Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes.
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.
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Jianbing
J
Jiang
Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Chi-Lin
CL
Kuo
Department of Chemistry, University of York , Heslington, York, YO10 5DD, U.K.
Liang
L
Wu
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Christian
C
Franke
Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Wouter W
WW
Kallemeijn
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Bogdan I
BI
Florea
Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Eline
E
van Meel
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Gijsbert A
GA
van der Marel
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Jeroen D C
JD
Codée
Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Rolf G
RG
Boot
Department of Chemistry, University of York , Heslington, York, YO10 5DD, U.K.
Gideon J
GJ
Davies
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Herman S
HS
Overkleeft
Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Johannes M F G
JM
Aerts