Co-reporter:Jianbing Jiang, Chi-Lin Kuo, Liang Wu, Christian Franke, Wouter W. Kallemeijn, Bogdan I. Florea, Eline van Meel, Gijsbert A. van der Marel, Jeroen D. C. Codée, Rolf G. Boot, Gideon J. Davies, Herman S. Overkleeft, and Johannes M. F. G. Aerts
ACS Central Science 2016 Volume 2(Issue 5) pp:351
Publication Date(Web):April 26, 2016
DOI:10.1021/acscentsci.6b00057
The development of small molecule activity-based probes (ABPs) is an evolving and powerful area of chemistry. There is a major need for synthetically accessible and specific ABPs to advance our understanding of enzymes in health and disease. α-Glucosidases are involved in diverse physiological processes including carbohydrate assimilation in the gastrointestinal tract, glycoprotein processing in the endoplasmic reticulum (ER), and intralysosomal glycogen catabolism. Inherited deficiency of the lysosomal acid α-glucosidase (GAA) causes the lysosomal glycogen storage disorder, Pompe disease. Here, we design a synthetic route for fluorescent and biotin-modified ABPs for in vitro and in situ monitoring of α-glucosidases. We show, through mass spectrometry, gel electrophoresis, and X-ray crystallography, that α-glucopyranose configured cyclophellitol aziridines label distinct retaining α-glucosidases including GAA and ER α-glucosidase II, and that this labeling can be tuned by pH. We illustrate a direct diagnostic application in Pompe disease patient cells, and discuss how the probes may be further exploited for diverse applications.
Co-reporter:Cécile M. J. Ouairy, Maria J. Ferraz, Rolf G. Boot, Marc P. Baggelaar, Mario van der Stelt, Monique Appelman, Gijsbert A. van der Marel, Bogdan I. Florea, Johannes M. F. G. Aerts and Herman S. Overkleeft
Chemical Communications 2015 vol. 51(Issue 28) pp:6161-6163
Publication Date(Web):19 Feb 2015
DOI:10.1039/C5CC00356C
Acid ceramidase is responsible for the ultimate step in the catabolism of (glyco)sphingolipids by hydrolysis of ceramide into sphingosine and free fatty acid. Deficiency in acid ceramidase is the molecular basis of Farber disease. Here we report the synthesis and characterization of an activity-based acid ceramidase probe.
Co-reporter:Mina Mirzaian, Patrick Wisse, Maria J. Ferraz, Henrik Gold, Wilma E. Donker-Koopman, Marri Verhoek, Herman S. Overkleeft, Rolf G. Boot, Gertjan Kramer, Nick Dekker, Johannes M.F.G. Aerts
Blood Cells, Molecules, and Diseases (April 2015) Volume 54(Issue 4) pp:
Publication Date(Web):1 April 2015
DOI:10.1016/j.bcmd.2015.01.006
Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5–9] 13C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r = 0.968, slope = 1.038). Next, we analysed GlcSph in 24 h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20 nM, range 0.11–8.92 nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples.In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.
Co-reporter:Cécile M. J. Ouairy, Maria J. Ferraz, Rolf G. Boot, Marc P. Baggelaar, Mario van der Stelt, Monique Appelman, Gijsbert A. van der Marel, Bogdan I. Florea, Johannes M. F. G. Aerts and Herman S. Overkleeft
Chemical Communications 2015 - vol. 51(Issue 28) pp:NaN6163-6163
Publication Date(Web):2015/02/19
DOI:10.1039/C5CC00356C
Acid ceramidase is responsible for the ultimate step in the catabolism of (glyco)sphingolipids by hydrolysis of ceramide into sphingosine and free fatty acid. Deficiency in acid ceramidase is the molecular basis of Farber disease. Here we report the synthesis and characterization of an activity-based acid ceramidase probe.