Beta-glucocerebrosidase activity in mammalian stratum corneum.
Beta-glucocerebrosidase activity in mammalian stratum corneum.
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DOI：
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发表时间：
1999-05
影响因子：
6.5
通讯作者：
Y. Takagi;E. Kriehuber;G. Imokawa;P. Elias;W. Holleran
中科院分区：
文献类型：
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作者：
Y. Takagi;E. Kriehuber;G. Imokawa;P. Elias;W. Holleran
尽管先前的研究已经证明了酶β-葡糖脑苷脂酶（GlcCer'ase）在哺乳动物角层（SC）中的膜结构成熟和表皮稳态的最后步骤中的关键作用，但GlcCer'ase活性和蛋白质的精确体内定位尚不清楚。在这里，我们开发了一种组织切片上的荧光原位测定（酶谱法）来阐明GlcCer'酶活性的体内分布，并进一步表征和定位SC GlcCer'酶活性。酶谱技术揭示了在小鼠和人SC中的颗粒上层和SC中较高的GlcCer'酶活性;该活性均被Conduritol B环氧化物（一种特异性GlcCer'酶抑制剂）抑制，并且是pH依赖性的;即，在pH 5.2时存在，在中性pH（7.4）时不存在或显著降低，这与已知的体外表皮GlcCer酶的最适pH一致。GlcCer'ase蛋白的免疫组织化学染色显示在人表皮的外层中增强的荧光信号，集中在颗粒层和下SC的顶端和边缘。此外，在来自单个表皮层的提取物中，GlcCer'ase活性存在于整个小鼠表皮中，其中在SC中具有最高活性，牛磺胆酸钠对SC活性的刺激大于10倍，而溴代糖醇B环氧化物则抑制SC活性。最后，从SC片制备的分离的膜偶联也显示出显著的GlcCer'酶活性。这些数据通过三种不同的技术将GlcCer酶活性定位于外表皮，并支持该酶在葡萄糖神经酰胺至神经酰胺的细胞外加工中的作用，这是渗透屏障成熟和功能所需的。
Although previous studies have demonstrated a crucial role for the enzyme beta-glucocerebrosidase (GlcCer'ase) in the final steps of membrane structural maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic sections (zymography) to elucidate the in vivo distribution of GlcCer'ase activity, and further characterized and localized the SC GlcCer'ase activity in vitro. The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was both inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal GlcCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein showed enhanced fluorescent signal in the outer layers of human epidermis, concentrated at the apex and margins of stratum granulosum and lower SC. Moreover, in extracts from individual epidermal layers, GlcCer'ase activity was present throughout murine epidermis, with the highest activity in the SC, peaking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by sodium taurocholate, and inhibited by bromoconduritol B epoxide. Finally, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer'ase activity. These data localize GlcCer'ase activity to the outer epidermis by three different techniques, and support the role of this enzyme in extracellular processing of glucosylceramides to ceramides, required for permeability barrier maturation and function.