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In-situ visualization of glucocerebrosidase in human skin tissue: Zymography vs activity-based probe labeling.

著者 van Smeden J , Dijkhoff IM , Helder RWJ , Al-Khakany H , Boer DEC , Schreuder A , Kallemeijn WW , Absalah S , Overkleeft HS , Aerts JMFG , Bouwstra JA
J Lipid Res.2017 Oct 12 ; ():.
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Epidermal 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. Here we report on two developed in-situ methods to localize active GBA in human epidermis: i) an optimized zymography method that is less labour 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. ii) a novel technique to visualize active GBA1 molecules by their specific labelling with a fluorescent activity-based probe (ABP MDW941). The latter method proved more robust and sensitive, provides higher resolution microscopic images, and is less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various β-glucosidases, MDW941 specifically labels 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 stratum corneum. With ABP-labelling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with reversible inhibitor isofagomine, irrespective of GBA expression. This inhibition affected the stratum corneum ceramide composition: mass spectrometry analysis revealed an inhibitor-dependent increase in glucosylceramide:ceramide ratio.
PMID: 29025868 [PubMed - as supplied by publisher]
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