Kuokkanen Elina, Smith Wesley, Mäkinen Marika, Tuominen Heidi, Puhka Maija, Jokitalo Eija, Duvet Sandrine, Berg Thomas, Heikinheimo Pirkko
Institute of Biotechnology, University of Helsinki, FIN-00014, Finland.
Glycobiology. 2007 Oct;17(10):1084-93. doi: 10.1093/glycob/cwm083. Epub 2007 Aug 6.
A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral alpha-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe(2+), Co(2+), and Mn(2+), and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe(2+) than Co(2+). Without activating cations the main reaction product was Man(8)GlcNAc, and Cu(2+) completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.
一种糖基水解酶家族38的酶，即中性α-甘露糖苷酶，被认为参与了细胞质中游离寡糖的水解，这些寡糖要么源自内质网错误折叠的糖蛋白，要么来自N-糖基化过程。尽管这种酶已从细胞质中分离出来，但通过亚细胞分级分离也发现它与内质网有关。我们通过免疫荧光显微镜研究了中性α-甘露糖苷酶的亚细胞定位，并用天然底物对人重组酶进行了表征，以阐明该酶的生物学功能。免疫荧光显微镜显示内质网、溶酶体和自噬体中不存在中性α-甘露糖苷酶，它以颗粒状分布在细胞质中。在光漂白后荧光恢复实验中，中性α-甘露糖苷酶在细胞质中的扩散比预期的两相扩散要慢。这一结果与免疫染色中的颗粒外观表明，部分中性α-甘露糖苷酶池以某种方式形成了复合物。纯化的重组酶是一种四聚体，活性的最适pH为中性。在Fe(2+)、Co(2+)和Mn(2+)存在的情况下，它将Man(9)GlcNAc水解为Man(5)GlcNAc，与其他生物体的中性α-甘露糖苷酶不同的是，人源酶被Fe(2+)激活的程度比Co(2+)更高。在没有激活阳离子的情况下，主要反应产物是Man(8)GlcNAc，而Cu(2+)完全抑制中性α-甘露糖苷酶。我们从酶-底物表征和亚细胞定位研究中得到的结果支持了中性α-甘露糖苷酶在可溶性细胞质寡甘露糖苷水解中的推测作用。
