Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
J Biol Chem. 2010 Jan 29;285(5):3360-70. doi: 10.1074/jbc.M109.068650. Epub 2009 Dec 2.
UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.
UDP-GlcNAc:溶酶体酶 N-乙酰氨基葡萄糖-1-磷酸转移酶是一种α(2)β(2)γ(2)六聚体，它介导溶酶体酸性水解酶上甘露糖 6-磷酸识别标记合成的第一步。我们采用了多种方法，包括分析缺乏γ亚基的小鼠和成纤维细胞中酸性水解酶的磷酸化情况，以及对重组α(2)β(2)γ(2)和α(2)β(2)形式的转移酶的动力学研究，探讨了α/β和γ亚基的功能。研究结果表明，α/β亚基除了介导转移酶的催化功能外，还能识别酸性水解酶的蛋白质决定簇。在小鼠脑中，α/β亚基以接近野生型水平的方式磷酸化约三分之一的酸性水解酶，但需要γ亚基才能对其余酸性水解酶进行最佳磷酸化。除了增强α/β亚基对一部分酸性水解酶的活性外，γ亚基还促进了这些底物的高甘露糖寡糖上第二个 GlcNAc-P 的添加。我们推测，γ亚基的甘露糖 6-磷酸受体同源结构域结合并以有利的方式将受体的高甘露糖糖链呈现给α/β催化位点。
