Proteomics Approach For Stress Response And Immune Function Investigation For Creatine Kinase In Amphioxus

Amphioxus, a cephalochordate, has been regarded as a basal lineage of chordate. Recently, the studies for the origin of the AIS focus on seeking evidence of the existence of adaptive immune system (AIS) in amphioxus. An attempt was made to identify the proteins relate to amphioxus immune response using immunological, proteomic, genetic and peotein-protein interaction technologies in the current study. Furthermore, CK (creatine kinase) was investigated for its ability of immune defence.We utilized proteomic methodologies to achieve altered proteins of amphioxus humoral fluid or muscle in response to salt stress, caudal trauma, bacterial infection and challenge. In this regard, several groups of animals were investigated and found that the groups immunized and challenged with the same bacteria had a lower death rate, indicating that the specific protection may be in dawn in this animals. On the other hand, fifteen altered proteins were identified, in which ribosomal protein S10, CAVPT, cathepsin, intermediate filament protein and CK were determined in more than two of the responses above. These results suggest that these proteins may play a important role in amphioxus immune response. More importantly, eleven protein spots were identified as CK in 2-DE maps, in which the spot with the lowest pI, CK1, changed remarkably.The amount and enzyme activity of CK showed a dynamic pattern of APP in amphioxus humoral fluid following caudal trauma. These results suggest that the CK elevation is an active response to caudal trauma rather than a passive release from muscle damage. Comparison of the nucleotide sequence of the cloned sequences (DQ508694) revealed an identity with Branchiostoma floridae and Branchiostoma belcheri ranging from 95 to 96%, respectively, indicating that the diversity of protein spots of CK did not result from the diversity of CK gene. Several CK in line might suggest that CK was post-translationally modified in the animals, although CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine and phosphoserine. The results that CK was pulled down by bacteria and agglutinated with bacteria indicated that CK bind to bacteria directly. CK enzyme activity was inhibited by both Gram-negative and -positive bacteria with dose-dependent manner. The inhibiting ability was related to bacterial species. The combination of CK with E. coli BW25113 was by OmpA, OmpX and OmpW, in which OmpA and OmpW inhibited directly CK enzyme activity. This inhibition was strong in OmpA than OmpW. Furthermore, OmpA and OmpW could bind to both domainC and domainN of CK, but OmpX was only bound to domainC.Wounded animals were significantly elevated with the use of CK gene silence following bacteria infection, indicating that CK play a important role in host defence. Change in CK activity was associated with amount of bacteria, showing enzyme activity elevation and less than 10% wounded rates in response to injection with about 20 bacteria, whereas decreased activity and 100% wounded frequency in response to injection with more than 20,000 bacteria In addition, bacterial growth was inhibited by high concentration of CK. These results indicate that CK may be a non-toll like receptor and effector in amphioxus defence.