P48.2 Protein Rgd Domain On The Biological Activity Of P48.2 Protein Polyclonal Antibody Preparation

p48.2 protein is a novel immune molecule which has a conserved motif of cytokine receptor typeⅠ.We cloned this gene from K562 leukemia cells,and submitted it to NCBI GenBank(DQ298450).p48.2 gene is localized at the flanking region of NFI gene on chromosome 17q11.2 that is frequently deleted in patients with neurofibrominlocus.The coding sequence for p48.2 gene is 1,317 base pair that encode a 438aa protein.We have demonstrated that over-expressed p48.2 arrested the cell cycle at G0/G1 phase.We found that p48.2 protein might be an important negative regulator in cell cycle.Therefore,in this study,we make a further exploration on the molecular mechanism conducted by p48.2.Bioinformatic analysis shows that the evolution of p48.2 is well conserved, homologues of p48.2 can be found in many vertebrate species,p48.2 is a kind of soluble cytoplasmic protein without nuclear localization signal,cell organelle localization signal and transmembrane domain.The analysis of functional domain shows that p48.2 protein has a typical FNⅢdomain.A RGD motif and WSXWS motif of typeⅠcytokine receptor are observed at the C-terminus of FNⅢdomain.RGD motif is known to be interacting recognition sequence between cell surface integrin and its ligand,RGD motif plays an critical role in cell-cell and cell-matrix adhesion,and mediates cell signal communications.One part of my study is to determine whether RGD motif is the important domain for the biological activity of p48.2.The following approches were employed:1) PCR site-directed mutagenesis was used to construct eukaryotic expression vector of RGD-to-RGE and RGD deletion mutants,and named pCMV-RGE and pCMV-DEL,respectively.2) pCMV-p48.2 and two mutant plasids were transfected transiently into 293T cells.Immunostaining analysis showed that both p48.2 and its mutant derivetives were distributed in cytoplasm and cell membrance,and RGD mutation didn't change the localization of p48.2.3) p48.2 gene and its mutant derivetives were subcloned into pEGFP-N1. GFP/DNA multi-parameter flow cytometry analyzed the cell cycle of 293T cell transfected with pEGFP-p48.2,pEGFP-RGE,pEGFP-DEL,p48.2 and mutants could cause G0/G1 phase arrest and reduce S phase population.4) pCMV-p48.2 and two mutants were transfected transiently into 293T cells,all of them could down-regulate cyclin D1 and D2 expressions by reversed transcription-PCR.These studies indicate that p48.2 protein is cytoplasm protein,over-expression of p48.2 in 293T cell could significantly arrest the cells at G0/G1 phase by down-regulating cyclin D1 and D2 expressions.Thus,p48.2 might represent a type of cytoplasmic protein functioning as a negative regulator at the G0/G1 phase by down-regulating cyclin D1 and D2.RGD mutation didn't change the localization of p48.2 and it also didn't release p48.2-mediated G0/G1 arrest and down-regulation of cyclin expressions.Therefore RGD motif is not necessary to p48.2-mediated cell cycle arrest.In another part of my study,p48.2 gene was also subcloned into pGEX-4T-1 vector.The GST-p48.2 fusion protein was expressed in E.coli and purified by GST affinity chromatography.Then we prepared the rabbit anti-p48.2 polyclonal serum with this purified soluble fusion protein.Indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis demonstrated that the p48.2 protein was recognized by rabbit polyclonal serum.In conclusion,we produced polyclonal serum against p48.2 protein.The antibody provides us a good tool for future functional characterization of p48.2 protein.