Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2

Abstract

The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2–derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.

Glucocerebrosidase mutations and synucleinopathies: Toward a model of precision medicine

“…[26][27][28][29][30] Furthermore, the use of peptides targeting helix 5 of LIMP-2 has been able to reduce α-syn levels by activating endogenous wild-type and mutant GCase. 31 These findings open a window for the design of small molecules targeting this domain to enhance LIMP-2/GCase interaction.…”

“…Small molecular chaperones such as ambroxol and isofagomine, which target misfolded GCase and increase GCase trafficking to lysosomes, reduce α‐syn burden, in both in vitro and in vivo disease models and may have utility as disease‐modifying agents . Furthermore, the use of peptides targeting helix 5 of LIMP‐2 has been able to reduce α‐syn levels by activating endogenous wild‐type and mutant GCase . These findings open a window for the design of small molecules targeting this domain to enhance LIMP‐2/GCase interaction.…”

Glucocerebrosidase: Functions in and Beyond the Lysosome

“…Following correct folding of newly formed GCase molecules in the ER, these bind to the membrane protein LIMP2 (lysosomal membrane protein 2) [72][73][74]. This binding is mediated by hydrophobic helical interfaces on both proteins [75]. Action myoclonus renal failure syndrome (AMRF) is a recessively inherited disease caused by mutations in LIMP2 [76].…”

The role of glucocerebrosidase in Parkinson disease pathogenesis

“…. Following translation of GCase at the ER, GCase is transported via the Golgi to lysosomes via the transporter protein LIMP2, undergoing several glycosylation modifications on the way . Analysis of the glycosylated forms of GCase in the cortex of control human brains suggested that lysosomal maturation of GCase was diminished in brains with higher amounts of α‐synuclein .…”