LOCALIZATION OF β-GLUCOCEREBROSIDASE ACTIVITY IN MAMMALIAN EPIDERMIS BY IN SITU ZYMOGRAPHY.

A critical, late step in epidermal barrier formation is the transformation of secreted lamellar body contents into mature extracellular lamellar unit structures. Although previous studies have demonstrated a crucial role for the enzyme β-glucocerebrosidase (GlcCer’ase) in the final steps of membrane structural maturation, the precise in vivo localization of GCase activity and protein is not known. Here, we characterized stratum corneum (SC) GlcCer’ase activity, and developed a fluorogenic in situ assay on histologic sections (zymography) to further elucidate the in vivo distribution of enzyme activity. In extracts from individual epidermal layers, GlcCer’ase activity was present throughout both murine and human epidermis, with the greatest activity in the SC, peaking in the lower-to-mid-SC. Moreover, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer’ase activity. Furthermore, with the zymographic technique, we confirmed the concentration of GlcCer’ase activity in upper stratum granulosum (SG) and SC, both in murine and human SC. In situ GlcCer’ase activity was both pH-dependent; i.e., present at pH 5.6, and absent or significantly reduced at neutral pH (7.6), consistent with the known pH optimum for epidermal GlcCer’ase in vitro; and it was inhibitable by conduritol B epoxide. Finally, the biochemical and zymographic results correlated with immunohistochemical staining for GlcCer’ase, which also showed maximal staining in the outer layers of human epidermis, concentrated at the apex and margins of SG and lower SC. These data demonstrate the localization of GlcCer’ase activity in 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.

Barrier properties formation, Beta-glucocerebrosidase, EpiDerm, Lamellar sheets, Lipids, ceramides, Zymography