LYSOSOMAL ENZYMES AND ASSOCIATED HUMAN GENETIC DISEASES

有超过40种已知的溶酶体酶和辅助蛋白协同作用，将复杂的分子分解成简单的成分。已知由32种不同溶酶体蛋白突变引起的人类遗传疾病谱强调了这些蛋白质在生物医学上的重要性。令人信服的证据表明，还存在其他尚未鉴定的溶酶体蛋白，其中一些缺陷极有可能是病因不明的遗传性疾病的基础。本提案的总体目标是发展和实施一种系统的方法，将导致溶酶体蛋白的鉴定和表征及其在人类遗传疾病中的作用。这种方法依赖于发现大多数溶酶体蛋白含有一种常见的翻译后修饰，甘露糖6-磷酸（Man-6-P）识别标记物，该标记物将它们与其他类型的蛋白质区分开来。有四个具体目标。具体目的1是鉴定人脑中存在的Man-6-P糖蛋白谱。这将鉴定出一些新的人类溶酶体蛋白，并创建一个以等电点和分子量分离的人类Man-6-P糖蛋白的二维参考数据库。具体目的2是通过比较患者样本与正常对照中Man-6-P糖蛋白的谱，确定在病因不明的人类遗传性疾病中存在缺陷的溶酶体蛋白。具体目标3是使用基因组方法确定可能与编码新型溶酶体蛋白的基因相关的候选疾病。特异性目的4是通过鉴定相应基因的分子缺陷来证实溶酶体蛋白参与病因不明的疾病。拟议的研究极有可能导致发现多种人类遗传性疾病的分子基础，并将是实现明确诊断和产前检测的关键步骤。此外，这些研究将导致以前未知的人类溶酶体蛋白的特征，并将为理解溶酶体在生物学和医学中的作用提供基础贡献。

There are over 40 known lysosomal enzymes and accessory proteins that function in concert to degrade complex molecules into simple components. The biomedical importance of these proteins is underscored by the spectrum of human genetic diseases known to result from mutations in 32 different lysosomal proteins. Compelling evidence indicates that additional lysosomal proteins not yet identified exist, and it is highly likely that defects in some of these underlie hereditary diseases of unknown etiology. The overall goal of this proposal is to develop and implement a systematic approach that will result in the identification and characterization of lysosomal proteins and their role in human genetic diseases. This approach relies on the finding that most lysosomal proteins contain a common post-translational modification, the mannose 6-phosphate (Man-6-P) recognition marker, which distinguishes them from other types of proteins. There are four specific aims. Specific aim 1 is to identify the spectrum of Man-6-P glycoproteins present in human brain. This will identify a number of novel human lysosomal proteins and create a two-dimensional reference database of human Man-6-P glycoproteins separated by isoelectric point and molecular weight. Specific aim 2 is to identify lysosomal proteins that are defective in human genetic diseases of unknown etiology by comparing the spectrum of Man-6-P glycoproteins present in patient samples with normal controls. Specific Aim 3 is to use genomic methods to identify candidate diseases that may be associated with the genes encoding novel lysosomal proteins. Specific Aim 4 is to confirm the involvement of lysosomal proteins in diseases of unknown etiology by identifying the molecular defect in the corresponding genes. The proposed studies are highly likely to lead to the discovery of the molecular basis for multiple human hereditary diseases, and will be a critical step that will enable definitive diagnosis and prenatal testing. In addition, these studies will result in the characterization of previously unidentified human lysosomal proteins and will provide fundamental contributions towards understanding the role of lysosomes in biology and medicine.