Abstract
Although previous studies have demonstrated a crucial role for the enzyme β-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.—Takagi, Y., E. Kriehuber, G. Imokawa, P. M. Elias, and W. M. Holleran. β-Glucocerebrosidase activity in mammalian stratum corneum. J. Lipid Res. 1999. 40: 861–869.
Highlights
Previous studies have demonstrated a crucial role for the enzyme -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
During the transition from the basal proliferating epidermal layer to the outer cornified layer, the phospholipid– neutral lipid mixture is replaced by ceramides, cholesterol, and free fatty acids [1,2,3,4], and the total amount of lipids increases
In the SC, these lipids are localized to the intercellular spaces, where they form lamellar membrane structures, which are essential for epidermal permeability barrier function
Summary
Previous studies have demonstrated a crucial role for the enzyme -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. Isolated membrane couplets, prepared from SC sheets, 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.—Takagi, Y., E. Lipids of the stratum corneum (SC) of skin comprise a distinctive mixture that is enriched in ceramides, cholesterol, and free fatty acids.
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