The Gene Evolution And Divergent Functions Of Two Isoforms Of Costimulatory Molecule B7-H3

T cell activation and function are regulated by costimulator molecules, which are mainly expressed on the surface of the antigen-presenting cells and could control the regulation of cellular immunologic response through increasing or decreasing the TCR signal interacting with ligands on the surface of T cells. According to the difference of structure costimulator molecular are classified with immunoglobulin superfamily and tumor necrosis factor (TNF) superfamily. B7 family members are immunoglobulin superfamily expressing on antigen presented cells including dentritic cells, monocytes, Langerhans cell, macrophage and B cells.B7-H3 is among the most recently discovered B7-like molecules, which was cloned by chapoval from DC cDNA library in 2001. The difference between B7-H3 and other B7 family was that mouse B7-H3 gene has only one isoform with two Ig-like domains (2IgB7-H3) with one IgV- and one IgC-like domain, whereas human B7-H3 has two isoforms: one form was with four Ig-like domains, two pairs of IgV and IgC named 4IgB7-H3 or B7-H3b. The other form was 2IgB7-H3 with one IgV and IgC in the extracellular portion. In most human tissues B7-H3b or 4Ig-B7-H3 is the major isoform, resulted from gene duplication with 98 percent homology in two pairs of IgV and IgC.Several studies initially demonstrated B7-H3 as a positive costimulatory molecule and an inducer for IFN-γand proliferation in human CD4+ T cells. However, other experiments showed that B7-H3 could take an important role as inhibitory costimulatory molecules. These contradictory results about b7-h3 function have a relation with the unknown receptor. Some researchers reported B7-H3 might interact with TLT-2 to induce the proliferation of T cells but other researchers thought that no evidence between the interaction of these two molecules. These contradictory results also have a relation with two isoforms, which might bind different receptors to take different roles just like B7.1 B7.2 with CTLA-4 and CD28.Many costimulatory molecules could exist in membrane and solube forms including CD28, CTLA-4, CD80, CD86、ICOSL、B7-H3 and B7-H4. Soluble molecules could be either shed following proteolytic cleavage or generated from alternative mRNA splice variants. We reported soluble B7-H3 for the first time in 2008 and its expression levels could help to diagnose sepsis or bacterial meningitis and correlate with non-small lung cancer and toxemia. The underlying mechanisms by which sB7-H3 is generated and whether soluble forms of both 2IgB7-H3 and/or 4IgB7-H3 isoforms can be produced remain unknown.In this program we performed for the first time a genomic survey of B7-H3 gene from teleost to mammal in various nucleinic acid and protein databases and analyze the gene structure and isoform expression through boinformatics methods then verified through molecular biology. Next we wanted to deduce the evolution model through the sequence blast and analysis to direct the divergent function between two isoforms. Further sequences analysis indicated that this duplication resulted in a new conserved region in the first IgC domain, which might disable 4IgB7-H3 from releasing soluble form unlike 2IgB7-H3 presenting both membrane and soluble forms. Through three-dimensional (3D) structure modeling and fusion-protein binding assays we discovered duplication isoform had a different structure and might bind another potential receptor on activated T cells, which were verified that two isoforms took different function on the activation of T cells and monocytes in vitro.The distribution of B7-H3 two isoforms in various invertebrates and the model of gene duplication【objective】To discuss the distribution of B7-H3 two isoforms in various invertebrates and answer the question of why there were two isoforms in human and only one isoform in mouse. Research the model of gene duplication about 4IgB7-H3 and its divergent function from 2IgB7-H3.【methods】Searching various B7-H3 sequences in various databases through bioinformatics methods and analyzing the number of structure domains and the splicing forms to deduce the distribution of B7-H3 in various species. PCR analysis after acquiring the RNA from representative species was performed to verify the prediction. We also constructed the phylogenetic tree analysis of all B7-H3 genes and the V domain and C domain sequences. The calculation of ration of dN and dS were also done.【Results】we acquired thirty-eight B7-H3 sequences and 4IgB7-H3 is also present in pigs, guinea pigs, cows, dogs, panda,megabat,African elephants and higher primate animals, which were verified by PCR analysis. Through phylogenetic tree analysis it was found that interspecies sequence clustering in higher primate animals and the ration of dN and dS were greater than1.【Conclusion】B7-H3 was earliest expressed in teleost with the form of 2IgB7-H3. 4IgB7-H3 is also present in pigs, guinea pigs, cows, dogs, panda,megabat,African elephants and higher primate animals, which is resulted from tandem exon duplication. The duplication model was not an independent one at least in rodent and higher primate animals and the aim of duplication was to product a new molecule to take new functions.The modeling and the potential receptor analysis of human 2IgB7-H3 and 4IgB7-H3【objective】To analyze the divergence of two B7-H3 isoforms in space structure through modeling the three-dimensional structure and the possibility of two isoforms to bind different receptors on activated T cells through protein competing-binding exprements.【methods】The modeling of the three-dimensional structure of these two proteins was performed by three automated homology modeling programs, Geno 3D, Swissmodel and Modeller after the B7-H3 sequences were removed its signal , transmembrane and cysto- sequences. The PBMC were isolated by Ficoll-Hypaque through gradient centrifugation from peripheral blood of healthy donors and were activated using PHA for 24h. Then cells were stained with biotinylated proteins or stained with biotinylated proteins after incubated with non-biotinylated proteins. Then cells were resuspended in PBS and analyzed by FACScan and CellQuest software【Results】Through PSI-Blast we acquired PD-L1 x-ray structure (3biSA) close to B7-H3 (36% identity over 222aa) and we based our modeling on this structure. Two isoforms have different 3D structure and both could bind on activated T cells, which could be blocked by the same protein and not interfered by another isoform.【conclusion】We model the 3D structure of 2IgB7-H3 and 4IgB7-H3, which have different structures. There are two potential receptors on activated T cells to bind two B7-H3 isoforms.The divergent functions of human 2IgB7-H3 and 4IgB7-H3 in biological function【objective】To research the divergent functions to activated T cells and monocytes mediated by LPS of human 2IgB7-H3, 4IgB7-H3 and mouse B7-H3 by gene transfected cells and fusion-proteins.【methods】The PBMC were isolated by Ficoll-Hypaque through gradient centrifugation from peripheral blood of healthy donors. Human T cells and monocytes were isolated from PBMC using a human T Cells or monocytes Enrichment Kit. Activated T cells were cocultured with different number of 2IgB7-H3 and 4IgB7-H3 transfected cells and different dose of fusion-protein.Proliferation was determined by using Cell Counting Kit-8 after 72h. To measure cytokine production, supernatants were harvested at 72h and samples were assayed by IL-2 and IFN-γELISA screening. Human monocytes were incubated with human 2IgB7-H3 and 4IgB7-H3 transfected cells and different dose of fusion-protein. Supernatants were harvested at 24h and samples were assayed by IL-6、TNF-α. The same protocol was carried on mouse T cells and monocytes.【Results】The proliferation levels of purified T cells activated with anti-CD3 and anti-CD28 Ab were enhanced in the presence of human 2IgB7-H3/L929 and mouse B7-H3/CHO and at the same time the secretion of IL-2 and IFN-γwas also increased. In contrast, stimulation of T cells in the presence of 4IgB7-H3 led to decreased proliferative responses. There was no obvious effect on monocytes with human 4IgB7-H3.【conclusion】There were two B7-H3 receptors on activated T cells. Human 2IgB7-H3 and mouse B7-H3 could bind costimulatory receptor to increase T cell and monocytes functions, while human 4IgB7-H3 could bind inhibitory receptor to decrease T cell activation.the mechanism of conserve amino acids“PQRSPT”in influencing 4IgB7-H3 secreting soluble form and carrying on its functions【objective】To analyze the difference of structure domains between human 4IgB7-H3 and 2IgB7-H3 with the highly repeating sequences, and to discuss the mechanism of this difference on the express of sB7-H3 and functions.【methods】The sequence difference between human 4IgB7-H3 and 2IgB7-H3 was analyzed by amino acids sequences alignment ,and the target gene losing the six conservative amino acids was obtained by splicing PCR. Then the target gene was inserted into the eukaryotic expressing vector pIRES2-EGFP after being digested with EcoR I and BamH I to construct the recombinant vector pIRES2-EGFP/4IgB7-H3-Del.The recombinants were transfected into L929 cells with Lipofect2000 Reagent, followed by G418 selection. RT-PCR, flow cytometry (FCM) and Western blot were used to detect the expression of B7-H3. Western blot and ELISA were used to detect the expression of soluble B7-H3. Activated T cells were cocultured with L929/4IgB7-H3-Del and proliferation was determined by using Cell Counting Kit-8 after 72-h. To measure cytokine production, supernatants were harvested at 72h and samples were assayed by IL-2 and IFN-γELISA screening【Results】The results of recombinant vectors enzyme digestion and DNA sequencing showed the target gene was inserted correctly into plasmid pIRES2-EGFP and the recombinant vector was constructed successfully. The stable cell line L929 cells transfected with vector pIRES2-EGFP/4IgB7-H3-Del was obtained successfully. Flow cytometry, ELISA and Western blot revealed that L929 cells transfected with vector pIRES2-EGFP/4IgB7-H3 could express B7-H3 protein on the cell surface but no soluble B7-H3 in the supernatant of transfected cells. By contrast, L929 cells transfected with vector pIRES2-EGFP/4IgB7-H3-Del could express B7-H3 both on the cell surface and in the culture supernatant. There was no obvious function of 4IgB7-H3-Del to activated T cells related to control.【conclusion】The stable cell line L929 /4IgB7-H3-Del was established successfully. Conservative amino acids (PQRSRT) might be an important motif about why 4IgB7-H3 expressed no soluble B7-H3 and inhibited activated T cells.In summary, we performed in this work, for the first time, a systemic analysis of the 2IgB7-H3 and 4IgB7-H3 expression in various vertebrates and discussed the duplication event model happened in most lineages of vertebrates. We found that there was a conserved region in 4IgB7-H3 during the duplication event, which might be an important motif to interfere this molecule disable soluble form and inhibiting the activation of T cells. Two isoforms of B7-H3 was discovered with different structure through homology modeling and have the possibility to bind different receptor to take functions, which was verified by experiments in vitro. Human 2IgB7-H3 and mouse B7-H3 could take part in the infection immune in membrane-bound or soluble form and upregulate the function of T cells and monocytes. In contrast, human 4IgB7-H3 could only inhibit T cell response through binding a different receptor, which needs to be verified through further experiments after the receptors were found.