Relative stabilities of wild-type and mutant glial fibrillary acidic protein in patients with Alexander disease

Abstract

Alexander disease (AxD) is an often fatal astrogliopathy caused by dominant gain-of-function missense mutations in the glial fibrillary acidic protein (GFAP) gene. The mechanism by which the mutations produce the AxD phenotype is not known. However, the observation that features of AxD are displayed by mice that express elevated levels of GFAP from a human WT GFAP transgene has contributed to the notion that the mutations produce AxD by increasing accumulation of total GFAP above some toxic threshold rather than the mutant GFAP being inherently toxic. A possible mechanism for accumulation of GFAP in AxD patients is that the mutated GFAP variants are more stable than the WT, an attribution abetted by observations that GFAP complexes containing GFAP variants are more resistant to solvent extraction. Here we tested this hypothesis by determining the relative levels of WT and mutant GFAP in three individuals with AxD, each of whom carried a common but different GFAP mutation (R79C, R239H, or R416W). Mass spectrometry analysis identified a peptide specific to the mutant or WT GFAP in each patient, and we quantified this peptide by comparing its signal to that of an added [¹⁵N]GFAP standard. In all three individuals, the level of mutant GFAP was less than that of the WT. This finding suggests that AxD onset is due to an intrinsic toxicity of the mutant GFAP instead of it acting indirectly by being more stable than WT GFAP and thereby increasing the total GFAP level.

Keywords: Alexander disease; MS; astrocyte; astrogliopathy; genetic disease; glial fibrillary acidic protein (GFAP); intermediate filament; mutant; protein stability.

Figure 1.

MS/MS spectra used for identifying peptides specific for WT or mutant GFAP. A–C, peptides are diagnostic for WT GFAP in an R79C patient (A), R416W mutant GFAP (B), and R239H mutant GFAP (C). In each panel, the first spectrum is for the unlabeled peptide and the second is for the corresponding ¹⁵N-labeled peptide. The ion m/z ratios were calculated using the Protein Prospector MS Product tool (http://prospector.ucsf.edu).⁴

Figure 2.

GFAP immunoblot of ¹⁵N-labeled GFAP proteins and patient total homogenate samples. For each ¹⁵N-labeled GFAP standard (lanes 1–3), 7.5 ng of purified standard was loaded on the gel. The amounts of total protein applied for the R239H, R416W, and R79C patient samples (lanes 5–7) were 200 ng, 4,600 ng, and 270 ng, respectively. These patient protein amounts reflected differences in GFAP content, which were determined by densitometric comparison of GFAP staining intensity in pilot gels with that of the ¹⁵N-labeled GFAP standards. The negative control (lane 4) had no protein loaded. The positions of relevant molecular mass markers run concurrently are indicated.

Figure 3.

Illustration of the quantitation method. Data shown are from the 40-h tryptic digest of the intact GFAP band from the run using 36 ng of ¹⁵N-labeled mutant 416W protein standard and 113 ng of unlabeled R416W patient GFAP (Table 1). A and B, elution chromatograms for detection of the product ions from the R416W mutant–specific peptide TVEMWDGEVIK that were used for quantitation. A, unlabeled patient GFAP. B, [¹⁵N]GFAP internal standard. C and D, data for the common peptide FADLTDAAAR. Note that, as expected, the unlabeled and ¹⁵N-labeled peptides coelute. The table insets in B and D list the product ions, the peak areas of their signals, and the ratios of the peak area of each unlabeled ion to that of the labeled ion. These ratios provide measures of the amount of mutant GFAP and total GFAP, respectively, in the patient sample relative to the ¹⁵N standard. The ratio of these two values is the fraction of total patient GFAP that is the mutant form. The actual calculations summarized in Table 1 include additional values from other common peptides. Data for each individual peak area are provided in Table S1.