A4GALT基因SNP與紅血球P1抗原表現之分子機制

Abstract

P1及Pk是兩種表現於細胞表面的醣抗原，此兩者醣抗原在細胞表現的變化已知和多種疾病有所相關。過去的研究已知，紅血球表面P1及Pk抗原的表現量因個人而不同，然而其分子遺傳機制尚未完全明瞭。α-1,4-Galactosyltransferase (α4Gal-T)為一種半乳糖基轉移酶，具有可合成P1抗原以及Pk抗原之酵素活性。在過去的研究指出，個體紅血球表現P1抗原之多及寡(又稱為P1及P2血型)由A4GALT之基因表現量高低所決定，其中P2表型的紅血球，其A4GALT的mRNA表現量和血球表面的P1抗原數量，皆會少於P1表型的紅血球。於是找出造成A4GALT基因表現量改變的因素，便能有助於釐清形成P1/P2表型之分子機制。 單核苷酸多型性(single nucleotide polymorphism，簡稱SNP)指的是由單個或少數核苷酸的改變所引起的DNA序列的改變，所造成物種間染色體基因組之多樣性。在我們研究室先前的研究中已經分別分析P1及P2表型個體之A4GALT基因的SNPs，發現其中的SNP rs2143918 (SNP5)及SNP rs5751348 (SNP6)對於A4GALT基因表現量的高低與P1/P2表型的形成有很大的關聯性。我們因此進而使用電腦預測可能與此二個SNP結合親和力呈現差異的轉錄因子，得到ETS2、AML1、EGR3、CACCC-binding factor (KLFs)、CEBPD等轉錄因子的預測。接著我們利用K-562細胞模型以及報導基因分析法(Reporter assay)去做進一步的檢驗，發現在K-562細胞中，只有EGR3可促進A4GALT的基因表現，同時刺激帶A4GALT SNP6高表現基因型(P1 allele)的報導基因載體的轉錄活性。這些結果顯示，EGR3轉錄因子可能扮演刺激不同A4GALT SNP6基因型對偶基因的角色。後續的實驗我們想進一步去證實EGR3在A4GALT SNP6 P1/P2異合子型的細胞株中，對於A4GALT不同基因型的表現是否有影響，以更進一步證實EGR3轉錄因子扮演差異性調控帶有不同SNP6基因型的A4GALT基因的表現，因此而造成P1及P2血型之不同。

P1 and Pk are two different carbohydrate antigens present on cell surfaces. The expressions of the P1 and Pk antigen on red blood cells (RBCs) are different among individuals; however, the molecular genetic mechanism leading to this difference is still unclear. The formations of the P1 and Pk antigens are determined by the activity of an α-1,4-galactosyltransferase, encoded from the A4GALT gene. According to previous studies, the common phenotypic polymorphisms of the P1+ and P1– phenotypes on RBCs, which are designated as the P1 and P2 blood groups, respectively, depend on the different A4GALT gene expression levels among individuals. In RBCs with the P2 phenotype, the expression level of A4GALT mRNA and the amount of the P1 antigen are both lower than the RBCs with the P1 phenotype. In our previous investigation, we have demonstrated two single-nucleotide polymorphisms (SNPs), rs2143918 (SNP5) and rs5751348 (SNP6), located in the intron 1 region of the A4GALT gene, are associated with the P1/P2 phenotypes. In the present study, we aim to demonstrate the factor leading to the different expression levels between the A4GALT genes with the P1 and P2 genotypes. Several transcription factors, including ETS2, AML1, EGR3, KLF, and CEBPD, were predicted in silico to have differential binding affinity to the SNP5 or SNP6 regions with the different P1 and P2 genotypes. In further investigations using reporter assay and K-562 erythryoleukemia cells as a study model, we found that ectopic expression of the EGR3 transcription factor leads to a significant induction of the transcriptional activity in the reporter construct with the A4GALT SNP6-P1 genotype and also promote the expression of the A4GALT gene in K-562 cells. These results highly suggested that EGR3 may play an important role in activating the expression of the A4GALT gene with the P1 genotype at the SNP6 position. In our further investigation, we plan to demonstrate whether EGR3 will differentially stimulate the expression the A4GALT genes with the P1 and P2 genotypes in a P1/P2 heterozygous cell line to substantiate the functional role of EGR3 transcription factor in the formation of the P1/P2 blood groups.