Dataset Information

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

ABSTRACT: Epidermal ?-glucocerebrosidase (GBA1), an acid ?-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: i) 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-?-D-glucopyranoside or resorufin-?-D-glucopyranoside; and ii) 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 ?-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.

SUBMITTER: van Smeden J

PROVIDER: S-EPMC5711493 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

van Smeden Jeroen J Dijkhoff Irini M IM Helder Richard W J RWJ Al-Khakany Hanin H Boer Daphne E C DEC Schreuder Anne A Kallemeijn Wouter W WW Absalah Samira S Overkleeft Herman S HS Aerts Johannes M F G JMFG Bouwstra Joke A JA

Journal of lipid research 20171012 12

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: <i>i</i>) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods u ...[more]

PMID: 29025868

Similar Datasets

Project description:Recent years have seen the emergence of conditionally activated diagnostics and therapeutics that leverage protease-cleavable peptide linkers to enhance their specificity for cancer. However, due to a lack of methods to measure and localize protease activity directly within the tissue microenvironment, the design of protease-activated agents has been necessarily empirical, yielding suboptimal results when translated to patients. To address the need for spatially resolved protease activity profiling in cancer, we developed a new class of in situ probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZP) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. AZPs were leveraged within a generalizable framework to design conditional cancer diagnostics and therapeutics and showcased in the Hi-Myc mouse model of prostate cancer, which models features of early pathogenesis. Multiplexed screening against barcoded substrates yielded a peptide, S16, that was robustly and specifically cleaved by tumor-associated metalloproteinases in the Hi-Myc model. In situ labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, premalignant Hi-Myc prostatic glands. Systemic administration of an in vivo imaging probe incorporating S16 perfectly classified diseased and healthy prostates, supporting the relevance of ex vivo activity assays to in vivo translation. We envision AZPs will enable new insights into the biology of protease dysregulation in cancer and accelerate the development of conditional diagnostics and therapeutics for multiple cancer types. SIGNIFICANCE: Visualization of protease activity within the native tissue context using AZPs provides new biological insights into protease dysregulation in cancer and guides the design of conditional diagnostics and therapeutics.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure