Characterization of the N370S Mutant of Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Table 2. Summary of guanidine-titration HDX-MS conditions of GCase. Conditions A [a] B [b] C [c] wt [mm] 3.0-R495H [mm] 3.0 3.0-N370S [mm] 3.0-3.0 No ligand +-IFG [mm]-214 (72 equiv) 214 (72 equiv) ABX [mm]-1150 (390 equiv) 1150 (390 equiv) GuHCl [m] 0.2-3.0 0.2-3.0 0.2-3.0 (0.5-5.4 with IFG) (0.5-5.4 with IFG) HEPES [mm] 50 50 50 pH read 7.0 7.0 7.0 DMSO [%] 0.3 0.3 0.3 D 2 O [%] 75 75 75 T [8C] 22 22 22 t inc [s] [d] 30 30 30 [a] Guanidine titration in the absence of ligand for wt, N370S, and R495H GCases. [b] Guanidine titration for R495H GCase in the IFG-or ABX-bound state. [c] Guanidine titration for N370S GCase in the IFG-or ABX-bound state. [d] Incubation time. 2162 www.chembiochem.org

Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment

“…similars with the patented drugs. For example, HDX-MS was used to characterize the structure of various versions of insulin (55,56) and several forms of glucocerebrosidase (brand name Cerezyme) for treatment of Gaucher disease (57). Likewise, HRF-MS was used to analyze conformations of therapeutic proteins including recombinant erythropoietin (EPO), interferon ␣-2A (IFN), granulocyte colony-stimulating factor (GCSF) (58).…”

Properties of wild-type and mutant glucocerebrosidases

“…In addition, the first (glucosylation) and second (deglucosylation) steps in the catalytic mechanism of GCase are defective as assessed in vitro [34]. These kinetic and catalytic abnormalies of the N370S enzyme are not reflected in the crystal structure of this mutant GCase as it has essentially a wild-type structure and function in the presence of the wild-type R495 or the R495H alteration [35,36]. Thus, these bases for the N370S defective function probably resides in altered flexibility and/or loop movement upon binding and cleavage of substrate [36].…”

“…These kinetic and catalytic abnormalies of the N370S enzyme are not reflected in the crystal structure of this mutant GCase as it has essentially a wild-type structure and function in the presence of the wild-type R495 or the R495H alteration [35,36]. Thus, these bases for the N370S defective function probably resides in altered flexibility and/or loop movement upon binding and cleavage of substrate [36]. The substrate preferences for the GCases derived from various mutant alleles has not been studied in vivo in humans because of the inaccessibility www.futuremedicine.com of tissues.…”

Applications of Hydrogen/Deuterium Exchange MS from 2012 to 2014

“…The effects of Endo H trimming of high mannose glycans from α-amylase were studied . Several forms of recombinant glucocerebrosidase, sold as Cerezyme, a treatment for Gaucher’s disease, were investigated by HDX MS, including mutants and ligand-bound forms. The effects of pegylation on the conformation and dynamics of granulocyte colony stimulating factor (G-CSF) were studied .…”