTCTP) and the mutant expression plasmid (pET30a-Mutant-SmTCTP) was respectively transformed into E. coli strain BL21, the antioxidant capacities of these three recombinant expression bacterial strains to H₂O₂ oxidant were compared. The results showed that, at relatively low level of H₂O₂ (0.22 mM), bacteria with the expression of wild-SmTCTP showed the highest antioxidant activity, then the bacteria with pET30a, and the lowest was the bacteria with mutant-SmTCTP expression; at higher concentration of H₂O₂ (0.44 mM), none of the above three strains can survive even with extremely high seeding density. This shows that, the Wild-SmTCTP protein has antioxidant activity, but relatively low. The concentration of 0.44 mM H₂O₂ is beyond their tolerance limits; Cys residues is indispensable to the antioxidant activity of SmTCTP protein because the absence of Cys residues in mutant-SmTCTP significantly reduced the antioxidant activity. However, it is unexpected that the antioxidant activity of mutant-SmTCTP was significantly lower than that of control vector pET30a, indicating that expressed mutant-SmTCTP protein is toxic to the host bacterial cell.To further validate the toxicity of mutant-SmTCTP to the host bacteria, we further examined the growth curves of the above three strains (pET30a, pET30a-wild-SmTCTP and pET30a-mutant-SmTCTP). The results obtained show that, wild-SmTCTP expression can promote the growth of BL21 strain, mutant-SmTCTP expression significantly inhibited the growth of BL21 strain; in another word, the lack of C-end Cys of SmTCTP also affected the bacterial growth.Finally, in order to confirm the effect of C-end Cys of SmTCTP on cell growth, we cloned the coding regions of wild-SmTCTP and mutant-SmTCTP into the mammalian expression vector pcDNA ? 3.1/V5-His respectively and transiently transfected into in vitro cultured fish cells - flounder gill cell line (FG) using Lipofectamine LTX transfer agent. After the pcDNA empty vector plasmid, pcDNA-wild-SmTCTP plasmid and pcDNA-mutant-SmTCTP plasmid were transformed into FG cells, respectivel, the growth of transfected FG cell were compared. The results obtained show that, the over-expression of wild-SmTCTP protein can promote the growth of FG cells, but mutant-SmTCTP inhibited the growth of FG cells, also indicating that the C-end Cys is related to the growth-promoting function of SmTCTP.To elucidate the role of the C-end Cys in the growth-promoting functions of in SmTCTP, we also predicted the three-dimensional crystal structure of SmTCTP protein. It was found that C-end Cys residue is located in the protein's surface. This tells us that Cysâ†'Ala mutation will not affect the spatial structure of protein. Therefore, it is more possible that mutation of the C-end Cys will influence the self-dimerization of SmTCTP, or the dimerization of SmTCTP protein and its receptors.In summary, we think that the -SH residue of Cys is reductive and provides the main antioxidant activity of SmTCTP, but SmTCTP has only one Cys residue, so the antioxidant activity of SmTCTP is relatively low; SmTCTP proteins may have similar growth-promoting mechanism as growth hormone, that SmTCTP protein first bind to receptors on the cell surface, then pulls the receptors together and activates them by the self-dimerization of SmTCTP.