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

N-葡聚糖与许多生物过程有关 包括细胞黏附。然而，N-葡聚糖在体内的确切作用是 很大程度上是不确定的。在这方面，由以下原因引起的遗传病 糖基化缺陷将作为阐明其作用的有用模型。 N-葡聚糖。迄今为止，这一类别中的两种人类遗传病是 为人所知。他们是碳水化合物缺乏症候群和HEMPAS (酸化血清阳性的遗传性红系多核 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，导致不完全糖基化 红系细胞中的多聚半乳糖胺聚糖。HEMPAS患者有贫血和 显示有肝硬变、糖尿病和胆结石，提示有缺陷 N-糖链的合成会影响这些组织。 为了更全面地了解复杂N-聚糖在发育和发育中的作用 疾病方面，我们建议进行以下研究。首先，我们将确定 两例HEMPA患者α-甘露糖苷酶II基因突变 案子。1例患者的α-MII基因表达显著降低。 另一例患者α-MII基因表达正常，而无α-MII基因表达。 测定了MII酶的活性。中国人α-MII基因突变的研究 这些案件将会得到裁决。第二，我们将定义底物 一种新的α-MillX的特异性和细胞类型特异性表达 我们最近鉴定出了MII同工酶。α-MiIX基因在 不同类型的细胞将被检查以解释组织特异性 海马斯病的临床表现。第三，我们将在 其中阿尔法-Mix基因被敲除了。这种菌株和阿尔法-MII 基因敲除小鼠品系将作为HEMPAS的动物模型。这个 基因敲除小鼠的糖基化模式和病理改变 与HEMPAS患者相比。最后，我们将评估 α-MiI和/或α-MiIX零突变对碳水化合物的影响 介导的细胞黏附。来自Alpha-Mill和/或Alpha-Miix小鼠的细胞将 检测它们与E-、P-和L-选择素的结合。 创造阿尔法MiI/阿尔法Mix基因敲除小鼠将提供材料 N-糖链在个体发育和出生后的功能评估 函数，以期更好地了解HEMPAS 疾病。此外，该提案还将探讨N-糖链的参与 体内碳水化合物介导的细胞黏附。

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.