-Glucocerebrosidase activity in mammalian stratum corneum

Although previous studies have demonstrated a crucial role for the enzyme b -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. b -Glucocerebrosidase activity in mammalian stratum corneum. J. Lipid Res. 1999. 40: 861–869. Supplementary key words b -glucocerebrosidase • b -glucosidase • epidermis • stratum corneum • Gaucher disease • glucosylceramide • ceramide • sphingolipid • permeability barrier • cutaneous Lipids of the stratum corneum (SC) of skin comprise a distinctive mixture that is enriched in ceramides, cholesterol, and free fatty acids. These lipids appear to provide the barrier against excess water loss and to limit the ingress of xenobiotics. The ceramide–cholesterol–free fatty acid mixture replaces a phospholipid–neutral lipid mixture during the transition of keratinocytes from the stratum granulosum to the SC (1–4). A number of lipid catabolic enzymes, including sphingomyelinase (5), phospholipase A (6), triacylglycerol hydrolase (5, 7), and steroid sulfatase (8) have been localized in sites where these transformations occur. Moreover, many of these enzymes also have been localized within epidermal lamellar bodies (9–11), suggesting that, although alternative delivery pathways may exist, most of these lipid hydrolytic activities in the SC result from the secretion of this organelle’s contents. A high concentration of glucosylceramides exists in the stratum granulosum (SG), but in the inner SC, glucosylceramides are eliminated while the ceramide content is markedly increased. This distribution pattern for glucosylceramide, along with other more direct evidence discussed below, suggests that the conversion of glucosylceramide to ceramide may be important for the maintenance of skin barrier function. For example, mucosal epithelia (12, 13) and cetacean epidermis (9) do not display a fully competent barrier, and the ratio of glucosylceramides to ceramides in these tissues remains high (14). Although a number of prior studies have reported epidermal b -glucosidase activity in vitro (12, 15–20), we found this activity is due specifically to b -glucocerebrosidase (GlcCer’ase) (21), which only hydrolyzes glucosylceramides to ceramides. Moreover, several studies indicate an important role for this specific enzyme in the epidermal permeability barrier: 1 ) inhibition of GlcCer’ase diminishes epidermal permeability barrier function in association with altered lamellar membrane formation (22, 23); and 2 ) depletion of GlcCer’ase in Gaucher disease and in transgenic, knockAbbreviations: GlcCer’ase, b -glucocerebrosidase; BrCBE, bromoconduritol-B epoxide; 4-MUG, 4-methylumbelliferylb d -glucoside; PBS, Dulbecco’s calciumand magnesium-free phosphate-buffered saline; SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale. 1 To whom correspondence should be addressed. at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom