Molecular interaction between the components in lysosomal sialidase complex

近年来的研究表明，哺乳动物溶酶体唾液酸酶与β-半乳糖苷酶和保护蛋白以复合物的形式存在。该保护性蛋白是30kDa和20kDa原体的48kDa异源二聚体，是β-半乳糖苷酶聚合以及唾液酸酶活性表达和稳定所必需的。为了了解β-半乳糖苷酶复合物的功能，我们利用Biacore表面等离子体共振技术研究了复合物中β-半乳糖苷酶组分之间的分子相互作用。每个β-半乳糖苷酶组分被分离和纯化如前所述。生物化的β-半乳糖苷酶单体（64kDa）与传感器尖端表面的链脲嘧啶基团偶联。检测保护蛋白中48kDa、30kDa和20kDa蛋白组分与固定在传感器尖端的β-半乳糖苷酶单体的相互作用。在酸性条件下，48kDa和20kDa蛋白与β-半乳糖苷酶单体具有较强的亲和力，而30kDa蛋白与β-半乳糖苷酶单体无相互作用。结果表明，β-半乳糖苷酶与保护蛋白中20kDa蛋白组分结合，形成β-半乳糖苷酶异聚复合物，而30kDa蛋白具有羧肽酶活性。为了阐明唾液酸酶与β-半乳糖苷酶复合物之间的分子相互作用，还需要进一步的研究。我们从海星（Asterina pectinifera）的卵巢中高度纯化了唾液酸酶。纯化酶的n端氨基酸序列与组织蛋白酶D具有较高的同源性。在纯化过程中，不仅唾液酸酶活性比活性提高，组织蛋白酶D活性也有所提高。两种酶在聚丙烯酰胺凝胶电泳上显示出不同的蛋白带。唾液酸苷酶是否与组织蛋白酶D相互作用还有待进一步研究。

Recent studies has shown that the mammalian lysosomal sialidase exists as a complex with β-galactosidase and protective protein. The protective protein is a 48kDa heterodimer of 30kDa- and 20kDa protomers and is required for multimerization of β-galactosidase as well as the expression and stabilization of sialidase activity. To understand the function of β-galactosidase complex, we examined the molecular interaction between β-galactosidase components in the complex using Biacore surface plasmon resonance. Each of the β-galactosidase components was separated and purified as described previously. The biotinized β-galactosidase monomer(64kDa) was coupled to a streptoavidine group on the senser tip surface. The 48kDa, 30kDa and 20kDa protein components in protective protein were tested for their interaction with β-galactosidase monomer fixed on the sensor tip. Among the components, 48kDa and 20kDa proteins had strong affinity with β-galactosidase monomer at acidic pH, but 30kDa protein did not show any interaction. This result suggests that β-galactosidase bind to 20kDa protein component in protective protein and forms a β-galactosidase hetero polymer complex, in contrast with 30kDa protein which possess carboxypeptidase activity. To elucidate the molecular interaction between sialidase and β-galactosidase complex, further studies are necessary.We highly purified a sialidase from the ovary of the starfish, Asterina pectinifera. The analysis of the N-terminal amino acid sequences of the purified enzyme showed high homology with that of cathepsin D. On the purification processes, the specific activities of not only sialidase activity but also cathepsin D activity were increased. The two enzymes showed distinct protein band on polyacrylamide gel electrophoresis. It is necessary to study more precisely whether sialidase interact with cathepsin D or not.