Transcript Ronnie Almonte PREP Poster 2011

A Novel Pharmaceutical Chaperone for Restoring Enzyme Activity in
Schindler Disease Mutants
Ronnie Almonte, Matthew Metcalf, Nathaniel E. Clark, Scott C. Garman
Department of Biochemistry and Molecular Biology
ABSTRACT
Lysosomes are organelles that rely on acid hydrolase enzymes and transport
proteins to break down waste materials and digest macromolecules for the cell.
Lysosomal storage diseases are a group of conditions where a deficiency in the
activity of one or more of these lysosomal proteins leads to a toxic accumulation of
their undegraded substrate(s) or binding partner(s). There are over 50 types of
LSDs, and as a group their incidence is approximately 1 in 7,000 people. No cures
exist for lysosomal storage diseases, and current treatments are based on
supplementing with exogenous enzyme (i.e. enzyme replacement therapy) to
compensate for endogenous deficiency.
Schindler disease is caused by loss-of-function mutations in NAGA, a gene encoding
a lysosomal exoglycosidase called α-N-acetylgalactosaminidase (α-NAGAL) that
cleaves terminal α-N-acetylgalactosamine (GalNAc) residues from glycopeptides
and glycolipids. Studies in vitro have shown that a chemical chaperone called 1deoxygalactonojirimycin (DGJ) can bind to the active site of α-galactosidase (αGAL), a homolog of α-NAGAL associated with Fabry disease, and increase the wildtype activity of particular missense mutations. Since α-NAGAL and α-GAL share 11
of the 13 residues that make up their corresponding active sites, we predict that the
DGJ equivalent for α-NAGAL, called 1,2-deoxy-2-acetamidodeoxygalactonijirimycin
(DGJNAc), can similarly restore the wildtype activity of some Schindler disease
mutants. We use western blot analysis, fluorescence spectrophotometry, and
flourescent microscopy to gain insight as to whether DGJNAc can restore some
wildtype activity in two documented missense mutations found in patients.
Enzyme Activity Assay
Electrodialysis
Substrate and Chemical Chaperone DGJNAc
EXPERIMENTAL DESIGN AND RESULTS
Activity With and Without DGJNAc, Before and After Dialysis
Flourescent Microscopy and Western Blot Analysis
INTRODUCTION
E367K without DGJNAc
E367K with DGJNAc
Lysosomal Storage Diseases and Schindler Disease
120%
100%
80%
60%
Before Dialysis
After Dialysis
40%
In Vitro Expression
20%
0%
Enzymes
WT (-)
WT (+)
CONCLUSIONS AND FUTURE DIRECTIONS
• Western blot analysis and flourescent microscopy show that DGJNAc may
increase the wildtype expression and trafficking in these mutants
• Electrodialysis protocol is not likely optimal, as we would expect an increase in
activity of WT with DGJNAc after dialysis
Human α-NAGAL Structure
Future Directions
• Repeat western blot with additional mutants
• Flourescence microscopy to visualize whether DGJNAc increases α-NAGAL
trafficking to the lysosome
•
Optimize electrodialysis protocol
S160C
ACKNOWLEDGEMENTS
• Dr. Scott Garman, Matthew Metcalf, Nathaniel E. Clark, Yadilette RiveraColón and all Garman Lab members
• Dr. Katayoun Chamany and the science department at Eugene Lang
College
• The Northeast Alliance for Graduate Education and the Professoriate
(NEAGEP)
• Jackeline Almonte, Raul Flores, and Annie Flynn Murray Holmes for their
love and support