Lysosomal neuraminidase - Catalytic activation in insect cells is controlled by the protective protein/cathepsin A
Lysosomal neuraminidase - Catalytic activation in insect cells is controlled by the protective protein/cathepsin A
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DOI：
10.1074/jbc.m007380200
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发表时间：
2000-12-01
影响因子：
4.8
通讯作者：
d'Azzo, A
中科院分区：
文献类型：
--
作者：
Bonten, EJ;d'Azzo, A
溶酶体n -乙酰- α -神经氨酸酶与保护蛋白/组织蛋白酶A （PPCA）和β -半乳糖苷酶复合物具有活性，与PPCA的相互作用对于神经氨酸酶的正确胞内路径和溶酶体定位至关重要，但其催化激活机制尚不清楚。为了研究这一过程，我们利用杆状病毒表达系统在细胞中共同表达神经氨酸酶和PPCA前体，从而获得了神经氨酸酶的高酶活性，34和20 kda的PPCA亚基都是激活所必需的。我们进一步证明，当单独表达时，神经氨酸酶前体保持二聚体（114 kDa），酶活性低，但当与PPCA和β -半乳糖苷酶共表达时，它与其他两种蛋白一起聚合成一个类似于1350 kDa的复合物。充分活性的神经氨酸酶与全长PPCA和β -半乳糖苷酶前体共沉淀。然而，当与单个PPCA亚基共表达时，神经氨酸酶仅与小的20 kda多肽共沉淀，因此该多肽必须包含神经氨酸酶结合位点。我们的发现表明，神经氨酸酶的激活依赖于其在酸性pH下的寡聚化，这是通过与PPCA的相互作用介导的。
Lysosomal N-Acetyl-alpha -neuraminidase is active in complex with the protective protein/cathepsin A (PPCA) and beta -galactosidase, The interaction with PPCA is essential for the correct intracellular routing and lysosomal localization of neuraminidase, but the mechanism of its catalytic activation is unclear. To investigate this process, we have used the baculovirus expression system to co-express neuraminidase and PPCA precursors in in sect cells, which resulted in high enzymatic activity of neuraminidase, Both the 34- and 20-kDa PPCA subunits were required for the activation. We further demonstrated that when expressed alone, the neuraminidase precursor remained dimeric (114 kDa) and had low enzymatic activity, but when co-expressed with PPCA and beta -galactosidase, it multimerized in a complex of similar to 1350 kDa, together with the other two proteins. The fully active neuraminidase co-precipitated with full-length PPCA and beta -galactosidase precursors. However, when co-expressed with the individual PPCA subunits, neuraminidase co-precipitated only with the small 20-kDa polypeptide, which therefore must contain a neuraminidase-binding site. Our finding suggests a model of activation of neuraminidase dependent on its oligomerization at acidic pH that is mediated by interaction with PPCA.