Vinogradova M V, Michaud L, Mezentsev A V, Lukong K E, El-Alfy M, Morales C R, Potier M, Pshezhetsky A V
Université de Montréal, Service de Génétique Médicale, Département de Pédiatrie, Hôpital Sainte-Justine, Montréal, Québec, H3T 1C5 Canada.
Biochem J. 1998 Mar 1;330 ( Pt 2)(Pt 2):641-50. doi: 10.1042/bj3300641.
Galactosialidosis is an inherited lysosomal storage disease caused by the combined deficiency of lysosomal sialidase and beta-galactosidase secondary to the deficiency of cathepsin A/protective protein, which is associated with sialidase and beta-galactosidase in a high-molecular weight (1.27MDa) complex. Clinical phenotypes of patients as well as the composition of compounds which are stored in patient's tissues implicate sialidase deficiency as the underlying pathogenic defect. The recent cloning and sequencing of lysosomal sialidase [Pshezhetsky, Richard, Michaud, Igdoura, Wang, Elsliger, Qu, Leclerc, Gravel, Dallaire and Potier (1997), Nature Genet. 15, 316-320] allowed us to study the molecular mechanism of sialidase deficiency in galactosialidosis. By Western blotting, using antibodies against the recombinant human enzyme, and by NH2-terminal sequencing, we showed that sialidase is synthesized as a 45.5 kDa precursor and after the cleavage of the 47-amino acid signal peptide and glycosylation becomes a 48.3 kDa mature active enzyme present in the 1.27 kDa complex. Transgenic expression of sialidase in cultured skin fibroblasts from normal controls and from galactosialidosis patients, followed by immunofluorescent and immunoelectron microscopy showed that in both normal and affected cells the expressed sialidase was localized on lysosomal and plasma membranes, but the amount of sialidase found in galactosialidosis cells was approximately 5-fold reduced. Metabolic labelling studies demonstrated that the 48.3 kDa mature active form of sialidase was stable in normal fibroblasts (half-life approximately 2.7 h), whereas in galactosialidosis fibroblasts the enzyme was rapidly converted (half-life approximately 30 min) into 38.7 and 24 kDa catalytically inactive forms. Altogether our data provide evidence that the molecular mechanism of sialidase deficiency in galactosialidosis is associated with abnormal proteolytic cleavage and fast degradation.
半乳糖唾液酸贮积症是一种遗传性溶酶体贮积病，由组织蛋白酶A/保护蛋白缺乏继发的溶酶体唾液酸酶和β-半乳糖苷酶联合缺乏引起，该蛋白与唾液酸酶和β-半乳糖苷酶在高分子量（1.27MDa）复合物中相关联。患者的临床表型以及贮存在患者组织中的化合物组成提示唾液酸酶缺乏是潜在的致病缺陷。最近溶酶体唾液酸酶的克隆和测序[Pshezhetsky、Richard、Michaud、Igdoura、Wang、Elsliger、Qu、Leclerc、Gravel、Dallaire和Potier（1997年），《自然遗传学》15卷，316 - 320页]使我们能够研究半乳糖唾液酸贮积症中唾液酸酶缺乏的分子机制。通过蛋白质免疫印迹法，使用针对重组人酶的抗体，以及通过氨基末端测序，我们表明唾液酸酶作为45.5 kDa前体合成，在47个氨基酸的信号肽裂解和糖基化后成为存在于1.27 kDa复合物中的48.3 kDa成熟活性酶。在来自正常对照和半乳糖唾液酸贮积症患者的培养皮肤成纤维细胞中进行唾液酸酶的转基因表达，随后进行免疫荧光和免疫电子显微镜检查表明，在正常细胞和受影响细胞中，表达的唾液酸酶都定位于溶酶体和质膜上，但在半乳糖唾液酸贮积症细胞中发现的唾液酸酶量减少了约5倍。代谢标记研究表明，48.3 kDa成熟活性形式的唾液酸酶在正常成纤维细胞中稳定（半衰期约2.7小时），而在半乳糖唾液酸贮积症成纤维细胞中，该酶迅速转化（半衰期约30分钟）为38.7 kDa和24 kDa催化无活性形式。总之，我们的数据提供了证据，表明半乳糖唾液酸贮积症中唾液酸酶缺乏的分子机制与异常的蛋白水解裂解和快速降解有关。
