Beta-glucocerebrosidase activity in mammalian stratum corneum.
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.
Takagi
Y
Y
Biological Science laboratories, Kao Corporation, Tochigi, Japan.
Kriehuber
E
E
Imokawa
G
G
Elias
P M
PM
Holleran
W M
WM
D000818:Animals; D004817:Epidermis / Q000033:anatomy & histology / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D005962:Glucosylceramidase / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008812:Mice, Hairless; D008819:Mice, Nude; D008856:Microscopy, Fluorescence
D005962:Glucosylceramidase