IN VITRO ROLE OF N-GLYCANS BY GENETIC ANALYSIS OF GOLGI ALPHA MANNOSIDASE

N-聚糖参与了大量的生物学过程 包括细胞粘附。 然而，N-聚糖在体内的确切作用是 大部分未定义。 在这方面， 糖基化缺陷将作为有用的模型，阐明的作用， N-聚糖。 迄今为止，这类人类遗传疾病中有两种是 知道的 它们是碳水化合物缺乏糖蛋白综合征和HEMPAS （遗传性成红细胞多核性，酸化血清阳性 裂解）。 HEMPAS是一种罕见的遗传性疾病，由高尔基体缺陷引起 α-甘露糖苷酶II，导致 红系细胞中的聚乳糖胺聚糖。 HEMPAS患者贫血， 显示肝硬化，糖尿病和胆结石，这表明， N-聚糖的合成影响这些组织。 为了更全面地了解复杂N-聚糖在发育和 针对这些疾病，我们提出以下研究建议。 首先，我们将确定 两种HEMPAS中α-甘露糖苷酶II（alphaMII）基因的突变 例 1例患者显示α-MII mRNA显著降低。 另一名患者表达了正常水平的α-MII mRNA，而没有α-MII mRNA。 检测MII酶活性。 人的α-MII基因突变 将对这些案件作出裁决。 其次，我们将定义衬底 特异性和细胞类型特异性表达的α-MillX，一种新的α- MII同工酶是我们最近鉴定的。 α-MIX在大肠杆菌中的表达 将检查各种细胞类型以解释组织特异性 HEMPAS疾病的表现。 第三，我们将创建一个小鼠品系， 其中α-MIX基因被敲除。 这种菌株和α-MII 基因敲除小鼠品系将用作HEMPAS的动物模型。 的 将对基因敲除小鼠的糖基化模式和病理学进行分析。 与HEMPAS患者相比。 最后，我们将评估 α-MII和/或α-MIX无效突变对碳水化合物影响 介导的细胞粘附。 来自α-Mill和/或α-MIX小鼠的细胞将 检查它们与E-、P-和L-选择素的结合。 创造α MII/α-MIX基因敲除小鼠将提供材料， 用于个体发育和出生后N-聚糖的功能评估 函数，并期望更好地了解HEMPAS 疾病 此外，该提案将探讨N-聚糖的参与 在体内碳水化合物介导的细胞粘附中。

N-glycans have been implicated in a large number of biological processes including cell adhesion. However, the exact role of N-glycans in vivo is largely undefined. In this regard, genetic diseases caused by glycosylation defects will serve as useful models for elucidating the role of N-glycans. To date, two human genetic diseases in this category are known. They are carbohydrate deficiency glycoprotein syndrome and HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis). HEMPAS is a rare genetic disease caused by a defective Golgi alpha-mannosidase II, leading to incomplete glycosylation of polylactosaminoglycans in erythroid cells. HEMPAS patients are anemic and show liver cirrhosis, diabetes and gall stones, suggesting that defective synthesis of N-glycans affects these tissues. To understand more fully the role of complex N-glycans in development and diseases, we propose the following studies. First, we will determine mutations in the gene of alpha-mannosidase II (alphaMII) in two HEMPAS cases. One patient showed significant reduction of alpha-MII mRNA. Another patient expressed normal levels of alpha-MII mRNA, while no alpha- MII enzymatic activity was detected. Mutation of the alpha-MII gene in these cases will be determined. Second, we will define substrate specificity and cell type-specific expression of alpha-MillX, a new alpha- MII isozyme we have recently identified. Expression of alpha-MIIX in various cell types will be examined to explain the tissue specific manifestation of HEMPAS disease. Third, we will create a mouse strain in which the alpha-MIIX gene is knocked-out. This strain and the alpha-MII gene knock-out mouse strain will be used as an animal model of HEMPAS. The glycosylation pattern and pathology of the gene knocked-out mice will be compared with those from HEMPAS patients. Finally, we will evaluate the effect of alpha-MII and/or alpha-MIIX null mutation on carbohydrate mediated cell adhesion. Cells from alpha-Mill and/or alpha-MIIX mice will be examined for their binding to E-, P-, and L-selectins. Creating alpha MII/alpha-MIIX gene knock-out mice will provide materials for functional assessments of N-glycans in ontogeny and post-natal function, with the expectation that to gain a better understanding HEMPAS disease. Moreover, this proposal will explore the involvement of N-glycans in carbohydrate mediated cell adhesions in vivo.