In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling

Abstract

Epidermal β-glucocerebrosidase (GBA1), an acid β-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: ) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-β-D-glucopyranoside or resorufin-β-D-glucopyranoside; and) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various β-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.

An overview of activity-based probes for glycosidases

“…Many glycosidase ABPs are cell permeable, allowing for their use in cells, tissues, and even whole organisms, enabling a variety of imaging applications (Figure 4a). van Smeden et al have shown that epidermal GBA in intact skin sections can be visualized by confocal microscopy following application of fluorescent cyclophellitol-derived ABPs [52]. Similar GBA imaging in human fibroblasts was demonstrated by van Meel et al, who also applied correlated light and electron microscopy (CLEM) to confirm specific staining of the lysosomal compartment [53].…”

Role of μ-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish

“…In man and mice, a complete deficiency of Gba1 is not compatible with terrestrial life due to altered skin permeability causing trans-epidermal water loss (37, 55). Recently the abundant presence of active GBA1 in the stratum corneum of human skin has been visualized by labelling with a specific fluorescent ABP and zymography (56). Fortuitously, different properties of fish skin and habitat allow generation of animals with a Gba1 deficiency.…”

Unravelling effects of relative humidity on lipid barrier formation in human skin equivalents

“…Activity-based probe assay using MDW941 was performed as reported earlier by van Smeden et al . [52].…”

“…This could be a consequence of elevated pH values at submerged or occluded conditions [42], which may affect the hydrolytic activities of β-glucocerebrosidase-1 (GBA) or acid sphingomyelinase (aSMASE). These enzymes convert glucosylceramide and sphingomyelin, respectively, to ceramide at the stratum granulosum (SG)–SC interface [16, 52]. Opposite to submerged conditions, in HSEs generated at reduced RH (50%), the expression of glucosylceramide synthase was upregulated [46], indicating that RH is associated with biosynthesis and processing of glucosylceramides.…”