Deficiency of Glucocerebrosidase Activity beyond Gaucher Disease: PSAP and LIMP-2 Dysfunctions

Abstract

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1. However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2-linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1- and SCARB2-mutant fibroblasts than PSAP-mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1-mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1-linked GCase deficiency.

Keywords: AMRF; Gaucher Disease; LIMP-2; glucocerebrosidase; prosaposin; saposin C.

Figure 1

(A) Western blot analysis of GCase expression in WT, LIMP2_PT1, and LIMP2_PT2 fibroblasts treated (+) or not (−) with Endo H or Endo F; (B) western blot analysis of GCase expression in WT, LIMP2_PT1, and LIMP2_PT2 fibroblasts treated (+) or not (−) with the proteasomal inhibitor MG132; (C) quantitation of GCase expression in LIMP2_PT1, and LIMP2_PT2 fibroblasts treated with the proteasomal inhibitor MG132; (D) western blot analysis of GCase expression in WT and PSAP_PT fibroblasts treated (+) or not (−) with Endo H; (E) western blot analysis of GCase expression in WT and PSAP_PT fibroblasts treated (+) or not (−) with the lysosomal proteases inhibitor cocktail lPIC; (F) quantitation of GCase expression of PSAP_PT fibroblasts treated with lPIC. Results are expressed as mean ± SD of three independent experiments. * p-value < 0.05; *** p-value < 0.001.

Figure 2

Filipin stained intracellular unesterified cholesterol of LIMP2_PT1, LIMP2_PT2, PSAP_PT fibroblasts, fibroblasts from a healthy control (WT), and Niemann–Pick type C-affected patient (NPC). Scale bar 100 µm.

Figure 3

GCase fate in a normal cell and in cells presenting GCase-deficient activity due to biallelic pathogenic variants of SCARB2, PSAP, or GBA1 genes. (A) In a normal cell, GCase is synthesized in the ER, subsequently processed in the Golgi, and eventually delivered to the lysosome by its transporter LIMP-2. In the lysosome, GCase catalyzes the degradation of GlcCer in the presence of its activator SapC. (B) In SCARB2-linked deficiency of GCase activity, GCase fate differs according to the cell type. In LIMP-2-deficient fibroblasts, wt GCase cannot reach the lysosome and is degraded via proteasome and possibly also released outside the cell ([15,17,53] and present study); consequently, GlcSph accumulates within lysosomes ([53]). In LIMP-2-deficient blood cells, wt GCase undergoes proteasomal degradation, is released outside the cells and, in small amounts, also somehow reaches the lysosome, where it seems able to degrade GlcCer to some extent, resulting in moderate plasma release of GlcSph ([15,16,17,21,22,23,53], and present study). In addition, due to the lack of one of its transporter LIMP-2, lysosomal cholesterol (Chol) efflux is partially impaired ([64], and present study). (C) In PSAP-linked deficiency of GCase activity, wt GCase can effectively reach the lysosome but is unable to actively degrade GlcCer, as SapC is missing. Thus, the wt GCase is degraded by lysosomal proteases and GlcCer and GlcSph are accumulating ([47,49,52], and present study). Likely due to the lack of SapD, low plasma levels of GlcSph are observed in comparison with GBA1-linked deficiency of GCase activity. In addition, since all Saps are lacking, other glycosphingolipids and cholesterol (Chol) are accumulating ([64] and present study). (D) In GD cells, GBA1 biallelic pathogenic variants lead to the progressive accumulation of GlcCer and its deacylated form GlcSph which is abundantly released outside the cells resulting in high plasma levels of this glycosphingolipid. According to the type of GBA1 variant, the loss of GCase activity may depend on nonsense-mediated decay of mutant GBA1 mRNA, proteasomal degradation of ER-retained mutant GCase protein, lysosomal degradation of mutant GCases that are delivered to lysosomes ([74,75,76,77]). Parts of the figures were drawn using pictures from Servier Medical Art. ServierMedical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/, accessed on 14 April 2024).