ANTI-GAL IGG ON HUMAN RED CELLS--A MODEL FOR CELL AGING

本建议的总体目标是进一步了解 抗Gal介导的人衰老红细胞的破坏，并研究 增加抗-Gal结合位点在人类细胞上的表达的可能性 可能导致自身免疫现象的发生。抗Gal是一种 我们发现天然抗体占人体循环IgG的1%， 并与Gal α 1> 3Gal β 1> 4GlcNac-R表位特异性相互作用。 它在人的一生中作为一种恒定的抗原性的结果而产生。 通过表达相似碳水化合物胃肠细菌的刺激 表位体内抗Gal与正常人血清中从头暴露的表位结合 衰老的红细胞和相似的表位上的大部分 β-地中海贫血和镰状细胞贫血患者的病理性红细胞 疾病几百个抗Gal IgG分子在体内与红细胞结合 发现In足以标记细胞的吞噬作用， 体外巨噬细胞。这表明，抗Gal在 通过与隐蔽的Gal α 1.3Ga 1相互作用破坏老化红细胞 β 1> AGlcNac-R表位在正常或 病理性红细胞老化 为了获得与抗Gal相互作用的分子的进一步信息，我们 研究了红细胞表面Gal α 1> 3Ga1 β 1> AG1cNAc-R表位的表达， 细胞、有核细胞和各种哺乳动物的分泌糖蛋白 物种，并观察到一个惊人的进化模式。Gal α 1> 3Ga 1 β 1> AG1cNAc-R残基在非灵长类哺乳动物中大量存在， 原猴和新世界猴，但它在细胞上检测不到， 旧大陆猴、猿和人类分泌的糖蛋白。没有 发现来自后者物种的该表位的减少是由于 酶α 1> 3半乳糖基转移酶的活性，在Golg中： Galbeta1> 4GlcNAc-R + UDP- Gal> Gal α 1> 3Gal β 1> 4GlcNAc-R + UDP。 我们的研究表明这种酶的抑制可能是 发生在旧世界的一个进化事件， 年前我们有间接证据表明，α 1> 3 半乳糖基转移酶基因在人类基因组中是保守的。 我们目前的基本假设是α 1> 3半乳糖基转移酶基因 在人类中很少表达，导致合成了隐蔽的Gal 红细胞上的α 1> 3Ga 1 β 1> AG 1 cNAc表位。随着细胞的老化， 这些表位被暴露并结合抗Gal，从而起到衰老的作用 抗原的因此，本项目的主要工作将是克隆 α 1> 3半乳糖基转移酶（来自牛源），并将其用作 探讨研究这种存在，表达和调节模式， 在包括红细胞在内的各种人类细胞中的基因。我们还 将检验α 1> 3半乳糖基转移酶升高 在人类细胞中的活性，由于基因编码的失调， 酶，可能导致由相互作用介导的自身免疫现象 在抗Gal和从头表达的Gal α 1> 3Ga1 β 1> AG1cNAc之间 表位这将特别在来自全身的B淋巴细胞中进行研究。 红斑狼疮患者。当获得cDNA探针时， 还将研究α 1> 3半乳糖基转移酶基因的表达 在多种其他自身免疫性疾病中， 反应技术最后，为了延续我们原来的建议，我们 将研究抗半乳糖苷酶和抗半乳糖苷酶之间的相互作用 抗体和增加表达的Gal α 1> 3Ga1 β 1> AG1cNAc表位， 在选定的血液系统中红细胞的加速破坏 紊乱

The overall objective of this proposal is to further our understanding of the anti-Gal-mediated destruction of human aging red cells and to study the possibility that increased expression of anti-Gal binding sites on human cells may result in the initiation of autoimmune phenomena. Anti-Gal is a natural antibody which we found to constitute 1% of circulating IgG in man, and to interact specifically with Gal alpha1>3Gal beta1>4GlcNac-R epitopes. It is produced in man throughout life as a result of a constant antigenic stimulation by gastrointestinal bacteria expressing similar carbohydrate epitopes. In vivo anti-Gal binds to a de novo exposed epitope on normal senescent red cells and to a similar epitope on large proportion of pathologic red cells in patients with beta-thalassemia and sickle cell disease. A few hundred anti-Gal IgG molecules bound in vivo to red cells in were found to be sufficient for labeling the cells for phagocytosis by macrophages in vitro. This suggested that anti-Gal plays a role in the destruction of aging red cells by interacting with cryptic Gal alpha1.3Ga1 beta1>AGlcNac-R epitopes exposed de novo in the course of normal or pathologic red cell aging. To gain further information on the molecules interacting with anti-Gal, we studied the expression of Gal alpha1>3Ga1 beta1>AG1cNAc-R epitopes on red cells, nucleated cells, and secreted glycoproteins of various mammalian species, and observed a striking evolutionary pattern. The Gal alpha1>3Ga1 beta1>AG1cNAc-R residue was found to be abundant in nonprimate mammals, prosimians, and New World monkeys, but it is undetectable on cells and secreted glycoproteins of Old World monkeys, apes, and human. The absence of this epitope from the latter species was found to result from diminished activity of the enzyme, alpha1>3 galactosyltransferase, which, in the Golg: apparatus, catalyzes the following reaction: Galbeta1>4GlcNAc-R + UDP- Gal>Gal alpha1>3Gal beta1>4GlcNAc-R + UDP. Our studies suggest that the suppression of this enzyme may be the result of an evolutionary event which occurred in the Old World 20-30 million years ago. We have indirect evidence suggesting that the alpha1>3 galactosyltransferase gene has been conserved within the human genome. Our basic current hypothesis is that alpha1>3 galactosyltransferase gene is sparingly expressed in man, resulting in the synthesis of cryptic Gal alpha1>3Ga1 beta1>AG1cNAc epitopes on red cells. Upon aging of the cells, these epitopes are exposed and bind anti-Gal, thus serving as a senescence antigen. Thus a major effort in this project will be to clone the cDNA of alpha1>3 galactosyltransferasde (from a bovine source) and to use it as a probe for studying the presence, expression and mode of regulation of this gene in various human cells including erythropoietic cells. Further, we will test the hypothesis that elevation in alpha1>3 galactosyltransferase activity in human cells, due to deregulation of the gene encoding for this enzyme, may result in autoimmune phenomena mediated by the interaction between anti-Gal and de novo expressed Gal alpha1>3Ga1 beta1>AG1cNAc epitopes. This will be studied particularly in B-lymphocytes from systemic lupus erythematosus patients. When the cDNA probe is obtained, the expression of the alpha 1>3 galactosyltransferase gene will also be studied in a variety of other autoimmune diseases by utilizing the polymerase chain reaction technique. Finally, in continuation of our original proposal, we will study the contribution of the interaction between the anti-Gal antibody and increasingly expressed Gal alpha1>3Ga1 beta1>AG1cNAc epitopes, to the accelerated destruction of red cells in selected hematologic disorders.