Metabolism of lysosomal sialidase and molecular basis of sialidosis

为了深入了解唾液酸中毒的发病机制，我们对4名无关的日本患者进行了分子研究，并确定了5个新的错义突变，分别引起氨基酸取代，P80 L，V217 M，W240 R，G243 R和P316 S。利用同源模建方法，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，P80 L和P316 S的转换引起大的构象变化，包括负责结合唾液酸羧酸基团的活性位点残基。推测V217 M、W240 R和G243 R取代影响了构建的模型的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。V217 M引起的预测变化小于W240 R或G243 R引起的变化，后者导致了剧烈的，广泛的变化。含有这些突变的过表达基因产物具有与野生型相同的分子量，尽管产物的量适度降低。一项生化研究表明，表达的唾液酸酶含有V217 M突变被部分转运到溶酶体，并显示残留的酶活性，虽然其他突变体被保留在内质网/高尔基体区域，并失去了酶活性。考虑到这些数据，我们推测V217 M置换可能与晚发性和中度临床病程的唾液酸沉积症表型密切相关

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