Study On The Relationship Between Novel Mutation Sites Of Alpha(1,2)Fucosyltransferase Gene And Its Functions

BackgroundThe H antigen is fucosylated oligosaccharides associated to the ABO system and the lewis blood group, which belongs to Hh blood group. Bombay and para-Bombay are rare phenotypes in which the H antigen is totally or partially absent . It has been identified that the mutation of FUT1 gene is responsibility for Bombay and para-Bombay phenotype. In the advanced study , by analyzing the full coding region of alpha (1,2) fucosyltransferase (FUT1) gene, our colleagues identified the novel mutation 682A>G+35OT in the para-Bombay phenotype, which induced to the substitution of the amino acid A12V and M228V, respectively. Some scholars presume that 35C>T is responsibility for para-Bombay, which is named as h₄ allele. According to the investigation for population frequency of h₄ allele by our colleagues and comparison with homology analysis for fifteen kinds of mammal, it can be presumed that 35C>T is a single nucleotide polymorphism, which may be not maintain the activity of alpha(1,2) fucosyltransferase and is irrelvant to para-Bombay phenotype. We proposed that 682A>G can result in the weak expression of H antigen. However, this propose need to confirm in vitro. In this study, our work will be focusing on identification the mechanism of novel mutation (682A>G+35C>T ) in vitro. Moreover , the relationship between novel mutation site of alpha- (1,2) fucosyltransferase gene and its protein functions will be elucidated , which will becontribute to revealing the effection of 35C>T, 682A>G on the expression of H antigen.Methods1. The proband of para-Bombay was identified by serological and molecular biological technology. The coding region of FUT1 gene was amplified by PCR from genomic DNA of proband, and purified PCR product fragment with gel extraction method, then PCR product of FUT1 was ligated into expression vector using TOPO TA cloning kit. In the end, We choosed the recombined plasmids colony and cultured , then used the plasmids DNA as template were sequenced by BigDye^TMTerminator Cycle Sequencing Kit.2. The COS-7 cells were cultured under routine conditions method. The recombined plasmids were transfected into COS-7 cells by liposome when the cells were in Log grownth phase in the plate wells under microscope.3. After screening by G418, we tested H antigen expression on the cell membrance of COS-7 by flow cytometry on the second, third, fourth and seventh day . The cells were stained by FITC-labeled antibody of anti-H.4. We tested the FUT1 mRNA in the cytoplasm of COS-7 by real-time PCR on the first, second and third day. Glucose-6-phosphate dehydrogenase gene is viewed as internal control and FUT1 is target gene. Standarded curves were acquired to analyze the mRNA quantitation of the samples.Results1. construct recombined plasmidsThe full coding region of alpha (1,2) fucosyltransferase (FUT1) gene was analyzed in the proband and normal control by polymerase chain reaction and PCR product shows the target band is 1094bp in 2% agarose gel electrophoresis with ethidium bromide . Then PCR products of FUT1 were ligated into expression vector using TOPO TA cloning kit. Recombined plasmids DNA were sequenced by BigDye^TMTerminator Cycle Sequencing Kit. In the end, we acquired threekinds of plasmids, including pcDNA3.1/V5-His-wild(35C+682A), pcDNA3.1/V5-His-35T, and pcDNA3.1/V5-His-35T-682G.2. the analysis of antigen after transfectionWe tested the H antigen by FITC-labeled antibody of anti-H. The results show that the quality of H antigen is dependent on the time, with the maximum expression on the fourth day. Moreover, with compared to pcDNA3.1/V₅-His-wild, the H antigen expression strength of the 35T and 682G+35T was 52.7% and 13.3%, respectively.3. the analysis of mRNA after transfectionWe analyze the quantitation mRNA of the samples by two-standard curves. Although the level of FUT1 mRNA is similarly dependent on the time, it achieves to the maximum level on the first day. Furthermore, the mRNA quantitation of the pcDNA3.1/V5-His-35T-682G transfection cells is obviously less than that of the wild allele on the first day.Conclusion1. We successfully constructed three recombined plasmids, including pcDNA3.1/V5-His-wild, pcDNA3.1/V5-His-C35T, and pcDNA3.1/V5-His-35T -682G and have established a transient expression system for alpha- (1,2) fucosyltransferase gene. We identified FUT1 mutation can result in weak expression of H antigen in vitro by protein antigen analysis.2. 35C>T allele can result in the partially weak expression of H antigen in vitro.3. We confirm that 682A>G can obviously result in the reduction of H antigen, the chang of Met228 may effect on the activity of alpha (1,2) fucosyltransferase.4. The recombined mutations of 682A>G+35OT is relevant to the transcription of FUT1.