Metabolism of lysosomal sialidase and molecular basis of sialidosis

为了深入了解唾液中毒的发病机制，我们对4例无关的日本患者进行了分子研究，并鉴定出5种新的错义突变，分别导致P80L、V217M、W240R、G243R和P316S氨基酸替换。利用同源性模型，构建了人溶酶体唾液酸酶的结构，并推导了这些突变引起的结构变化。结果表明，P80L和P316S的翻转引起了大的构象变化，包括负责结合唾液酸羧酸基的活性位点残基。推断出V217M、W240R和G243R的取代会影响所构建模型中有限区域的分子表面结构，涉及与保护蛋白/组织蛋白酶a的相互作用。V217M引起的预测变化小于W240R或G243R引起的预测变化，后者导致剧烈而广泛的改变。含有这些突变的过表达基因产物的分子量与野生型相同，但产物的数量略有减少。生化研究表明，表达的含有V217M突变的唾液酸酶部分被转运到溶酶体并显示出残留的酶活性，尽管其他突变体保留在内质网/高尔基区并失去了酶活性。考虑到这些数据，我们推测V217M替换可能与晚发和中度临床病程的唾液中毒表型密切相关

To gain insight into the pathogenesis of sialidosis, we performed molecular investigations of four unrelated Japanese patients, and identified five novel missense mutations causing amino acid substitutions, P80L, V217M,W240R, G243R and P316S, respectively. Using homology modeling, the structure of human lysosomal sialidase was constructed and the structural changes caused by these mutations were deduced. The results showed that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The V217M, W240R and G243R substitutions were deduced to influence the molecular surface structure of a limited region of the constructed models, involving in the interaction with the protective protein/cathepsin A. The predicted change due to V217M was smaller than that caused by W240R or G243R, the latters resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed sialidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although other mutants were retained in the endoplasmic reticulum/Golgi area and had lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis with a late onset and moderate clinical course