紅血球分化過程I醣分支結構形成的調控機制

Abstract

人類紅血球組織抗原I 和i 為以N-acetyllactosamine（Galβ1-4GlcNAc，LacNAc） 為單位，重複連結所形成的醣抗原結構。兩者的不同在於，i 抗原為直鏈形的醣結 構，而I 為具有β1-6 GlcNAc 分支的醣抗原結構。胎兒的紅血球表面表現大量的直 鏈形i 抗原，僅有少數的I 分支結構。過去的研究證實，胎兒於出生前其紅血球表 面持續保持i 抗原結構，不生成大量的I 分支，為保護胎兒不因與母體ABO 血型 不合而引發溶血症的重要機制之ㄧ。然而新生兒出生後，紅血球表面的i 抗原即逐 漸轉換成分支結構的I 抗原；此表現的調控機制至今仍不清楚。I 醣抗原形成的決 定酵素為I 基因座所表現的I β-1,6-N-acetylglucosaminyltransferase（IGnT），此酵 素可將直鏈形結構i 抗原轉換成分支的I 抗原結構。經由轉錄以及splicing 作用後， I 基因座會表現IGnTA、IGnTB 和IGnTC 三種transcripts；其蛋白質產物皆具有形 成I 分支結構的酵素活性。我們過去的研究已證明，紅血球表面I 抗原的生成是由 I 基因座的IGnTC 基因負責。 本論文選用erythroleukemia 細胞株K-562 做為紅血球分化模型，以探討調控I 抗原表現的分子機制。我們的研究證實，以sodium butyrate 誘發K-562 細胞進行 紅血球分化過程中，轉錄因子CCAAT/enhancer binding protein α（C/EBPα）透過調 控IGnTC 基因的表現，促使細胞表面I 抗原的生成。後續染色體免疫分析法以及 免疫沉澱法的結果顯示，C/EBPα 磷酸化狀態的改變可能影響其與IGnTC 基因轉錄 調控區域的結合能力，進而調控IGnTC 基因的表現。我們進一步以成人和胎兒 CD34+血球幹細胞為模型，藉由體內自然分化以及體外培養誘發紅血球分化過程， 以驗證紅血球表面I 抗原表現的調控機制；並以lentiviral 表現系統將C/EBPα 轉錄 因子表現於成人及胎兒CD34+細胞中；結果顯示，其調控機制亦如同K-562 細胞 分化模型所顯現，C/EBPα 為紅血球分化過程中調控I 抗原表現的決定因子。而實 驗結果皆顯示，調控C/EBPα 的後轉譯修飾作用的機制才可能是調控IGnTC 基因 表現，進行促成紅血球表面I 抗形成的機制。然而C/EBPα 於紅血球分化過程中的 iii 後轉譯修飾及其形成機制，以及此機制在成人和胎兒之間可能的異同，有待進一 步研究；釐清此機制，才可進一步回答胎兒紅血球表面保持直鏈的i 醣結構，而在 出生後，轉換成I 醣分支結構的詳盡機轉。

The human histo-blood group I and i antigens are characterized as branched and straight chains of N-acetyllactosamine (Galβ1-4GlcNAc, LacNAc) repeat, respectively. Fetal and neonatal erythrocytes predominantly express i antigen, with only a small amount of I. It has been recognized that the presence of the straight-chain i antigen on fetal erythrocytes surface is one of the important mechanisms to prevent the hemolytic disease due to ABO incompatibility pregnancy. After birth, the i antigen of erythrocyte is gradually converted to the branched I antigen; however, the regulatory mechanism for the I branching formation awaits elucidation. Conversion of the straight-chain i to the branched I antigen is catalyzed by the enzyme encoded by the I locus, I-branching β-1,6-N-acetylglucosaminyltransferase (IGnT). The human I locus expresses three IGnT transcripts, IGnTA, IGnTB, and IGnTC, which have different exon 1, but identical exon 2 and exon 3 regions. In our previous study, we have demonstrated that IGnTC is the intrinsic gene responsible for the I antigen expression on erythrocytes. In this thesis, the erythroleukemia cell line K-562 was used as a model to study the regulatory mechanism of I antigen expression during erythroid differentiation. Our results demonstrated that the transcription factor CCAAT/enhancer binding protein α (C/EBPα) stimulated the expression of IGnTC gene in sodium butyrate-induced erythroid differentiation of K-562 cells, and consequently enhanced the I antigen expression on the cell surfaces. The results obtained from chromatin immunoprecipitation (ChIP) and immunoprecipitation (IP) analysis, suggested that the phosphorylation status of C/EBPα may affect its binding affinity onto the 5’ regulatory region of IGnTC gene and thus regulates the expression of IGnTC gene. Human adult and cord CD34+ hemopoietic stem cells were employed to further investigate the role of C/EBPα in the I antigen expression during erythroid differentiation, and lentiviral expression system was used to introduce C/EBPα into adult and cord CD34+ cells. The v results demonstrated that the transcription factor C/EBPα played the determining role in the regulation of the I antigen expression during erythropoiesis. Furthermore, the results suggested that the function of C/EBPα in the IGnTC gene expression and the subsequent I antigen formation may be determined by the post-translational modification status of C/EBPα. The post-translational modification and the mechanism leading to this modification on C/EBPα during erythroid differentiation await further investigation, and it is of significance to further reveal the possible difference of this mechanism between adult and fetal erythropoiesis processes. Only after the elucidation these mechanisms of the post-translational modification on C/EBPα the mechanisms leading to the predominant expression of straight-chain i antigen on fetal erythrocytes and the i-to-I conversion after birth could be answered in more detail.