Interacting Proteins Of Gp150 On Dictuostelium Discoideum During Multicellular Development

Dictyostelium discoideum is a unicellular eukaryote, which can take bacteria as food and multiply as a unicellular organism under favorable conditions. Upon starvation, a pathway consists of aggregation, mound, slug, and culmination stages induces the formation of a fruiting body which has a head of spores supported on a stalk of vacuolated cells. Spores await dispersal and germinate in favourable conditions for amoeboid growth. The genome of Dictyostelium discoideum is haploid, that is a major advantage compared with other model systems because it can generate a rich variety of mutants. The multi-cellular development process is relatively short (24h) and the regulatory signaling pathways are familiar with those in metazoan development, Dictyostelium discoideum is an excellent system in which to study cell mobility, cell-cell signal transduction, cell type differentiation, development and so on.Gp150 which encoded by lagC gene is a membrane glycoprotein and plays an important role in regulating cell differentiation and multicellular development. The mutant type strain AK127, which the lagC gene is absent, can't complete multicellular development and the development arrests at the aggregate stage. This suggested that gp150 protein is very important to the development of Dictyostelium discoideum. Distribution of gp150 is different in prestalk cells and prespore cells. Gp150 mainly exists in prestalk cells, while there is nearly no gp150 existing in prespore cells. Adhesion molecules are the candidates participating in intra- and inter- cellular signal transduction. So far, our knowledge of gp150's signaling pathway is still very limited.In order to screen the interacting proteins of gp150, protein gp150 (lagC C-terminal) was expressed and polyclonal antibody of gp150 C-terminal was prepared and identified. The recombinant plasmid pET-32a(+) with lagC C-terminal and 6 His tagged was expressed in E. coli BL21 (DE3) host cells. The fusion protein with 6xHis was purified by Ni2+ affinity chromatography column and then was used to immune rabbits for preparing polyclonal antibody. The titer and specificity of the rabbit's antiserum were measured by ELISA and Western blotting, respectively. The results showed that the fusion protein was successfully expressed and polyclonal antibody was also successfully obtained. The specificity of antibody was proved by Western blotting analysis of expression product of gp150. The potency of the polyclonal antibody was as high as 1:128000. The polyclonal antibody was used in Co-immunoprecipitation and seven gp150-intertacting proteins were captured, whose molecular weight is from 30KD to 15KD.We choose two clear protein bands for identifing by mass spectrometer.The result shows that the two protein is 40S ribosomal protein S3 and 40S ribosomal protein S24, which play important roles in regulation of cell cycle, cell development and apoptosis. The data suggests that gp150 can interact with RPS3 and RPS24 for regulating cell differentiation and development during multicellular development of Dictyostelium discoideum.This research lays the foundation for the study of gp150's role in the signal conduction of Dictyostelium discoideum. It also contributes to the study of the whole signal pathway of Dictyostelium discoideum.