Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

中文翻译：

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拟南芥AtGCD3蛋白是一种葡萄糖基神经酰胺酶，可优先水解长酰基链的葡萄糖基神经酰胺。

细胞膜含有许多脂质，其中一些例如鞘脂具有重要的结构和信号传导功能。常见的鞘脂糖基神经酰胺（GlcCer）存在于植物，真菌和动物中。作为主要的植物鞘脂，GlcCer参与脂质微区的形成，而GlcCer的调节是适应压力的关键。尽管已经阐明了GlcCer的生物合成途径，但对GlcCer的分解代谢知之甚少，并且尚未鉴定出植物的GlcCer降解酶（葡糖基神经酰胺酶（GCD））。在这里，我们确定了AtGCD3，这是人非溶酶体葡糖基神经酰胺酶的四个拟南芥同源基因之一，作为植物GCD。我们发现重组AtGCD3对于荧光脂质C6-NBD GlcCer具有较低的Km，并优先水解从拟南芥叶中纯化的长酰基链GlcCer。对哺乳动物葡糖基神经酰胺酶抑制剂的测试表明，人β-葡糖苷酶2的特异性抑制剂N-丁基脱氧野oxy霉素对人AtGCD3的抑制作用比人β-葡糖苷酶1的高露醇β-环氧化物的特异性抑制剂更有效。我们还发现AtGCD3中的Glu-499和Asp-647对GCD活性至关重要。GFP-AtGCD3融合蛋白主要位于质膜或内质网膜上。在gcd3突变体中未观察到明显的生长缺陷或鞘脂含量的变化。我们的结果表明AtGCD3是一种植物葡糖基神经酰胺酶，通过优先水解长酰基链GlcCers参与GlcCer分解代谢。