Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein.
Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein.
复制标题
重新捕获细胞外 GM2 激活蛋白的两种机制：该蛋白主要分泌形式的证据。
作者：
B. Rigat;Wen Wang;A. Leung;D. Mahuran
GM 2激活蛋白是一种小的单体蛋白，含有一个Asn连接的糖基化位点。其唯一经证实的体内功能是作为溶酶体β-氨基己糖苷酶A水解GM 2神经节苷脂的底物特异性辅因子。然而，我们和其他人已经表明，它可以作为一个一般的糖脂转运蛋白在中性pH值的体外。任何其他可能的体内功能都需要一些新合成的激活剂分子不靶向溶酶体。激活剂的溶酶体靶向机制尚未最终确定。虽然早期的报告表明，它可能是通过甘露糖-6-磷酸受体，另一个最近的报告表明，缺陷的人类细胞可以重新捕获非糖基化，细菌产生的激活剂，这表明它使用的替代靶向途径。在这里，我们表明，甘露糖-6-磷酸途径可能是主要的细胞内，生物合成途径的溶酶体，以及高亲和力的再捕获途径的细胞外液的激活蛋白的内吞作用。此外，我们表明，存在第二个较低的亲和力重新捕获途径，需要其天然的蛋白质结构，是碳水化合物的独立性，并可能不涉及其在质膜结合鞘糖脂的能力。最后，我们的文件，新合成的前体激活蛋白的池中含有大部分的分子与复杂的类型的寡糖，它不能包含一个功能性的甘露糖-6-磷酸靶向信号。这些分子构成正常人成纤维细胞中蛋白质的分泌形式。
The GM2 activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo function is to act as a substrate specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at neutral pH in vitro. Any other possible in vivo functions would require that some of the newly synthesized activator molecules not be targeted to the lysosome. The lysosomal targeting mechanism for the activator has not been conclusively identified. While earlier reports suggested that it is likely through the mannose-6-phosphate receptor, another more recent report demonstrated that deficient human cells could recapture nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mannose-6-phosphate pathway is likely the major intracellular, biosynthetic route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Additionally, we show that there exists a second lower affinity recapture pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphingolipids in the plasma membrane. Finally, we document that the pool of newly synthesized precursor activator protein contains a majority of molecules with a complex-type oligosaccharide, which cannot contain a functional mannose-6-phosphate targeting signal. These molecules makeup the secreted forms of the protein in normal human fibroblasts.