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Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis.

Gaspar P, Kallemeijn WW, Strijland A, Scheij S, Van Eijk M, Aten J, Overkleeft HS, Balreira A, Zunke F, Schwake M, Sá Miranda C, Aerts JM - J. Lipid Res. (2013)

Bottom Line: Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients.We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels.Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

View Article: PubMed Central - PubMed

Affiliation: Lysosome and Peroxisome Biology Unit (UniLiPe), Institute of Molecular and Cell Biology (IBMC), University of Oporto, Oporto, Portugal.

ABSTRACT
Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

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Visualization of GBA with fluorescent ABPs MDW941 and MDW933. A: Detection of GBA in homogenates of cultured fibroblasts (30 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. AMRF 1, patient 1 (LIMP2 W178X/W178X); AMRF 2, patient 2 (LIMP2 W178X/W178X); AMRF 3, patient 3 (LIMP2 W178X/W178X); Car, carrier of AMRF (LIMP2 W178X/WT); GD, type 2 GD patient; Rec. GBA, recombinant GBA (Cerezyme). B: Fluorescence microscopy. Fibroblasts labeled in vivo with MDW933 and DAPI [Upper micrograph, control fibroblasts (WT); lower micrograph, AMRF patient]. The scale bar represents 20 μm. C: Detection of GBA in homogenates of leukocytes (50 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. D: Detection of GBA in homogenates of cultured macrophages (20 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging.
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fig1: Visualization of GBA with fluorescent ABPs MDW941 and MDW933. A: Detection of GBA in homogenates of cultured fibroblasts (30 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. AMRF 1, patient 1 (LIMP2 W178X/W178X); AMRF 2, patient 2 (LIMP2 W178X/W178X); AMRF 3, patient 3 (LIMP2 W178X/W178X); Car, carrier of AMRF (LIMP2 W178X/WT); GD, type 2 GD patient; Rec. GBA, recombinant GBA (Cerezyme). B: Fluorescence microscopy. Fibroblasts labeled in vivo with MDW933 and DAPI [Upper micrograph, control fibroblasts (WT); lower micrograph, AMRF patient]. The scale bar represents 20 μm. C: Detection of GBA in homogenates of leukocytes (50 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. D: Detection of GBA in homogenates of cultured macrophages (20 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging.

Mentions: To detect active GBA molecules, homogenates of fibroblasts from three AMRF patients (LIMP2 W178X/W178X), two AMRF carriers (LIMP2 W178X/WT), two control subjects, and one type 2 GD patient (GBA L444P/L444P) were labeled with MDW941 (Inhibody Red) and subjected to SDS-PAGE. We demonstrated earlier that MDW941 specifically labels lysosomal GBA and no other retaining β-glucosidases in humans (GBA2 and GBA3) (8, 28). Actually, cultured fibroblasts contain no GBA3 and very little GBA2 (29). GBA, labeled after incubation of fibroblasts with MDW941, was detected by fluorescence imaging of the slab gel (Fig. 1A). Fibroblasts of LIMP2-deficient AMRF patients showed almost no active GBA. The active GBA was similarly reduced in cells from the type 2 GD patient. In the case of cells from the AMRF carrier, a normal amount of active GBA was detected.


Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis.

Gaspar P, Kallemeijn WW, Strijland A, Scheij S, Van Eijk M, Aten J, Overkleeft HS, Balreira A, Zunke F, Schwake M, Sá Miranda C, Aerts JM - J. Lipid Res. (2013)

Visualization of GBA with fluorescent ABPs MDW941 and MDW933. A: Detection of GBA in homogenates of cultured fibroblasts (30 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. AMRF 1, patient 1 (LIMP2 W178X/W178X); AMRF 2, patient 2 (LIMP2 W178X/W178X); AMRF 3, patient 3 (LIMP2 W178X/W178X); Car, carrier of AMRF (LIMP2 W178X/WT); GD, type 2 GD patient; Rec. GBA, recombinant GBA (Cerezyme). B: Fluorescence microscopy. Fibroblasts labeled in vivo with MDW933 and DAPI [Upper micrograph, control fibroblasts (WT); lower micrograph, AMRF patient]. The scale bar represents 20 μm. C: Detection of GBA in homogenates of leukocytes (50 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. D: Detection of GBA in homogenates of cultured macrophages (20 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927471&req=5

fig1: Visualization of GBA with fluorescent ABPs MDW941 and MDW933. A: Detection of GBA in homogenates of cultured fibroblasts (30 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. AMRF 1, patient 1 (LIMP2 W178X/W178X); AMRF 2, patient 2 (LIMP2 W178X/W178X); AMRF 3, patient 3 (LIMP2 W178X/W178X); Car, carrier of AMRF (LIMP2 W178X/WT); GD, type 2 GD patient; Rec. GBA, recombinant GBA (Cerezyme). B: Fluorescence microscopy. Fibroblasts labeled in vivo with MDW933 and DAPI [Upper micrograph, control fibroblasts (WT); lower micrograph, AMRF patient]. The scale bar represents 20 μm. C: Detection of GBA in homogenates of leukocytes (50 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging. D: Detection of GBA in homogenates of cultured macrophages (20 μg): labeling followed by SDS-PAGE and detection by fluorescence imaging.
Mentions: To detect active GBA molecules, homogenates of fibroblasts from three AMRF patients (LIMP2 W178X/W178X), two AMRF carriers (LIMP2 W178X/WT), two control subjects, and one type 2 GD patient (GBA L444P/L444P) were labeled with MDW941 (Inhibody Red) and subjected to SDS-PAGE. We demonstrated earlier that MDW941 specifically labels lysosomal GBA and no other retaining β-glucosidases in humans (GBA2 and GBA3) (8, 28). Actually, cultured fibroblasts contain no GBA3 and very little GBA2 (29). GBA, labeled after incubation of fibroblasts with MDW941, was detected by fluorescence imaging of the slab gel (Fig. 1A). Fibroblasts of LIMP2-deficient AMRF patients showed almost no active GBA. The active GBA was similarly reduced in cells from the type 2 GD patient. In the case of cells from the AMRF carrier, a normal amount of active GBA was detected.

Bottom Line: Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients.We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels.Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

View Article: PubMed Central - PubMed

Affiliation: Lysosome and Peroxisome Biology Unit (UniLiPe), Institute of Molecular and Cell Biology (IBMC), University of Oporto, Oporto, Portugal.

ABSTRACT
Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

Show MeSH
Related in: MedlinePlus