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

这项建议的总体目标是加深我们对 抗半乳糖介导的人衰老红细胞的破坏及其机制的研究 人抗半乳糖结合位点表达增加的可能性 细胞可能导致自身免疫现象的启动。反Gal是一种 我们发现的天然抗体占人类循环免疫球蛋白的1%， 并与Gal alpha1&gt；3Gal beta1&gt；4GlcNac-R表位特异性相互作用。 作为一种持续的抗原性，它在人的一生中都会产生 表达相似碳水化合物的胃肠道细菌的刺激作用 表位。体内抗Gal与正常的从头暴露表位结合 衰老的红细胞和很大比例上的类似表位 β-地中海贫血和镰状细胞病患者的病理红细胞 疾病。体内数百个抗Gal免疫球蛋白分子与红细胞结合 In被发现足以标记细胞的吞噬作用。 巨噬细胞体外培养。这表明，抗Gal在 通过与神秘的Gal Alpha1.3Ga1相互作用破坏老化的红细胞 β1；AGlcNac-R表位在正常或变态过程中重新暴露 病理性红细胞老化。 为了获得关于与抗半乳糖相互作用的分子的进一步信息，我们 Galα1&gt；3Ga1β1&gt；AG1cNAc-R表位在红色表面的表达 多种哺乳动物的细胞、有核细胞和分泌的糖蛋白 物种，并观察到一种显著的进化模式。Gal alpha1&>3Ga1 在非灵长类哺乳动物中发现了丰富的β1和AG1cNAc-R残基， 原猴和新大陆猴子，但在细胞和 东半球猴子、猿和人类分泌的糖蛋白。《缺席》 后一物种的这一表位的减少是由于 Alpha1&Gt；3半乳糖基转移酶的活性，在Golg中： 装置，催化以下反应：Galbeta1&gt；4GlcNAc-R+UDP- Gal&gt；Gal alpha1&gt；3Gal beta1&gt；4GlcNAc-R+UDP。 我们的研究表明，这种酶的抑制可能是 发生在旧大陆的一个进化事件，两三千万 几年前。我们有间接证据表明字母1和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α1和3半乳糖基转移酶基因 在人类身上很少表达，导致合成了神秘的Gal 红细胞表面的α1&gt；3Ga1β1&gt；AG1cNAc表位。在细胞老化时， 这些表位被暴露并与抗Gal结合，从而起到衰老的作用 抗原。因此，该项目的主要工作将是克隆人的cdna α1和3半乳糖转移酶(来自牛源)，并将其用作 对其存在、表达及调控方式研究的探讨 基因存在于包括造血细胞在内的各种人类细胞中。此外，我们 将检验阿尔法1和3半乳糖基转移酶升高的假设 人类细胞中的活性，这是由于编码这一基因的基因的放松调控 酶，可能导致相互作用所介导的自身免疫现象 在抗Gal和从头表达Galα1和Gt；3Ga1β1和Gt；AG1cNAc之间 表位。这一点将被研究，特别是在系统性B淋巴细胞 红斑狼疮患者。当获得cDNAs探针时， 还将研究α1&GT；3半乳糖基转移酶基因的表达。 通过利用聚合酶链在各种其他自身免疫性疾病中 反应技术。最后，为了继续我们最初的提议，我们 将研究反GAL之间的相互作用的贡献 抗体和日益表达的Galalalpha1&gt；3Ga1 Beta1&gt；AG1cNAc表位， 与选定血液病患者红细胞的加速破坏有关 精神错乱。

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.