title: Studies of pH-dependent self-association of a recombinant form of arylsulfatase A with electrospray ionization mass spectrometry and size-exclusion chromatography.
abstract: Arylsulfatase A is an endogenous enzyme that is responsible for the catabolism and control of sulfatides in humans. Its deficiency results in the accumulation of sulfatides in the cells of the central and peripheral nervous system leading to the destruction of the myelin sheath and resulting in metachromatic leukodystrophy (MLD), a neurodegenerative lysosomal storage disease. A recombinant human form of this glycoprotein (rhASA) is currently under development as an enzyme replacement therapy. At neutral and alkaline pH, this protein exists as a homodimer but converts to an octameric state in the mildly acidic environment of the lysosome, and a failure to form an octamer results in suboptimal catalytic activity (most likely due to a diminished protection from lysosomal proteases). Despite the obvious importance of the rhASA oligomerization process, its mechanistic details remain poorly understood. In this work, we use size exclusion chromatography (SEC) and electrospray ionization mass spectrometry (ESI MS) to monitor the dimer-to-octamer transition as a function of both solution pH and protein concentration. While SEC clearly shows different profiles (i.e., retention time differences) for rhASA when the chromatography is performed at neutral and lysosomal pH, consistent with changing oligomerization states, no resolved peaks could be observed for either octamer or dimer when analyzed at intermediate pH (5.5-6.5). This could be interpreted either as the result of a rapid dimer-to-octamer interconversion on the chromatographic time scale or as a consequence of the presence of previously unidentified intermediate species (e.g., tetramer and/or hexamer). In contrast, ESI MS provides strong evidence of the dimer-to-octamer transition state that occurs when the analysis is performed within a narrow pH range (6.0-7.0). Octamer assembly was shown to be a highly cooperative process with no intermediate states that are populated to detectable levels. A tetrameric state of rhASA exists at equilibrium with a dimer at neutral pH but does not appear to be involved in the octamer assembly process.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts, United States.
authors.forename: Rinat R
authors.initials: RR
authors.lastname: Abzalimov
authors.forename: Cedric E
authors.initials: CE
authors.lastname: Bobst
authors.forename: Paul A
authors.initials: PA
authors.lastname: Salinas
authors.forename: Philip
authors.initials: P
authors.lastname: Savickas
authors.forename: John J
authors.initials: JJ
authors.lastname: Thomas
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D002553:Cerebroside-Sulfatase / Q000032:analysis / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D002553:Cerebroside-Sulfatase