A Novel Isoform Of Lymphoid Enhancer Factor-1

Lymphoid enhancer factor-1(LEF-1) is a member of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of high mobility group (HMG) transcription factors, in q23-q25 of chromosome 4. Since LEF-1 contains aβ-catenin binding domain at N terminal, interacting withβ-catenin as a downstream mediator of Wnt signal transduction pathway, it has a close relationship with cell differentiation and tumorigenesis. It is known that other members of LEF/TCF family all have different isoforms, and in their C termini may generate an extremely important alternative domain necessary for the repression and/or activation of a select subset of Wnt target genes. In this study, we investigated bioinformatics features of LEF-1 C terminal to predict a novel isoform, prepared a monoclone antibody specifically against the novel isoform of LEF-1, mapped the epitope of monoclone antibody and primarily found its expression in vitro and in vivo. This would be useful to further explore the structure and biological role of the LEF-1.The aim of this study is to explore whether there is an isoform of LEF-1, to study the possible cause of its formation, to identify its functional alteration in comparison with the wild type, and to prepare a specific monoclone antibody against it, to investigate its expression in cultured cell lines and clinical samples. The project was studied in following 4 parts.1.Bioinformatic analysis of LEF-1(1) In GenBank, human LEF-1 is stopped by"UGA"stop codon. However, in some tissues, UGA may encode an amino acid --- selenocysteine (Sec) .This phenomenon is called"readthrough", carried out with translation at the same time, and Sec is called the 21st amino acid. In eukaryotic organism, the mechanism of encoding Sec is complicated. It is necessary of selenocysteine insertion element (SECIS), a loop-stem structure formed in 3'-UTR of mRNA, in this procedure. Using RNA structure software, we also found a loop-stem structure in 3'-UTR of human LEF-1 mRNA.(2) Analysis of the extended protein of LEF-1 isoform (eLEF-1). The alignment of the extended sequence and a family of HMG box proteins was carried out with the ClustalW with 30.7% sequence identity and 49.3% sequence similarity. A homology model has been built based on the structures of HMG box proteins. The predicted model shares high degree of structural similarity with HMG box proteins. The molecular dynamic simulations indicated the stability of the DNA/model complex over 1ns simulation time. It is suggested that the predicted LEF-1 isoform may have a new HMG box through which it can interact with DNA and correlate with cell differentiation and tumorigenesis.2.Preparation of the monoclonal antibody against eLEF-1.After LEF-1 cDNA was amplified from Jurkat cell line and cloned into prokaryon expression vector PQE40, the recombinant was transformed and induced with IPTG. eLEF-1-PQE40 expressed as inclusion body. After denatured with urea, the fusion protein was purified through ion exchange chromatography and affinity chromatograph. And then Balb/c mice were immunized with dislysed eLEF-1-PQE40. Through 2 times of cell fusion, screen of monoclonal antibodies, cloning, we obtained 6 stable monoclonal antibodies, one of which was named B8. After preparation of ascitic fluid and purification, we obtained purified monoclonal antibody B8. Meanwhile, the specificity of monoclonal antibody was tested with pET32a-eLEF-1. B8 is a specific monoclonal antibody against eLEF-1.3.Epitope identification of the monoclonal antibody B8.Different length gene fragments of eLEF-1 were subcloned in frame into PQE40.These construct plasmids were transformed and induced, and then were used to identify antigenic epitope of LEF-1 carboxyl terminus recognized by B8 with immunoblotting. B8 recognizes the antigenic epitope(37～54 amino acid). In addition, we synthesized polypeptide, using competive ELISA, identified the antigenic epitope was 40~48 amino acid (RPQRNTDIN), which is specific through NCBI BLAST alignment.4.Expression of the LEF-1 isoform. Utilizing B8, combined with wide type LEF-1 rabbit monoclonal antibody, it was shown in confocal test that two fluorescence colors had be merged in breast cancer cell line MDA-MB-435, MDA-MB-231 and MCF-7, suggested that it exists a novel readthrough LEF-1 isoform. Within 6 breast cancer cell lines, LEF-1 isoform expressed in 5 breast cancer cell lines by western blotting, more in MDA-MB-435; besides, in 4 breast cancer histological sections, we also found this isoform by IHC in 3 sections, two weakly positive, one strong positive; and it may have relationship with metastasis of breast cancer.In conclusion, in this study, we analyzed LEF-1 existed a novel isoform generated from readthrough with application of bioinformatics; prepared a monoclonal antibody B8 specifically against eLEF-1, and identified its antigenic epitope; investigated the expression of LEF-1 isoform in cultured cell lines and clinical samples, and the possible relationship with metastasis of breast cancer. This isoform of LEF-1 may help us to know further about the structure and role of LEF-1.