ADP-ribosylation is a post-translational modification of certain proteins by addition of the ADP-ribose moiety of NAD to a target protein by ART (ADP-ribosyltransferase) activity. The two different ARTs can be classified in terms of ADP-ribosylated production, mono-ART and poly-ART. The enzymatic activities are extremely variable within these ARTs if compared between prokaryotes and eukaryotes. The ARTs’ enzymatic activities are different even from one species to another within eukaryotes. The ADP-ribosylation of the certain proteins generally changes the proteins’ biological functions significantly. For example, cholera toxin and the related heat-labile enterotoxin of Escherichia coli ADP-ribosylate G proteins in epithelial cells in the intestine and alter critical metabolic pathways of the host leading to the death of the host. The differently classified eukaryotic ARTs have different cellular localizations. More than five different ecto-ARTs, designated ART1-ART5, which distantly resemble ADP-ribosylating bacterial toxins, have been cloned from human and other mammals. ART1-ART4 are glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, while, ART5 is a secretory soluble enzyme. There are a series of ARTs throughout mammalian tissues. Different tissues or systems have different ARTs. Although these ARTs have been studied for a long time, the full biological functions of these ARTs are not completely understood. One of the most well understood ARTs is ART 2 expressed in rats, the BBDR and BBDP strains. These two different strains have almost the same genetic background except for a few loci in their chromosomes. One of these genes is ART 2. BBDR rats express ART 2 on the surface of T cells while BBDP rats do not, and BBDR is resistant to diabetes type I while BBDP is prone to diabetes type I.
There is little doubt that ART 2 is involved in modulation of diabetes type I development in animal models like the Bio-Breeding rat, BBDP stain. However, the molecular mechanism through which ART2 plays a role in modulation of diabetes type I has not yet been uncovered. In order to understand the deeper mechanism through which ART 2 is involved in the modulation of diabetes type I, a hypothesis is proposed in this thesis that ART 2 is one of the immune regulators involved in the modulation of diabetes type I and that prevention of diabetes type I is through the enzymatic activity of ART2. This enzymatic activity may be regulated by T cells through protein-protein interaction at the T cell membrane micro-environment or vice versa, the enzymatic activity of ART 2 also may regulate immune function in a tissue specific way, for instance, at the pancreatic islet. Although the expression and the functional roles of ART 2 in human, rat, and mouse are different, understanding the role of ART 2 in any of human, rat, or mouse will strongly support the final understanding, curing and prevention of human diabetes type I. Therefore, the mouse ART 2.1 was chosen in this study and the systematic studies were carried out.
Recombinant mouse ART 2.1 is an ADP-ribosyltransferase, which transfers ADP-ribosyl moiety of NAD to certain targets in a reducing reagent dependent reaction. In this study, the functional and structure differences between the membrane-bound ART 2.1 and soluble ART 2.1 were illustrated and the possible signaling pathway of ART 2.1 in T cell development and activation was proposed from the results of these studies. In particular, the following points were newly uncovered in this thesis studies. The recombinant mouse ART 2.1 containing lipid complex was isolated from recombinant ART 2.1 expressing COS1 cells. The membrane-bound mouse ART 2.1 ADP-ribosylated the high molecular weight histone more efficiently than the one with low molecular weight, while soluble mouse ART 2.1 ADP-ribosylated in a ratio equivalent to the amount of each histone fraction. ADP-ribose partially promoted the enzymatic activity of ART 2.1, while reducing reagent DTT was absolutely necessary for the transferase’s activity of ART 2.1. The conformation of membrane-bound ART 2.1 was different from soluble ART 2.1. This was presumably due to the interaction of ART 2.1 with other molecules in membrane. The GPI bond between ART 2.1 and plasma membrane was one of the factors involved in maintaining the conformation of membrane-bound ART2.1, because GPI bond digestion by PI-PLC released the ART 2.1 from cell surface and the enzymatic activity of the released ART 2.1 was similar to that of soluble ART2.1. The conformation of membrane-bound ART 2.1 could be shifted into the conformation of soluble ART 2.1 by isolating the ART 2.1 from the lipid environment with detergents, like Triton X 100. Besides lipid components, several proteins were found in the ART 2.1 lipid complex, these proteins were displayed in 2D gel electrophoresis. Based on such experimental information, the lollipop model of ART 2.1 was excluded and the flip down model of ART 2.1 was proposed. A T cell signaling pathway for ART 2.1 was also proposed based on the information obtained from this study.
생쥐ADP-ribosyltransferase (ART) 2.1 은 NAD 의 ADP-ribose 부분을 수용체에 결합시키는 효소로서T 세포 표면에 Glycosylphosphatidylinosyltol (GPI) bond를 형성하여 존재한다. 본 연구에서는 세포막 지질체 혼합물에 있는 ART 2.1과 용해성 ART 2.1의 구조와 기능의 차이를 밝혀 T 세포의 분화와 활성화의 신호전달체계를 밝히고자 하였다. 재조합 생쥐 ART 2.1 염기 서열을 COS1 세포에서 발현시켜, 생쥐 ART 2.1 효소 세포막 지질체 혼합 물질을 분리하고 ART 2.1 염기 서열 중 신호 펩타이드 뒷부분의 GPI 연결 신호 펩타이드를 제거한 재조합 효소를 대장균에서 발현시켜 용해성 ART 2.1를 정제하였다. 세포막 표면에 발현시킨 ART 2.1효소 기능과 대장균에서 발현시킨 용해성 ART 2.1효소의 기능을 비교한 결과 세포막 표면에 발현한 ART 2.1효소는 ADP-ribose 를 큰 분자량의 histone 에 전달한 양이 작은 분자량의 histone에 전달한 양보다 많았다. 반면, 용해성 ART 2.1는 histone 분자량과 상관없이 큰 분자량의 histone 과 작은 분자량의 histone에 비슷한 양으로 ADP-ribose를 전달하였다. 이 원인을 연구한 결과 GPI bond 외 여러 요인들이 작용한 것으로 추정된다. PI-PLC로 GPI bond를 제거한 결과 세포막 표면으로부터 ART 2.1가 떨어지면서 이ART 2.1은 용해성 ART 2.1과 비슷한 기질 특이성을 보였다. 세포막표면 ART 2.1의 GPI bond 를 유지시키고 Triton-X 100로 처리했을 때 세포막 표면에 발현시킨 ART 2.1 은 위와 같이 용해성 ART 2.1과 유사한 기질 특이성을 보였다. 이 결과로 세포 표면에 발현시킨 ART 2.1 은 GPI bond 로 세포막과 연결되어 있을 뿐만 아니라 GPI bond 와 세포막의 다른 지질체와 상호작용하면서 세포표면의 ART 2.1의 입체구조를 유지시키고 있음을 알 수 있었다. 세포 표면의 ART 2.1가 여타의 단백질과 상호작용을 하는지 조사하기 위하여 생쥐 ART 2.1 효소 세포막 지질체 혼합체을 2D 전기영동으로 전개한 결과 10여종의 다른 단백질이 생쥐 ART 2.1 효소 세포막 지질체 혼합체에 포함되어 있었다.
결론적으로 생쥐 ART 2.1 효소는 세포막 표면에 GPI bond로 정착하여 세포막의 지질 및 단백질들이 생쥐 ART 2.1과 상호작용을 하고 있음을 알 수 있었다. 이러한 상호작용들은 생쥐 ART 2.1의 효소 특이성에 중요한 요인으로 작용할 것이다. 기능적으로는 ART 2.1 이 지질 및 단백질들과의 상호작용을 통하여 T 세포의 신호 전달에 관련될 가능성을 시사하며 이 체계의 이상이Type I 당뇨병의 병인과 관련된다고 추정된다.
