The Biological Function Of The Molecular Mechanisms Of Motif "PQRSPT" In The Human B7-H3 Gene

Effective activation of T cells during an immune response requires two different signals. Costimulatory signals play an important role in regulating the balance of immune responses. B7-H3 has been identified in recent years as a new member of the B7 superfamily. Two different splicing isoforms of human B7-H3 are respectively reported: 2IgB7-H3 and 4IgB7-H3. Extracellular immunoglobulin of 2IgB7-H3 generates a 2Ig domain (V1-C2) transcript, but extracellular immunoglobulin of 4IgB7-H3 generates a 4Ig domain (V1-C1-V2-C2) transcript. Studies suggest that 4IgB7-H3 is the major isoform of B7-H3 expression, but the differences between them remain to be investigated.Researchs indicate that both membrane and soluble form of a number of B7-CD28 superfamily members have been found. A natural form of sB7-H3 has been found in healthy donors by Zhang GB et al. And we found sB7-H3 is only from 2IgB7-H3. It appears that the 4IgB7-H3 isoform is the result of a gene duplication of two exons encoding the IgV–IgC domains of the 2IgB7-H3 molecule. Sequence alignment of 2IgB7-H3 and 4IgB7-H3 shows that their homology of the amino acid sequence is over 95%, and"PQRSRT"motif specifically located in terminal of the first IgC-like domain was observed in human 4IgB7-H3, but not in 2IgB7-H3. In our study, we obtained the 2IgB7-H3-Add gene adding the expression of"PQRSPT"motif and the 4IgB7-H3-Del gene deleting the expression of"PQRSPT"motif by PCR, and established the stable cell lines L929/2IgB7-H3-Add and L929/4IgB7-H3-Del. And we studied the mechanism of motif"PQRSPT"in the biologcial fuctions of human B7-H3.Part 1 The construction of 2IgB7-H3-Add and 4IgB7-H3-Del gene transfected cell linesThe full-length human 2IgB7-H3 and 4IgB7-H3 genes coding region were respectively cloned from the human 2IgB7-H3-Add and 4IgB7-H3-Del gene transfected cell lines by RT-PCR. Specific primers with 5'phosphorylation were designed to amplify the short fragments respectively. Then two target genes were obtained by linking two fragments with the T4 DNA ligase and PCR, then inserted into the eukaryotic expression vector pIRES2-EGFP to construct the recombinant pIRES2-EGFP/2IgB7-H3-Add and pIRES2-EGFP/4IgB7-H3-Del after double digestion with EcoR I and BamH I Each correct recombinant plasmid was transfected into murine L929 cells after induction with LipfectamineTM2000 reagent, followed by G418 selection. RT-PCR results showed that 2IgB7-H3-Add and 4IgB7-H3-Del gene were respectively integrated into the genome of L929 cells and could transcribe successfully. Flow cytometry results indicated that both GFP and B7-H3 proteins were stably expressed in the two stable cells'surface. Western Blot analysis showed that L929/2IgB7-H3-Add cells could successfully express 2IgB7-H3 protein, and L929/4IgB7-H3-Del cells could successfully expressed 4IgB7-H3 protein. These results suggested that the stable cell line L929 cells transfected with recombinant vector pIRES2-EGFP/2IgB7-H3-Add and pIRES2-EGFP/4IgB7-H3-Del were obtained successfully respectively.Part 2 The biological function of the molecular mechanisms of motif"PQRSPT"in the human B7-H3 geneThe culture supernatant of the transgenic cells was collected for ELISA and Western Blot analysis, the result showed that transgenic cells L929/2IgB7-H3 and L929/4IgB7-H3-Del could express soluble B7-H3 protein, but no soluble B7-H3 protein in the culture supernatant of transgenic cells L929/Mock, L929/2IgB7-H3-Add and L929/4IgB7-H3. It was demonstrated that conservative amino acids sequence (PQRSRT) might be an important motif about why human 4IgB7-H3 couldn't release soluble B7-H3. Meanwhile, the effect of transgenic cells on peripheral blood T cells proliferation and cytokine production in vitro was researched by methods of CCK8 and ELISA. The results showed that L929/2IgB7-H3 cells could obviously promote the proliferation of T cells activated by anti-CD3 with anti-CD28 mAbs. The up-regulation on the cytokine production such as IL-2 and IFN-γcould be detected by ELISA. By contrast, L929/2IgB7-H3-Add cells increased expression of"PQRSPT"motif had no effect on the proliferation of T cells and the cytokine secretion. The results also showed that L929/4IgB7-H3 cells could obviously inhibit the proliferation of T cells activated by anti-CD3 with anti-CD28 mAbs. The down-regulation on the cytokine production could be detected by ELISA. By contrast, L929/4IgB7-H3-Del cells deleted expression of"PQRSPT"motif had no effect on the proliferation of T cells and the cytokine production. It was revealed that the conservative amino acids sequence"PQRSPT"might be a functional motif in B7-H3, which played a major role in changing the B7-H3 biological function.In conclusion, two stablely transgenic cells L929/2IgB7-H3-Add and L929/ 4IgB7-H3-Del have been constructed successfully. The detection of sB7-H3 in the culture supernatant of transgenic cells demonstrats that the"PQRSPT"motif in human 4IgB7-H3 closely related to the releasing of sB7-H3. Furthermore, studies on T cells proliferation in vitro show that both L929/2IgB7-H3-Add and L929/ 4IgB7-H3-Del cells have no effect on the proliferation of T cells and cytokine production. It is indicated that the"PQRSPT"motif may play an important role in the inhibition of 4IgB7-H3. This study provides a new thread on interpreting the mechanism of sB7-H3 expression and establishs the molecular basis on studying the difference between two isoforms of B7-H3.