Analysis Of Promoter Sequence And Single Nucleotide Polymorphism Site Of Human CD226 Gene

CD226,also named platelet and T cell activation antigen 1 (PTAl),was discovered by Burns in 1985. PTA1 mainly expressed on activated T cell, NK cell, megakaryocyte and platelet. Previous study indicated that IL-2,TNF- a and PMA could upregulate the expression of PTA1 on T lymphocytes and TGF- P down-regulate its expression. It is interesting that PMA plus calcium ionophore A23187 can inhibit PMA-induced PTA1 expression, and this effect can't be reversed by calcmeurin inhibiter FK506. PTA1 mAbs can inhibit CTL activation and differentiation in mixed lymphocyte culture system when added at the beginning of the culture but can induce platelet activation and aggregation in the Fc dependent manner. In 1997, PTA1 cDNA was cloned from cDNA library of TPA activated Jurkat cells, which belongs to immunoglobulin superfamily (IgSF) with two V-like domains of extracelluar region of PTA1. In the same year, the PTA1 homologous cDNA of gibbon and monkey were cloned in our lab. Homology analysis showed that PTA1molecule is highly conserved among human, gibbon and monkey, sharing the same sequence about 93%~95% at the protein level. PTA1 and monoclone antibodies FMU1-7 were named as CD226 in 7th Workshop and Conference on Human Leucocyte Differentiation Antigens(HLDA). Recently we have cloned mouse CD226 cDNA and found different isoforms.Promotor ,in broad sense, consists of transcription start site, binding site of RNA polymerase (promoter in narrow sense) and upstream regulation sequence. The most efficient regulation of gene occurs at transcription level by regulating the interaction between transcription factors and upstream regulation sequence. Thus, to investigate promoter of a target gene will be helpful to predicate the principle of molecule regulation, biological function of molecule and even involving pathogenesis of some diseases. With the accomplishment of Human Genome Project (HOP) and development of computer technique, bioinformatics is becoming more and more important scientific branch, which have applied to searching new gene, analysing structure of gene, structure prediction of RNA and protein and sequence analysis.In this study we analyse promoter sequence and single nucleotide polymorphism site of human CD226 gene. It has been found that there are two putative promotors, PI and P2, which locate in -375bp and -130bp respectively. Both of them have two INF- Y response elements( Y -IRE) and one common binding site for PEAS, Ets-1 and PU.l in the 3' flank. In addition PI contains GC box and TATA-box, while P2 has GAGA-box. We also found binding sites of some transcription factors such as AP-1, Ets-1, Spl, P300, PU.l, PEA3 and CBP, which may be involved in regulation of CD226 gene expression. We confirmed one single nucleotide polymorphism (SNP) site in the exon 7 of CD226 gene with AGT-GGT polymorphism of 307 codon. Among ten volunteers, 4 cases are serine and 6 cases are glycine at this codon. The biological significance of this SNP needs to be investigated.A set of monoclone antibodies FMU1-7 against CD226 have been made in our lab, immunolized by natural CD226 protein. FMU1-5 recognize domain 1 of extracelluar region CD226 molecule while and FMU6-7 fordomain 2.It has been demonstrated that only FMU1-3 can bind to T cell and regulate T cell and NK cell function. We investigate the relationship between structure and function of CD226 on platelet and found some similar results to T cell. FMU1-3 can bind to platelet and FMU1 and FMU2 also can induce platelet activation and aggregation.