STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

鞘脂增多症是一组严重的疾病 由鞘脂降解的遗传缺陷引起。在 为了研究发病机制和新的治疗方法， 通过全基因组研究， 基因敲除技术我们已经应用了这个 技术，建立泰-萨二氏病、山德霍夫病和 GM 2激活剂缺乏症在一些鞘脂病中，如 戈谢病，在小鼠中有效建模需要 引入微妙的突变变化。戈谢病是 由编码溶酶体酶的基因突变引起 葡糖脑苷脂酶（GC）。戈谢病的三种临床类型 已根据存在（类型2和3）定义，或 无（1型）中枢神经系统疾病和严重程度 临床表现疾病的临床过程与 GC基因携带的突变类型为了 产生点突变的小鼠， 我们设计了一种高效的 一步突变法--单插入突变法 程序（SIMP）-将人类疾病突变插入 小鼠GC基因。使用SIMP，已经产生了携带 要么是非常严重的RecNciI突变， 疾病或与3型相关的不太严重的L444 P突变 疾病RecNciI突变纯合子小鼠几乎没有GC 酶活性和积累的葡萄糖神经酰胺在大脑中， 肝脏相比之下，L444 P突变纯合子小鼠 更高水平的GC活性和未检测到的 葡萄糖神经酰胺在大脑和肝脏。2型和3型小鼠均死亡 出生后48小时内表皮渗透性受损 葡萄糖神经酰胺代谢缺陷引起的屏障 表皮目前的工作旨在引入遗传 这些修改将使戈谢小鼠能够在 新生儿期

The sphingolipidoses are a group of severe disorders caused by inherited defects in the degradation of sphingolipids. In order to study pathogenesis and new treatment approaches we developed models of some of these disorders by total gene knockout technology in the mouse. We have applied this technology to establish models of Tay-Sachs, Sandhoff disease and the GM2 activator deficiency. In some of sphingolipidoses, such as Gaucher disease, effective modeling in the mouse requires the introduction of subtle mutational changes. Gaucher disease is caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC). Three clinical types of Gaucher disease have been defined according to the presence (type 2 and 3) or absence (type 1) of central nervous system disease and severity of clinical manifestations. The clinical course of the disease correlates with the type of mutation carried by the GC gene. In order to produce mice with point mutations that correspond to the clinical types of Gaucher disease, we have devised a highly efficient one-step mutagenesis method -- the Single Insertion Mutagenesis Procedure (SIMP) -- to insert human disease mutations into the mouse GC gene. Using SIMP, mice have been generated carrying either the very severe RecNciI mutation that can cause type 2 disease or the less severe L444P mutation associated with type 3 disease. Mice homozygous for the RecNciI mutation had little GC enzyme activity and accumulated glucosylceramide in brain and liver. In contrast, the mice homozygous for the L444P mutation had higher levels of GC activity and no detectable accumulation of glucosylceramide in brain and liver. Both the type 2 and 3 mice died within 48 hrs of birth of a compromised epidermal permeability barrier caused by defective glucosylceramide metabolism in the epidermis. Current work is aimed at introducing genetic modifications that will allow the Gaucher mice to survive past the neonatal period.