Role of LIMP-2 in the trafficking of Acid β-Glucosidase

Acid β-glucosidase (GCase) is a lysosomal enzyme defective in most cases of Gaucher disease (GD). GCase, encoded by the GBA gene, is targeted to the lysosomes through its interaction with the lysosomal integral membrane protein type 2 (LIMP-2). To date, more than 300 mutations of the GBA gene have been reported, most of them lead to the synthesis of misfolded proteins that are retained in the endoplasmic reticulum (ER) and rapidly degradated by endoplasmic reticulum associated degradation (ERAD). It has been proposed that mutations in the SCARB2 gene, encoding LIMP-2, may modify the phenotypic expression of GD. However, the nature of the GCase-LIMP-2 interaction has not been characterized in detail and the impact of GBA mutations on this interaction has not been explored. Recently, mutations in the human gene encoding LIMP-2 (SCARB2) were found to be responsible for action myoclonus-renal failure syndrome (AMRF), a disorder characterized by a mistargeting of GCase. The main feature of AMRF is the presence of progressive myoclonus epilepsy, a frequent sign found in patients affected by the chronic neurological form of GD. However, even if AMRF and GD affect the same metabolic pathway and share some neurological features, they present with different clinical and biochemical phenotypes, suggesting that an alternative lysosomal targeting pathway may be active in different tissues. In this study we investigated the impact of GBA mutations on LIMP-2-GCase association and the role of LIMP-2 in the trafficking of GCase to the lysosomes in different human tissues. First, we analyzed the impact of 9 novel GBA alleles, found in 8 GD Italian patients (P159S, N188I, E235K, P245T, W312S, S366R, W381C, [N188S;G265R], [E326K;D380N]) and two frequent GBA mutations (N370S and L444P) on GCase enzymatic activity and binding to LIMP-2. All mutants were inactive except for the P159S, which retained a 15% of wild type activity. The presence of N188I, [N188S;G265R] and W381C mutations significantly impaired the ability of GCase to associate with LIMP-2, suggesting that these residues may be important for LIMP-2-GCase interaction. In addition, we performed an in vitro functional analysis of a SCARB2 mutation (E471G) recently described to be associated with myoclonic epilepsy in a family affected by GD. The obtained results showed that the mutation do not affect the binding of LIMP-2 to GCase. However, in the presence of this mutation very low levels of LIMP-2 protein could be detected, suggesting that the mutation would severely impact LIMP-2 protein expression and/or protein degradation. Then, we evaluated the role of LIMP-2 in the trafficking of GCase in different human tissues, by comparing the GCase enzymatic activity and intracellular localization in cells derived from a patient affected by AMRF, patients affected by GD and healthy controls. The results strongly suggest that LIMP-2 is critical for the lysosomal targeting of GCase in human fibroblasts. In these cells LIMP-2 deficiency led to the retention of GCase in the ER and its degradation, which in turn, caused a severe impairment in GCase activity. A LIMP-2 dependent mechanism was also observed for the targeting of human recombinant GCase (hrGCase) to the lysosomes in human fibroblasts. Instead, LIMP-2 deficient macrophages and lymphoblasts retained a quite high GCase residual activity and digestion with endo-H clearly showed that in these cells, a significant fraction of GCase is able to leave the ER and probably reaches the lysosomes. These data suggest the existence of a LIMP-2 independent mechanism for the lysosomal targeting of GCase in blood cells. Finally, in a model of human LIMP-2 deficient neurons, obtained by neuronal differentiation of adult stem cells derived from human fibroblasts (hSKIN-MASC) of a patient affected by AMRF, GCase enzymatic activity was almost undetectable and GCase did not localize within the lysosomes. Furthermore, an accumulation of GM2 gangliosides was observed within lysosomes of these cells. These observations suggest that LIMP-2 is critical for the trafficking of GCase in neuronal cells and support the hypothesis that the neurological signs present in AMRF patients are caused by the lack of functional GCase in lysosomes with subsequent storage of glycosphingolipid material.