Ion mobility–mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy

Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine–Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility–mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft–mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.

The misfolding of membrane proteins is implicated in the mechanisms of multiple debilitating diseases such as cystic fibrosis and retinitis pigmentosa (1–4). Specific membrane protein mutations are often associated with disease states, with variant forms exhibiting altered stability and cellular trafficking (5). Unfortunately, due to the challenges associated with preparing and handling pure, highly concentrated membrane protein samples, detailed structural information on such targets is often lacking, especially for disease mutant forms. Furthermore, as some membrane proteins associated with misfolding-based diseases have hundreds of mutations of interest (3), there is a clear need for high-throughput methods to assess disease mutation-induced changes in membrane protein stability and structure.Peripheral myelin protein 22 (PMP22) is such a membrane protein, for which misfolding and trafficking of mutant variants have been implicated in disease (6). PMP22 is a tetra-span integral membrane glycoprotein predominately expressed in Schwann cells, which are the principal glial cells of the peripheral nervous system (PNS), where they produce myelin (7–9). In addition to accounting for ∼5% of the protein found in the myelin sheath surrounding PNS nerve axons, PMP22 is thought to regulate intracellular Ca²⁺ levels (10), apoptosis (11), linkage of the actin cytoskeleton with lipid rafts (12), formation of epithelial intercellular junctions (13), myelin formation (14), lipid metabolism, and cholesterol trafficking (15). Dysregulation and misfolding of PMP22 has been identified as a key factor in multiple neurodegenerative disorders, such as Charcot-Marie-Tooth disease types 1A and E, as well as Dejerine–Sottas syndrome (6, 16–18). Like a number of other disease-linked membrane proteins (19), the trafficking of PMP22 is known to be inefficient, with only 20% of the wild-type (WT) protein reaching its final plasma membrane destination in Schwann cells (16, 20). Previously, it has been shown through a range of biophysical analyses that disease-associated PMP22 mutations lower thermodynamic protein stability as the root cause of reduced trafficking and loss of protein function; however, the mechanism by which destabilization of PMP22 causes mistrafficking is still not well understood (6). Additionally, a high-resolution structure of PMP22 has not yet been published.Native mass spectrometry (MS) has recently been demonstrated to overcome sample purity and concentration barriers to reveal critical details of membrane protein structure and function (21–23). Through the use of nano-electrospray (nESI), intact membrane proteins are ionized within detergent micelles or other membrane mimetics (24–27), which can then be removed from the membrane protein ions within the instrument. This method has been used to elucidate oligomeric state (28–30), complex organization (31, 32), and lipid interactions (33–35) of diverse membrane proteins. The addition of ion-mobility separation–mass spectrometry (IM-MS) provides data on the orientationally averaged size of analytes (36) and enables collision induced unfolding (CIU) experiments (37). In CIU, the energies experienced by gas-phase protein ions are increased in a stepwise fashion causing gas-phase protein unfolding to occur. These dynamic measurements have been shown to be sensitive to ligand binding (38, 39), glycosylation (40, 41), and disulfide bonding (40) in soluble proteins, as well as selective lipid and small molecule binding in membrane proteins (42–45). While CIU can clearly capture subtle structural changes in membrane proteins (43, 45, 46) and soluble mutant protein variants (47, 48) its ability to characterize membrane protein variants is only beginning to be explored.Here, we demonstrate the ability of native MS and CIU to detect key differences in the gas-phase stability and homodimer complex formation of PMP22 variants, together leading to insights into the mechanism of PMP22 dysregulation in disease. We quantify the propensity of PMP22 to dimerize across WT and seven disease-associated point mutations. We find that mutations associated with severe disease states form significantly more dimer than WT. Through CIU, we quantify the stability of gas-phase monomeric and dimeric PMP22 and find that variants bearing mutations associated with severe neuropathy exhibit the lowest relative monomer conformational stability. Interestingly, we also observe that dimers formed by various disease mutant forms of PMP22 are all more stable than WT PMP22 dimeric complexes. We continue by comparing our results to previously published biophysical datasets and find that our monomeric PMP22 gas-phase stability values correlate well with cellular trafficking data (6). Finally, we probe the effects of solubilization agents on PMP22 by characterizing its dimerization within sphingomyelin and cholesterol rich (SCOR) bicelles (49). We find that dimeric PMP22 complexes persist within SCOR bicelles and that the mutants resulting in the most severe disease phenotypes form higher population of dimer than WT. We conclude by describing a possible mechanism of PMP22 dysregulation in severe neurodegenerative diseases by which PMP22 monomers are destabilized, leading to dimers that traffic much less efficiently to the plasma membrane than WT PMP22.