The FASEB Journal article fj.15-271148. Published online May 27, 2015.

The FASEB Journal • Research Communication

Role of LIMP-2 in the intracellular trafficking of b-Glucosidase in different human cellular models Erika Malini, Stefania Zampieri, Marta Deganuto, Milena Romanello, Annalisa Sechi, Bruno Bembi, and Andrea Dardis1 Regional Coordinator Centre for Rare Diseases, University Hospital Santa Maria della Misericordia, Udine, Italy Acid b-glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)-2. In humans, LIMP-2 deficiency leads to action myoclonus-renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, they are different from clinical and biochemical points of view, suggesting that the role of LIMP-2 in the targeting of GCase would be different in different tissues. Besides, the role of LIMP-2 in the uptake and trafficking of the human recombinant (hr)GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multipotent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LIMP-2, whereas in blood cells, GCase is partially targeted to lysosomes by a LIMP-2-independent mechanism. Although hrGCase cellular uptake is independent of LIMP-2, its trafficking to the lysosomes is mediated by this receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.—Malini, E., Zampieri,S.,Deganuto,M.,Romanello,M.,Sechi,A.,Bembi,B., Dardis, A. Role of LIMP-2 in the intracellular trafficking of b-Glucosidase in different human cellular models. FASEB J. 29, 000–000 (2015). www.fasebj.org

LYSOSOMAL INTEGRAL MEMBRANE PROTEIN TYPE 2 (LIMP-2) is a ubiquitously expressed type III transmembrane glycoprotein (1) mainly located in endosomes and lysosomes. Based on homology, LIMP-2 has been defined as a member of the cluster of differentiation (CD)36 family of scavenger receptor proteins (2, 3). Among its known functions is its involvement in intracellular trafficking from the endoplasmic reticulum (ER) to the lysosomes of acid b-glucosidase (GCase), a lysosomal enzyme

responsible for the degradation of glucosylceramide (GlcCer) into ceramide and glucose (4). LIMP-2-related sorting of GCase is pH dependent. The neutral pH of the ER allows GCase to associate with LIMP-2, whereas the acidic pH of the endosomal and lysosomal compartments leads to their dissociation. The region of LIMP-2 responsible for the interaction with GCase includes 3 helices in domain II that form a bundle (5, 6), whereas a highly conserved 11-amino-acid sequence within GCase is involved in its binding to LIMP-2 (7). Recently, it has been demonstrated that LIMP-2 binds the mannose-6-phosphate receptor (M6PR) via mannose 6 phosphate (M6P). Furthermore, GCase, LIMP-2, and M6PR form a heterotrimeric complex in vitro, suggesting that M6PR contributes indirectly to the lysosomal targeting of GCase by binding the LIMP-2 component of the GCase/LIMP-2 complex (6). However, these results contrast with published findings showing that GCase is targeted to the lysosomes in a M6PR-independent mechanism (4). The actual role of M6PR on GCase trafficking to the lysosomes should be further investigated. In humans, LIMP-2 deficiency due to mutations in the SCARB2 gene causes action myoclonus–renal failure (AMRF) syndrome [Online Mendelian Inheritance in Man (OMIM) 254900; National Center for Biotechnology Information, Bethesda, MD, USA], an autosomal recessive disorder characterized by progressive myoclonus epilepsy (PME) and renal failure (8–14). GCase deficiency, caused by mutations in the GBA gene, leads to Gaucher disease (GD), the most common lysosomal storage disorder, characterized by the accumulation of GlcCer and other glycosphingolipids (GSLs) within the lysosomes (15). Three major clinical variants of GD have been identified based on the absence [type 1 (GD1); MIM 230800] or presence [type 2 (GD2); acute neuronopathic, MIM 230900, and type 3 (GD3); subacute neuronopathic, MIM 231000] of primary CNS involvement. Because AMRF syndrome and GD affect the same metabolic pathway, it is not unexpected that they would share some common clinical features. However, they

Abbreviations: AMRF, action myoclonus-renal failure; CD, cluster of differentiation; CHAT, choline acetyltransferase; co-IP, coimmunoprecipitation; EBV, Epstein-Barr virus; EEA, early endosome antigen; endo-H, endoglycosidase-H; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERT, enzyme replacement therapy. FCS, fetal calf serum; (continued on next page)

1 Correspondence: Regional Coordinator Centre for Rare Diseases, University Hospital Santa Maria della Misericordia, Udine, P.le Santa Maria della Misericordia 15, 33100, Udine, Italy. E-mail: [email protected] doi: 10.1096/fj.15-271148 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

ABSTRACT

Key Words: lysosomal syndrome

disease • Gaucher

0892-6638/15/0029-0001 © FASEB

disease • AMRF

1

present several clinical and biochemical differences, suggesting that an alternative lysosomal targeting pathway is active in some tissues. In fact, whereas GCase activity is absent or very low in both fibroblasts and leukocytes in patients with GD, patients with AMRF syndrome show absent or very low GCase activity in fibroblasts (less than 10% of controls), close to the values observed in fibroblasts of patients with GD, but normal or slightly reduced GCase activity in leukocytes, suggesting that even in the absence of LIMP-2, at least part of GCase protein reaches the lysosomes in these cells. In addition, patients with AMRF syndrome do not show the presence of lipid-laden macrophages in bone marrow (Gaucher cells), a hallmark of GD. Consistently, patients with AMRF syndrome do not show elevated activity of chitotriosidase in plasma, which is a marker of macrophage activation in patients with GD (11, 16, 17). Furthermore, splenomegaly, hepatomegaly, thrombocytopenia, bleeding tendencies, anemia, and skeletal pathology, the most common systemic features of GD, likely caused by defective cells of mononuclear phagocyte lineage (18), are not present in patients with AMRF syndrome. These data suggest that, in the cells of some tissues, in particular blood cells, GCase is transported to the lysosomes by a LIMP-2-independent mechanism. This hypothesis is further supported by recent published data showing that LIMP-2-deficient white blood cells contain considerable amounts of active GCase (19). Besides these differences, PME, a main feature of AMRF syndrome, may be present in a subgroup of patients affected by the chronic neuronopathic form, GD3 (20), suggesting that LIMP-2 is essential for the trafficking of GCase in the CNS. However, the intracellular fate of GCase in human cells other than fibroblasts carrying SCARB2 mutations has rarely been studied. Therefore, we have evaluated the role of LIMP-2 in the trafficking of GCase in different cell types derived from a patient affected by AMRF syndrome. MATERIALS AND METHODS Patients’ cell lines Fibroblasts and blood cells were obtained from a patient affected by AMRF syndrome. Clinical and biochemical findings have been described by our group (9). Fibroblasts from a patient affected by (continued from previous page) GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCase, acid b-glucosidase; GD, Gaucher disease; GlcCer, glucosylceramide; GSL, glycosphingolipid; HEK293, human embryonic kidney cells; HLA-DR, human leukocyte antigen-D related; hrGCase, human recombinant acid b-glucosidase; hSKIN-MASC, human skin fibroblast-multipotent adult stem cell; KDR, kinase insert domain receptor; LAMP, lysosome-associated membrane protein; LIMP, lysosomal integral membrane protein; M6P, mannose-6-phospate; M6PR, M6P receptor; m-CSF, macrophage colony-stimulating factor; MAP, mitogen-activated protein; NGS, normal goat serum; NP40, Nonidet P40; PBMC, peripheral blood mononuclear cell; PME, progressive myoclonus epilepsy; PNGase F, endoglycosidase F; pTER, phosphotriesterase related; TetR, Tet repressor; shRNA, short hairpin RNA; WT, wild-type

2

Vol. 29

September 2015

GD2 and immortalized lymphoblasts from patients with GD3 were provided by the Cell Line and DNA Biobank from Patients Affected by Genetic Diseases (Instituto Giannina Gaslini, Genoa, Italy). Blood cells were obtained from 4 patients affected by GD1 and 1 patient affected by GD3, who were under observation at the Regional Centre for Rare Diseases. Written consent was obtained from the subjects or from caregivers or guardians on behalf of the minors involved in the study. The fibroblasts were cultured and maintained in DMEM (Life Technologies-Gibco, Paisley, United Kingdom) containing 10% fetal calf serum (FCS) and penicillin/streptomycin, in a humidified atmosphere of 5% CO2 at 37°C. Lymphocyte transformation Lymphocytes (1 3 106) were grown in 500 ml of RPMI medium (Life Technologies-Gibco) containing 15% FCS and penicillin/ streptomycin, along with 300 ml Epstein-Barr virus supernatant prepared from marmoset cell line B95-8 and with cyclosporine (0.1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA). After 10-15 d, typical outgrowths were clearly visible. Lymphoblasts were then cultured in RPMI medium containing 15% FCS and penicillin/ streptomycin, in a humidified atmosphere of 5% CO2 at 37°C. Monocyte isolation and differentiation Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats by density centrifugation. Monocytes were isolated by magnetic separation with CD14+ microbeads, according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14+ cells were treated with human macrophage colony-stimulating factor (hM-CSF; Miltenyi Biotec) for 7 days, to induce differentiation into macrophages. Stem cell selection, culture, and neuronal differentiation Stem cell–enriched cultures were obtained from already established skin fibroblast cultures at early passages (P1, P2, and P3), as previously described (21–23). After at least 3 passages in selective medium, the stem cells were detached and stained with the following primary conjugated antibodies: CD13, CD49a, CD49b, CD49d, CD90, CD73, CD44, CD45, human leukocyte antigen-D related (HLA-DR), CD34, and CD271 (BD Biosciences, Franklin Lakes, NJ, USA); CD105 and kinase insert domain receptor (KDR; Serotec, Oxford, United Kingdom); and CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of cells expressing all the antigens was determined by fluorescenceactivated cell sorting (FACS) analysis (CyAn; Beckman Coulter, Brea, CA, USA). Properly conjugated isotype-matched antibodies were used as negative controls. For neurogenic differentiation, stem cells obtained after 3 passages in selective medium, were seeded at a density of 40,000 cells/plate onto 100 mm plates or on coverslips coated with fibronectin and induced to differentiate according to the protocol published by Bergamin et al. (23). Inducible silencing of GCase expression in the SH-SY5Y cell line An inducible model of GCase deficiency was generated in SHSY5Y neuroblastoma cells by using RNA interference mediated by short hairpin (sh)RNA. Oligonucleotides used to target GBA (sequences will be provided upon request) were cloned into the phosphotriesterase-related (pTER) vector (24), which produces a tetracycline (doxycycline)-responsive promoter.

The FASEB Journal x www.fasebj.org

MALIN ET AL.

The SH-SY5Y cells were first transfected with pcDNA6/TR (Invitrogen, Carlsbad, CA, USA), to generate stable tetracyclineregulated Tet repressor (TetR)–expressing cell clones that were selected by incubation with 2.5 mg/ml blasticidin (Life TechnologiesInvitrogen). A clone expressing the TetR at the highest level was selected for transfection with pTER-shRNA for GBA and subjected to selection with 50 mg/ml phleomycin (Zeocin; Life TechnologiesInvitrogen). Cell clones transfected with the empty pTER vector or with the pTER/scrambled vector were used as negative controls. For inducible siRNA experiments, doxycycline (1 mg/ml) was added to the cell culture medium for 7 d. GCase enzymatic activity assay Cells were lysed in H2O containing a protease inhibitor cocktail and sonicated for 15 s. Concentration of proteins in the samples was determined with the Bradford reagent (Bio-Rad, Hercules, CA, USA). GCase activity was determined with the fluorogenic substrate 4-methylumbelliferyl-b-D-glucopyranoside (Sigma-Aldrich).

Western blot Cells were lysed at 4°C in cell lysis buffer TNN [100 mM TrisHCl (pH 8), 250 mM NaCl, 0.5% Nonidet (NP)40, and 0.1 mM DTT], with complete protease inhibitor cocktail (Roche, Basel, Switzerland). To analyze GCase protein in the cultured medium, cells were incubated in serum-free medium for 24 h. The supernatants were concentrated 303 by Microcon centrifugal filter devices (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. Samples were electrophoresed through a 10% SDS-polyacrylamide denaturing gel and blotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk in TBST (0.01 M Tris HCl, 0.15 M NaCl, and 0.1% Tween) and incubated with the appropriate primary antibody: anti-GCase clone 2E2 (Abnova, Taipei, Taiwan); anti-LIMP-2 (Novus Biologicals, Littleton, CO, USA); anti-actin (SigmaAldrich); anti-lysosome-associated membrane protein (LAMP)-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-early endosome antigen (EEA)-1 (Abcam, Cambridge, United Kingdom). The membranes were then incubated with the appropriate secondary antibody and developed with ECL detection reagents (Thermo Fisher Scientific, Rockford, IL, USA). Immunoprecipitation of GCase For immunoprecipitation (IP) of GCase, fibroblasts, and immortalized lymphocytes were grown in serum-free medium for 24 h. The supernatants were concentrated as described above and precleared with 30 ml protein A/G (Santa Cruz Biotechnology) in the presence of IP buffer [100 mM NaCl, 10 mM HEPES (pH 8) 1 mM MgCl2, and 0.5% NP40 (v/v)] supplemented with protease inhibitor cocktail. The supernatants were incubated overnight at 4°C with anti-GCase and 30 ml protein A/G, followed by 3 washes with IP buffer. The proteins were eluted for 10 min at 100°C in SDS sample buffer, separated on 10% SDS-PAGE, and transferred onto nitrocellulose membranes (Bio-Rad).

Endoglycosidase F and endoglycosidase-H digestion Total cellular extracts were subjected to overnight incubation at 37°C with either endoglycosidase-H (endo-H; Roche) or N-endoglycosidase-F (PNGase F; Roche), according to the manufacturer’s instructions. Digestion samples were then subjected to Western blot analysis, as described above. Immunofluorescence microscopy Cells were fixed for 20 min with 4% paraformaldehyde in PBS and permeabilized for 5 min with 0.1% Triton X-100 in PBS. After they were blocked with 5% normal goat serum (NGS) in PBS, the slides were incubated overnight at 4°C with primary antibody in 2% NGS. The primary antibody was revealed by Alexa-Fluor 555- or 488-labeled secondary antibodies (Life Technologies-Molecular Probes, Eugene, OR, USA) at 1:600 dilution in 2% NGS. Images were obtained with a live-cell–imaging system consisting of a DMI 6000B microscope connected to a DFC350FX camera (Leica Microsystems, Wetzlar, Germany). Primary antibodies: anti-LAMP1 (1:100), anti-GCase clone 2E2 (1:50), anti-NeuN (1:50; Millipore), anti-tubulin b3 (1:1000; Covance, Inc., Princeton, NJ, USA) and, anti-mitogen-activated protein (MAP)-2 (1:50; Millipore). For the analysis of LIMP-2 on cell surfaces, HeLa cells transfected with Myc-tagged LIMP-2 construct (see below) were fixed and incubated with an anti-Myc antibody (Cell Signaling Technology, Danvers, MA, USA), without permeabilizing the cells. In all cases, cell nuclei were stained by Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Real-time PCR First-strand cDNA synthesis and quantitative RT-PCR of choline acetyltransferase (CHAT), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and dopamine active transporter (DAT) were performed with a LightCycler 480 Real-Time PCR System and LightCycler 480 SYBR Green I Master Mix (Roche) (23). GAPDH was used as the internal control for normalization. LightCycler 480 Basic software (Roche) used the second derivative maximum method to identify the crossing point. Uptake of hrGCase and correction of GCase activity Control fibroblasts were incubated with different concentrations (50 nM to 2 mM) of Imiglucerase (Cerezyme; Genzyme, Cambridge, MA, USA) and velaglucerase alfa (VIPRIV; Shire, Jersey, United Kingdome) for 4 h, to determine the working concentration for further assays. GD and AMRF cells and control fibroblasts were incubated with 200 nM to 1.6 mM velaglucerase for 4 h, and intracellular GCase was analyzed by Western blot or for enzymatic activity. Velaglucerase was labeled and purified with the Alexa Fluor 555 protein-labeling kit (Life Technologies-Invitrogen), according to the manufacturer’s instructions. Fibroblasts were incubated with Alexa Fluor 555-labeled hrGCase (15 nM) for different times ranging from 0 to 120 min. Lysotracker-Green (75 nM; Life Technologies-Molecular Probes) was added for the last 30 min. The cells were fixed in 4% paraformaldehyde in PBS and examined on a fluorescence microscope. Endosome and lysosome fractionation

Treatment with the proteasomal inhibitor MG132 The cells were treated with vehicle or 0.2 mM of the proteasomal inhibitor MG132 (Sigma-Aldrich) for 96 h.

ROLE OF LIMP-2 IN GCASE TRAFFICKING

Lysosomes and endosomes of fibroblasts treated for 4 h with 1.6 mM hrGCase were purified by differential centrifugation, as described by Schr¨oter et al. (25).

3

fibroblasts WT

Figure 1. Expression of LIMP-2 in different cell types. Western blot analysis of LIMP-2 protein expression of skin fibroblasts and neuronlike cells (N3) (left) and immortalized lymphoblasts (right) from a healthy control subject and a patient with AMRF syndrome. Actin was used as the loading control.

AMRF

lymphocytes

N3 WT

AMRF

WT

AMRF Limp-2

70 KDa

Actin 15 μg of total lysates

SCARB2 cloning and site directed mutagenesis SCARB2 cDNA was cloned into the pcDNA4-myc/His vector (Life Technologies-Invitrogen) The c.1087 C.A (H363N) mutation was introduced in the wild-type (WT) full-length cDNA by site-directed mutagenesis, with the QuikChange (Stratagene, Cedar Creek, TX, USA), according to the manufacturer’s instructions. The oligonucleotides used for mutagenesis will be provided upon request. The integrity and accuracy of the constructs were controlled by direct sequencing. The WT cDNA of GBA cloned into pcDNA3 was already available in our laboratory (26). Coimmunoprecipitation Human embryonic kidney (HEK)293 cells cotransfected with GCase and Myc-tagged LIMP-2 constructs were lysed in coimmunoprecipitation (co-IP) buffer: 100 mM NaCl, 10 mM HEPES (pH 8), 1 mM MgCl2, and 0.5% NP40 (v/v) supplemented with protease inhibitor cocktail. The lysates were incubated on ice for 30 min and then centrifuged at 10,000 g for 15 s. Protein concentration was determined by the Bradford method using the Bio-Rad Protein Assay. For IP of Myc-tagged LIMP-2, 200 or 20 mg total protein (for Western blot and enzymatic activity analysis, respectively) was incubated overnight at 4°C with Sepharoseimmobilized mouse anti-Myc antibody (Cell Signaling Technology) followed by 3 washes with co-IP buffer (pH 8) or 0.1 M acetate buffer (pH 4.2). Proteins were eluted for 10 min at 100°C in SDS sample buffer, separated by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). After the washing step with co-IP buffer (pH 8), GCase activity was measured by resuspending the beads in water and assaying directly by using the method described above. Statistical analysis Data were analyzed with Student’s t test. Statistical significance was set at P , 0.05.

RESULTS

50 μg of total lysates

results with those obtained in cells derived from patients with GD, in whom GCase activity was deficient in all tissues. Whenever possible, cells from patients affected by GD2 or -3 (depending on the availability), in which GCase activity was completely absent or very low, were used. The patient affected by AMRF syndrome presented the following SCARB2 mutations: c.1087 C.A (H363N) and c.424-2 A.C (9, 10). RT-PCR analysis of the SCARB2 mRNA followed by sequencing of the generated product showed that the c.424-2 A.C mutation led to the expression of an aberrant mRNA that degraded rapidly (data not shown). This result indicates that the LIMP-2 protein present in the patient’s cells was generated exclusively from the c.1087 C.A (H363N) mutated allele. The c.1087 C.A (p.H363N) mutation has been characterized in vitro by Blatz et al. (27). These authors showed that the mutation leads to the synthesis of an LIMP-2 protein that is retained in the ER and degraded via ERAD. However, this mutant protein still binds GCase in vitro, with an even higher affinity than WT protein. We first analyzed the expression levels of LIMP-2 by Western blot in immortalized lymphocytes, fibroblasts, and a neuronal model derived from human skin multipotent adult stem cells (hSKIN-MASCs) from a patient with AMRF syndrome and healthy control subjects. LIMP-2 was ubiquitously expressed in cells from the healthy controls (Fig. 1). However, whereas the abundance of LIMP-2 was similar in fibroblasts and neuronlike cells, it was undetectable in immortalized lymphoblasts in the same experimental conditions (data not shown). In these cells, LIMP-2 was detected only when 50 mg of total protein extracts was loaded on the gel and after the exposure time of the membranes was increased. These data indicate that the levels of LIMP-2 expression in immortalized lymphoblasts are much lower than in fibroblasts or neuronlike cells. As expected, LIMP-2 was barely detected in all cells from the patient with AMRF syndrome. Trafficking of GCase in human fibroblasts

To evaluate the role of LIMP-2 in the trafficking of GCase, we analyzed cells derived from a patient affected by AMRF syndrome diagnosed in our laboratory and compared the

Cultured fibroblasts derived from skin biopsies of a patient with AMRF syndrome were used to determine GCase

TABLE 1. GCase enzymatic activity in fibroblasts

Patient

AMRF syndrome GD2

4

Vol. 29

September 2015

Genotype

Residual activity (% of control)

c.1087 C.A (p. H363N)/c.424-2 A.C c.508 C.T (p.R131C)/c.508 C.T (p.R131C)

2.27 6 0.55 1.75 6 0.91

The FASEB Journal x www.fasebj.org

MALIN ET AL.

B A

100

AMRF

GD 2

Endo H

-

+

-

-

+

-

-

+

-

PNGase F

-

-

+

-

-

+

-

-

+

% GCase expression

WT

GCase

59 KDa

Actin

80 60 40 20 0

*** WT

ARMF

*** GD 2

C LAMP-1

GCase

merge

WT 10μm 10μm

10μm

10μm

10μm

10μm

10μm

AMRF 10μm

GD 2 10μm 10μm

10μm

10μm

Figure 2. Intracellular localization of GCase in AMRF syndrome and GD2 fibroblasts. Cell lysates prepared from skin fibroblasts of a healthy donor (WT) and patients (GD and AMRF syndrome) were subjected to overnight endo-H and PNGase F digestion. A) Western blot analysis with anti-GCase and -actin antibodies. B) Densitometric quantification of GCase in undigested samples. GCase signal intensity was normalized to that of actin. C) Coimmunostaining for the lysosome marker LAMP-1 (red) and GCase (green). Blue: DAPI-stained nuclei. Merge: overlaid images of GCase and LAMP-1 signals. Data are the means 6 SD of results in 3 independent experiments.***P , 0.001 vs. WT.

enzymatic activity and intracellular localization. Fibroblasts from a healthy control subject and a patient with GD2 were used for comparison. As previously reported (9, 16), GD2 and AMRF syndrome fibroblasts showed very low GCase activity (Table 1). To confirm whether the impairment of the enzymatic activity is caused by the mislocalization of GCase, we evaluated the glycosylation pattern of the protein by treating the samples with endo-H and PNGase F. Endo-H is a specific endoglycosidase that can distinguish between high mannose (.4 mannose residues) and a mature N-glycan complex (28, 29). The removal of mannose residues to generate the final core of 3 mannose residues is performed in the mid-Golgi. Therefore, endo-H can distinguish between glycoproteins that have not reached the mid-Golgi ROLE OF LIMP-2 IN GCASE TRAFFICKING

(endo-H sensitive) and mature (endo-H resistant) glycoproteins. PNGase F removed all asparagine-linked glycans. Total GCase protein levels were highly reduced in patients with AMRF syndrome or GD2, compared with levels in healthy controls (Fig. 2A, B). It is worth noting that the antiGCase antibody used in this analysis recognized a multiband pattern corresponding to differently glycosylated forms of GCase expressed in normal fibroblasts (30). In both AMRF syndrome and GD2 fibroblasts, the lowermolecular-weight band, corresponding to the mature form of GCase was not detected. In normal cells, most GCase was endo-H resistant or was partially digested by this enzyme, indicating that the protein reached the midGolgi where it was being processed or had already passed the mid-Golgi and probably reached the lysosome. In 5

B

MG132 0.2μM 96h

WT -

AMRF -

+ GCase

59 KDa

Actin

C

100

% GCase expression

A

Cell extracts Supernatants 80

*

60

GCase

40

**

20 0

WT

ARMF

Actin

ARMF+MG132 0.2μM 96h

E Extracellular Gcase abundance (relative to number of cultured cells)

D

59 KDa

Supernatants WT

AMRF GCase

*

IP anti-GCase; WB anti-GCase

80 70 60 50 40 30 20 10 0

WT

AMRF

Figure 3. Intracellular GCase in MG132-treated AMRF syndrome fibroblasts and in the culture medium. A) AMRF syndrome fibroblasts were treated with 0.2 mM MG132 for 96 h and subjected to Western blot analysis with anti-GCase and -actin antibodies. B) Densitometric quantification of GCase. GCase signal intensity was normalized to that of actin. C) Western blot analysis of GCase in cell extracts and of supernatants of healthy control (WT) and AMRF syndrome cultured fibroblasts, with anti-GCase and -actin antibodies. D) IP of GCase in supernatants of cultured fibroblast. Protein complexes were immunoprecipitated with antiGCase antibody, washed with IP buffer, separated by SDS-PAGE, and detected by Western blot analysis with anti-GCase antibody. *Heavy chain of the anti-GCase antibody. E) Densitometric quantification of GCase, as in (B). B, E) Data are the means 6 SD of results in 3 independent experiments. *P , 0.05, **P , 0.01 vs. WT.

contrast, in fibroblasts from both AMRF syndrome and GD2, GCase was entirely endo-H sensitive, indicating that the protein was almost completely retained in the ER. To further confirm the obtained results, intracellular GCase was analyzed by immunofluorescence. An SHSY-5Y cell line, in which GCase expression was specifically knocked out by GBA mRNA silencing, was used as a negative control to test antibody specificity (Supplemental Fig. S1). In normal cells, GCase accumulated in punctate lysosomal structures and colocalized with the lysosomal marker LAMP-1. In contrast, and in line with the results obtained by Western blot, almost no GCase protein was detected in fibroblasts from the patients with GD2 or those with AMRF syndrome (Fig. 2C). The extremely reduced levels of GCase protein and the pattern obtained after the endo-H digestion in the AMRF syndrome fibroblasts suggest that GCase is retained in the

ER of these cells and is subsequently degraded by the ubiquitin-proteasomal system (ERAD). However, the reduction of GCase levels may also be caused by an increased secretion of the protein into the extracellular media. Therefore, to analyze the fate of GCase in AMRF syndrome fibroblasts, we used Western blot to determine the abundance of GCase in cells treated with MG132, a proteasomal inhibitor, and in the conditioned culture medium, concentrated 303 (see Materials and Methods). MG132 treatment resulted in an increase in GCase (Fig. 3A, B), whereas no GCase protein was detected in the culture medium of normal or AMRF syndrome cells (Fig. 3C). However, when GCase was analyzed by IP in the concentrated conditioned medium, it was detected in the supernatants of both normal and AMRF syndrome cells (Fig. 3D, E), indicating that very low amounts of GCase (below the detection limit of the Western blot assay) were normally

TABLE 2. GCase enzymatic activity in immortalized lymphocytes

Patient

AMRF syndrome GD3 GD3 GD3

6

Vol. 29

September 2015

Genotype

c.1087 C.A (p.H363N)/c.424-2 A.C c.1342G.C (p.D409H)/c.971G.C (p.R285P) c.882T.G;c.1342G.C (p.H255Q;D409H)/ c.754T. A (p.F213I) c.1448 T.C (p.L444P)/c.1448 T.C (p.L444P)

The FASEB Journal x www.fasebj.org

Residual activity (% of control)

28.23 6 1.10 0.97 6 0.44 3.54 6 0.87 0.68 6 0.29

MALIN ET AL.

TABLE 3. GCase enzymatic activity in macrophages

Patient

AMRF syndrome GD1 GD1 GD1 GD1 GD3

Genotype

Residual activity (% of control)

c.1087 C.A (p.H363N)/c.424-2 A.C c.1226 A.G (p.N370S)/c.1226 A.G (p.N370S) c.1226 A.G (p.N370S)/c.508 C.T (p.R131C) c.259 C.T (p.R48W)/c.1448 T.C (p.L444P) c.259 C.T (p.R48W)/c.1448 T.C (p.L444P) c.1448 T.C (p.L444P)/c.1448 T.C (p.L444P)

31.5 11.6 13.0 12.5 14.1 3.6

secreted by cultured fibroblasts, and the secretion levels were not significantly affected in the AMRF syndrome cells.

of the Western blot assay) were normally secreted by immortalized lymphocytes. However, the secretion levels were not significantly affected in AMRF syndrome cells.

Trafficking of GCase in blood cells In all patients with AMRF syndrome described to date, GCase activity has been absent or severely reduced in fibroblasts, whereas it has been nearly normal or slightly reduced in leukocytes (9, 16). Therefore, to gain further insight into GCase trafficking in blood cells, we prepared lymphoblasts by immortalizing lymphocytes isolated from PBMCs of 1 patient with AMRF syndrome and 3 with GD3. In lymphoblasts from the patient affected by AMRF syndrome, GCase activity was found in ;30% of WT cells (Table 2). As expected, in lymphoblasts from all 3 patients with GD3, GCase activity was almost absent. Likewise, the analysis of GCase activity in macrophages obtained through the induced differentiation of monocytes obtained from 1 patient with AMRF syndrome, 5 patients with GD, and healthy donors, showed that GD macrophages displayed very low enzyme activity (,15% of WT activity), whereas AMRF syndrome macrophages showed high residual activity, reaching ;30% of that in WT cells (Table 3). It is worth noting that cells from only 1 patient with GD3 were available for this study; most of the cells studied derived from patients with GD1. As expected, GD1 macrophages displayed some degree of residual activity, whereas the GCase activity was almost completely absent in the GD3 macrophages. GCase protein levels were reduced in AMRF syndrome-immortalized lymphocytes when compared to normal cells (Fig. 4A, B). In normal control cells, after endo-H treatment ;90% of GCase was resistant, indicating that most protein had left the ER. In contrast, ;50% of GCase was endo-H resistant in AMRF syndrome cells, indicating that, even in the presence of mutated LIMP-2, ;50% of the protein passes the midGolgi (Fig. 4C). Still, the reduction in the total amount of GCase protein in AMRF syndrome cells indicated that, in these cells, GCase is, at least in part, degraded or secreted into the extracellular medium. Therefore, the abundance of GCase was analyzed in cells treated with MG132 and in the conditioned culture medium. MG132 treatment resulted in an increase in GCase, whereas no GCase protein was detected in the culture medium of normal or AMRF syndrome cells (Fig. 4D, E). Once again, when GCase was analyzed in the concentrated conditioned medium by IP it was detected in the supernatants of both normal and AMRF syndrome cells (Fig. 4F, G), indicating that very low amounts of GCase (below the detection limit ROLE OF LIMP-2 IN GCASE TRAFFICKING

Trafficking of GCase in a human neuronal model obtained by differentiation of hSKIN-MASCs PME is one of the main features of AMRF syndrome, suggesting that LIMP-2 would be essential for the targeting of GCase to the lysosomes in the CNS. However, the study of the molecular bases of neurodegeneration in humans is challenging because of the lack of suitable cellular models. We have recently described a method of generating a human neuronal model of Niemann Pick C disease through the induction of differentiation of hSKIN-MASCs (23). hSKIN-MASCs exhibit a mesenchymal stem cell immunophenotype, express pluripotent state–specific transcription factors, and are clonogenic and multipotent. Thus, we applied the same method to obtain a neuronal model of AMRF syndrome, as well as a GCase deficiency through differentiation of hSKIN-MASCs obtained from a patient with AMRF syndrome and one with GD2, respectively. As expected, the hSKIN-MASCs isolated from cultured fibroblasts of patients and healthy controls displayed an antigenic pattern characteristic of mesenchymal stem cells (Table 4). After differentiation, a large fraction of the cells became positive for markers of the neuronal lineage. In fact, they expressed tubulin-b3, NeuN, and MAP2, markers of mature neurons (Fig. 5A–J). Furthermore, as previously described, the cells expressed markers of CHAT neurons (Fig. 5K). GCase enzymatic activity and cellular localization were analyzed in the neuronal model in cells derived from the patient with AMRF syndrome and compared with cells derived from a patient with GD2 and healthy controls. GCase activity was severely impaired in both the AMRF syndrome- and GD2-derived cells (Table 5). To investigate the localization of GCase in this neuronal model, we performed an endo-H assay. Similar to the results obtained in fibroblasts, total GCase protein levels were highly reduced in both AMRF syndrome- and GD2derived cells compared to levels in healthy control cells (Fig. 6A, B), and the low amount of detected protein was almost completely retained in the ER (endo-H sensitive). The results were further confirmed by immunofluorescence. As expected, whereas GCase colocalized with the lysosomal marker LAMP-1 in healthy control cells, in AMRF syndrome cells, it was barely detected (Fig. 6C). 7

WT

Endo H PNGase F

-

+ -

fibroblasts

AMRF

+

-

+ -

AMRF

+

-

+ -

+ GCase

59 KDa

Actin

E

MG132 WT 0.2μM 96h -

AMRF + GCase

59 KDa

Actin

% GCase expression

D

100 80

**

60 40 20 0

WT

100 80

*

60 40 20 0

WT

AMRF

F Cell extracts Supernatants

80 60 40

59 KDa

GCase

20 0

WT

ARMF

G

H Supernatants WT

AMRF

*

100

AMRF

GCase

*

IP anti-GCase; WB anti-GCase

Actin

ARMF+MG132 0.2μM 96h

Extracellular Gcase abundance (relative to number of cultured cells)

lymphocytes

C % EndoH resistant fraction

B % GCase expression

A

3 2.5 2 1.5 1 0.5 0

WT

AMRF

Figure 4. Analysis of GCase in ARMF-immortalized lymphocytes. A) Cell lysates of immortalized lymphocytes from a healthy donor (WT) and a patient with AMRF syndrome were subjected to overnight digestion with endo-H or PNGase F, and Western blot analysis was performed with anti-GCase and anti-actin antibodies. Cell extracts from AMRF syndrome fibroblasts digested with endo-H and PNGase F were included for comparison. B) Densitometric quantification of GCase in undigested samples. GCase signal intensity was normalized to that of actin. C) The endo-H resistant fraction was determined by scanning the blots and measuring the intensity of each band. The GCase-resistant fraction was calculated by subtracting the intensity of the endoH-sensitive fraction (in the endo-H+ lane) from that of the total GCase in the same lane. D) AMRF syndrome-derived cells were treated with 0.2 mM MG132 for 96 h and subjected to Western blot analysis, with anti-GCase and -actin antibodies. E) Densitometric quantification of GCase. GCase signal intensity was normalized to that of actin. F) Western blot analysis of GCase in cell extracts and supernatants of healthy donor (WT) and AMRF syndrome cultured immortalized lymphoblasts, with anti-GCase and -actin antibodies. G) IP of GCase in supernatants of cultured lymphocytes. Protein complexes were immunoprecipitated with anti-GCase antibody, washed with IP buffer, separated by SDS-PAGE, and detected by Western blot analysis with anti-GCase antibody. *Heavy chain of the anti-GCase antibody. H) Densitometric quantification of GCase. GCase signal intensity was normalized to the number of cultured cells. B, C, E, H) Data are the means 6 SD of results in 3 independent experiments. *P , 0.05, ** P , 0.01 vs. WT.

Trafficking of hrGCase in human fibroblasts It has been demonstrated that enzyme replacement therapy (ERT) with hrGCase is effective in the treatment of peripheral symptoms of GD. hrGCase enters the cells via the mannose receptor (31, 32) and is primarily distributed on the surface of macrophages and dendrites, but is also expressed in skin cells such as human dermal fibroblasts and keratinocytes. To analyze whether LIMP-2 also plays a role in the lysosomal targeting of exogenous hrGCase, we first evaluated the uptake of hrGCase in AMRF syndrome fibroblasts treated for 4 h with 1.6 mM of the enzyme. This concentration was chosen to ensure the saturation of mannose receptor. Cultured GD2-derived fibroblasts were used for comparison. 8

Vol. 29

September 2015

The recombinant enzyme was internalized by both AMRF syndrome and GD2 fibroblasts (Fig. 7A). These data suggest that hrGCase enters the cells mainly via a LIMP-2independent mechanism. In line with this finding, LIMP-2 protein was not detected by immunofluorescence in the plasma membrane of cells transfected with Myc-tagged WT LIMP-2, suggesting that, in agreement with previous published data (31, 32), LIMP-2 would be poorly expressed on the cell surface (Supplemental Fig. S2). We evaluated the fate of internalized hrGCase in normal, AMRF syndrome, and GD2 fibroblasts by analyzing the internalization and intracellular localization of Alexa Fluor 555-labeled rhGCase. After the fibroblasts were treated with fluorescent rhGCase, the recombinant protein was efficiently taken up by normal, AMRF syndrome,

The FASEB Journal x www.fasebj.org

MALIN ET AL.

TABLE 4. Surface immunophenotype of hSKIN-MASCs isolated from fibroblast cultures Phenotype

CD49d CD49a CD49b CD133 CD271 CD44 CD105 CD90 CD73 CD34 CD45 KDR CD13 ,llHLA-DR

WT

96.2 6.7 99.3 0.37 0.33 99.2 89.3 98.7 98.7 1.2 0.4 1.12 99.4 0.6

6 6 6 6 6 6 6 6 6 6 6 6 6 6

AMRF

4.0 0.4 0.9 0.08 0.05 0.3 4.8 1.0 1.0 1.4 0.09 1.1 0.5 0.01

94.5 8.65 97.6 1.2 1.74 97.6 82.8 91.1 95.9 2.6 0.2 3.1 99.1 0.3

6 6 6 6 6 6 6 6 6 6 6 6 6 6

1.8 3.1 1.6 1.1 1.7 2.3 5.8 8.5 4.3 1.5 0.1 0.1 0.9 0.07

GD 2

98.4 39.63 99.4 0.68 0.7 99.3 78.0 97.2 99.8 0.33 0.16 2.67 99.8 0.1

6 6 6 6 6 6 6 6 6 6 6 6 6 6

2.1 4.2 0.1 0.6 0.3 0.5 4.35 1.3 0.01 0.5 0.1 1.6 0.04 0.09

and GD2 fibroblasts (Fig. 7B). However, whereas in normal and GD2 cells, the recombinant enzyme presented a lysosomal localization, as shown by its colocalization with the lysosomal marker LysoTracker (Life TechnologiesMolecular Probes), in AMRF syndrome cells the Alexa Fluor 555 signal did not colocalize with this lysosomal marker. These results clearly show that the targeting of hrGCase to lysosomes is impaired in AMRF syndrome cells, suggesting that LIMP-2 is a key player in the transport of hrGCase to the lysosomal compartment. To further confirm these data, we isolated lysosomes and endosomes from AMRF syndrome fibroblasts treated for 4 h with hrGCase and analyzed the GCase content in each fraction by Western blot. LAMP-2, a lysosomal marker and EEA1, an endosomal marker, were used to assess the purity of each fraction. Lysosomal and endosomal fractions isolated from GD2 fibroblasts treated with hrGCase were used for comparison. A significant enrichment of each subcellular fraction was achieved after purification (Fig. 7C). In line with the results presented above, most of the internalized enzyme in GD cells was located within the lysosomes, whereas in AMRF syndrome cells it was mostly retained in endosomes (Fig. 7D, E). Taken together, the data indicate that the intracellular trafficking of hrGCase is, at least in part, dependent on LIMP-2. GCase enzymatic activity was measured in cells treated with 200 nM to 1.6 mM hrGCase. A dose-dependent rescue of GCase activity was observed in both the GD2 and AMRF syndrome cells. However, whereas the maximum correction of enzyme activity in AMRF syndrome cells barely reached 20% of the endogenous activity detected in normal fibroblasts, in GD2 cells it reached 80% of the activity of control fibroblasts (Fig. 7F). This result was unexpected because, in AMRF syndrome cells, hrGCase did not reach the lysosomes but entered the cell. Thus, because the assay used to assess GCase activity measures total activity independent of its intracellular localization, we should have been able to detect it in the cellular extracts. It is worth noting that, as stated above, the AMRF syndrome cells used in this study carried the c.424-2 A.C mutation and the c.1087 C.A (p.H363N) mutation in the SCARB2 gene. Because the c.424-2 A.C allele was not ROLE OF LIMP-2 IN GCASE TRAFFICKING

expressed in the cells, the only LIMP-2 protein species present in the fibroblasts bore the p.H363N mutation, which leads to the synthesis of an LIMP-2 protein that is retained in the ER, where it still binds GCase with an even higher affinity than WT protein (27). The in vitro enzymatic assay is performed at acidic pH, resembling the lysosomal pH, at which GCase dissociates from LIMP-2 to perform its function. Therefore, we hypothesized that in AMRF syndrome cells, even at the acidic pH used to measure the enzymatic activity in vitro, p.H363N-mutatnt LIMP-2 and hrGCase remain, at least in part, associated. To test this hypothesis we performed a co-IP assay. Myctagged WT and p.H363N-mutant LIMP-2 were coimmunoprecipitated with WT GCase overexpressed in HEK293 cells. The immunoprecipitates were washed at neutral or acidic pH to dissociate the LIMP-2/GCase complex. In acidic conditions, the GCase dissociated from the complex WT LIMP-2, whereas it remained associated with mutated LIMP-2 (Fig. 8A). Furthermore, the enzymatic activity of GCase was measured in the coimmunoprecipitate (washed at neutral pH and therefore containing both LIMP-2 and GCase proteins). An interesting finding was that the enzymatic activity in the presence of the p.H363N mutant was 10 times lower than the activity in the presence of the WT protein (Fig. 8B). DISCUSSION A few years after the identification of LIMP-2 as the receptor of GCase, mutations in the SCARB2 gene encoding LIMP-2 were found to cause AMRF syndrome, characterized by progressive myoclonic epilepsy with associated renal failure. This disorder typically presents with neurologic symptoms that overlap with those frequently observed in patients affected by GD3. These phenotypic similarities are not unexpected, considering that LIMP-2 deficiency leads to a mistargeting of GCase and the consequent lack of GCase protein within the lysosomes. On the contrary, patients with AMRF syndrome do not show the massive occurrence of lipid laden macrophages, and they do not show consistently elevated plasma chitotriosidase activity (a marker of macrophage activation). In addition, they present normal or slightly reduced GCase activity in leukocytes but absent or reduced activity in fibroblasts (9, 16). These differences at the biochemical and clinical level suggest that the role of LIMP-2 in the transport of GCase to the lysosomes would not be the same in all tissues and that alternative lysosomal targeting pathways are probably active in some cell types. However, until now, the role of LIMP-2 in the trafficking of GCase to the lysosomes in various human tissues has not been characterized in detail. Therefore, several cell types from a patient with AMRF syndrome were analyzed, and the results were compared with those obtained in cells from patients with GD in whom GCase activity was deficient in all tissues. Our data confirmed previous findings that LIMP-2 is essential for the lysosomal targeting of GCase in fibroblasts (16). Indeed, in fibroblasts from the patient with AMRF syndrome, GCase was completely retained in the ER (endo-H sensitive) and partially degraded via ERAD. 9

WT

AMRF

GD 2

Tubulin beta 3

NeuN

G

H

I

MAP2

25 μm

J

25 μm

Tubulin beta 3

80

K 0.25

relative mRNA expression of chat

% positive cells

NeuN MAP2

60

40

20

0

25 μm

0.2 0.15 0.1 0.05 0

WT

AMRF

GD 2

UNDIFF.

DIFF.

Figure 5. Neural differentiation of hSKIN-MASCs obtained from cultured fibroblasts. Representative fluorescence images of the neuron-specific markers (A–C) tubulin b3 (green), (D–F) NeuN (green), and (G–I) MAP2 (red) in differentiated healthy, AMRF syndrome, and GD2 cells. Blue: DAPI-stained nuclei. J) Quantitative evaluation of the percentage of cells expressing the three markers. At least 100 cells were counted for each cell line. K) Relative expression of CHAT mRNA in hSKIN-MASCs and differentiated cells. The relative abundance of CHAT mRNA was analyzed by real-time PCR before (undifferentiated) and after (differentiated) neuronal differentiation. Data were normalized by the expression of GAPDH. J, K) Data are the means 6 SD of results in 3 independent experiments.

Instead, in immortalized lymphoblasts derived from the same patient, 50% of total GCase protein was endo-H resistant; indicating that about half of total GCase passes the mid-Golgi and probably reaches the lysosome. Independent of its intracellular localization, the total amount of GCase protein was significantly reduced in both AMRF syndrome-derived cell types (Figs. 2B, 4B), particularly in the fibroblasts. Still, the abundance of total GCase

was higher in AMRF syndrome fibroblasts than in immortalized lymphoblasts. In contrast, GCase activity was higher in AMRF syndrome lymphoblasts (;30% of normal activity) than in fibroblasts. It could be hypothesized that the activity detected in lymphoblasts derives from the GCase that escapes the ER degradation and reaches the lysosomes. Nevertheless, this result is unexpected, because the assay used to measure GCase activity does not differentiate

TABLE 5. GCase enzymatic activity in neuronlike cells

Patient

AMRF syndrome GD2

10

Vol. 29 September 2015

Genotype

Residual activity (% of control)

c.1087 C.A (p. H363N)/c.424-2 A.C c.508 C.T (p.R131C)/c.508 C.T (p.R131C)

2.88 6 0.09 1.64 6 0.33

The FASEB Journal x www.fasebj.org

MALIN ET AL.

A

B

Endo H PNGase F

-

+ -

AMRF +

-

+ -

100

GD 2 +

-

+ -

+

59 KDa

% GCase expression

WT

GCase Actin

80 60 40

0

C

GCase

**

20

WT

ARMF

*** GD 2

merge

LAMP-1

WT

10μm

10μm

10μm

10μm

AMRF 10μm 10μm

10μm

10μm

GD 2 10μm

10μm

10μm

10μm

Figure 6. Intracellular localization of GCase in AMRF syndrome and GD2 neuronlike cells. A) Cell lysates prepared from neuronal differentiated healthy donor, GD, and AMRF syndrome cells were subjected to overnight endo-H and PNGase F digestion. Western blot analysis was performed with anti-GCase and -actin antibodies. B) Densitometric quantification of GCase in undigested samples. GCase signal intensity was normalized to that of actin. Data are the means 6 SD of results in 3 independent experiments. C) Coimmunostaining for the lysosome marker LAMP-1 (red) and GCase (green). Blue: DAPI-stained nuclei. Merge: overlaid images of GCase and LAMP-1 signals. **P , 0.01, ***P , 0.001 vs. WT.

between ER and lysosomal enzyme. However, co-IP experiments showed that, at the acidic pH used in vitro to assess the enzymatic activity, GCase and mutant LIMP-2 remained at least partially associated, leading to a significant reduction of the detected enzymatic activity in vitro. Taken together, these findings suggest that, in the AMRF syndrome fibroblasts, GCase was completely retained in the ER and mostly degraded by ERAD. In these cells, the small amount of undegraded ER protein remained associated with LIMP-2, even in the acidic conditions used for the in vitro assessment of the enzymatic activity. Instead, in immortalized lymphoblasts, part of GCase reached the lysosome by a LIMP-2-independent mechanism. Therefore, it is likely that the activity detected in blood cells (30% of WT) derives from GCase that reaches the lysosome and is not associated with LIMP-2. ROLE OF LIMP-2 IN GCASE TRAFFICKING

It is also possible that the endo-H-resistant protein in these cells was detected as a result of a reuptake of secreted GCase. However, this hypothesis is unlikely, because the main fate of mistargeted GCase protein in AMRF syndrome cells (both fibroblasts and lymphoblasts) seems to be proteasomal degradation rather than secretion into the extracellular medium. It is also important to highlight that the p.H363N-mutant LIMP-2 expressed in the patient’s cells was mainly retained in the ER where it still bound GCase with high affinity. This may influence the fate of the mistargeted GCase in cells derived from this particular patient. Indeed, in cells from LIMP-2 knockout mice, GCase is mainly secreted into the extracellular medium (4). It would be interesting to analyze the final fate of mistargeted GCase in cells from patients carrying different types of SCARB2 mutations. 11

WT

hrGCase

-

AMRF -

D

GD2

+

-

GCase

59 KDa

Actin

B

hrGCaseAlexa555

LysoTracker green

Endosomal fraction 1.6

+

merge

Gcase protein level

A

1.2

0.8

0.4

0

GD2

NT

E

AMRF

Lysosomal fraction 0.8

1h

1h

GD 2

1h

AMRF

Gcase protein level

WT 0.6

**

0.4

0.2

0

GD2

F Endosomal fraction GD2

AMRF

Lysosomal fraction GD2

AMRF EEA1

130 KDa

100 KDa

Lamp-1

GCase activity (% of Control)

C

AMRF

250

**

200

150

*

100

50

*

*

**

**

*

0

59 KDa

GCase

Figure 7. Uptake of hrGCase in fibroblast cells. A) Western blot analysis of GCase protein internalized after treatment with 1.6 mM hrGCase for 4 h in AMRF syndrome- and GD2-derived fibroblasts Protein extracts from healthy fibroblasts were the loading control. B) Uptake of Alexa Fluor 555-labeled hrGCase. Healthy control and AMRF syndrome- and GD2-derived fibroblasts were grown on coverslips and incubated with 15 nM fluorescent rhGCase for 1 h (red); Lysotracker-Green (75 nM; Molecular Probes-Life Technologies) was added for the last 30 min (green). Untreated cells (NT) were the control. Blue: DAPI-stained nuclei. Merge: overlaid images of rhGCase and Lysotracker signals. C) Western blot analysis of internalized hrGCase in lysosomal and endosomal compartments after treatment with 1.6 mM hrGCase for 4 h of AMRF syndrome- and GD2-derived fibroblasts. D) Densitometric quantification of GCase in the endosomal and lysosomal fractions. GCase signal intensity normalized to those of EEA1 and LAMP-1, respectively. P , 0.01 vs. GD2. E) GCase activity after uptake of hrGCase. D, E) Data are the means 6 SD of results in 3 independent experiments. *P , 0.05; ** P , 0.01 vs. nontreated cells.

In agreement with data published by Gaspar et al. (19) LIMP-2-deficient macrophages retained a high residual activity (30% of that in normal controls). It was not possible to obtain enough cells to analyze the intracellular localization of GCase in this cell type. However, considering the 12

Vol. 29 September 2015

high residual activity (which was almost identical with the activity retained by the lymphoblasts) and the results indicating that the main effect of this LIMP-2 mutation would be the ER retention of GCase and degradation, it is likely that, also in macrophages, at least part of total GCase

The FASEB Journal x www.fasebj.org

MALIN ET AL.

B

A

Wash buffer

pH 8

100

LIMP-2 (H363N) + GCase

pH 4.2

pH 8

pH 4.2 LIMP2 -myc

IP anti-Myc; WB anti-Myc GCase

GCase activity (% of co-IP GCase wt -LIMP-2 wt)

LIMP-2(wt) + GCase

80 60 40

**

20 0

IP anti-Myc; WB anti-GCase

GCase wt-LIMP2 wt GCase wt-LIMP2 H363N

Figure 8. Effect of H363N mutation on GCase binding and activity. A) Co-IP assay: protein complexes obtained from HEK293 cells expressing myc-tagged LIMP-2 constructs (WT or H363N), together with GCase, were immunoprecipitated with anti-myc antibody, washed with co-IP buffer (pH 8) or with 0.1 M acetate buffer (pH 4.2), separated by SDS-PAGE, and detected by Western blot analysis with anti-GCase and -LIMP-2 antibodies. B) Enzymatic activity of GCase in the immunoprecipitated complex: protein complexes obtained from HEK293 cells expressing myc-tagged LIMP-2 constructs (WT or H363N), together with GCase, were immunoprecipitated with anti-myc antibody and washed with co-IP buffer (pH 8). GCase activity was measured directly on beads. Data are the means 6 SD of results in 3 independent experiments. **P , 0.01 vs. WT.

escapes ERAD and probably reaches the lysosomes, even in the presence of a mutated LIMP-2 protein. This hypothesis is supported by the observation that patients with AMRF syndrome did not present Gaucher cells in bone marrow, showed normal levels of chitotriosidase activity, and did not present typical visceral symptoms of GD. In light of these data, it is reasonable to hypothesize that these cells express membrane proteins other than LIMP-2 that mediate the intracellular transport of GCase from the ER to the lysosomes. It will be interesting to look at other receptors involved in the sorting of lysosomal proteins that may be active in different tissues. In the human neuronal model of LIMP-2 deficiency used in this study, the levels of GCase protein were significantly reduced, and the low amount of detected protein was entirely endo-H sensitive, indicating that it is almost completely retained in the ER. Taken together, these results indicate that the neuronal model of AMRF syndrome presents a very low amount of intracellular GCase protein that does not reach the lysosomes, leading to a severe impairment of GCase activity. These observations support the hypothesis that the neurologic signs in patients with AMRF syndrome are caused by the lack of functional GCase within the lysosomes, which in turn, would be the likely cause of storage of undigested GSL within this organelle. However, further studies are needed to confirm this hypothesis. In addition, we studied the trafficking of exogenous hrGCase to elucidate the role of LIMP-2 in the uptake and targeting of the recombinant enzyme used for ERT in patients with GD. We demonstrated that although hrGCase enters the cells by a LIMP-2-independent mechanism, probably via the mannose receptor (33, 34), LIMP-2 is necessary for the intracellular trafficking between endocyte compartments. However, even if we have demonstrated that LIMP-2 is involved in the trafficking of hrGCase to the lysosomes in fibroblasts, alternative mechanisms in cells of mononuclear phagocyte origin, the main target tissue of ERT in GD, cannot be excluded. Further studies to identify these mechanisms are ongoing. Besides shedding some light on the bases of the phenotypic differences between GD and AMRF syndrome, our ROLE OF LIMP-2 IN GCASE TRAFFICKING

findings may have important implications regarding the role of LIMP-2 in the pathogenesis and treatment of GD. Indeed, LIMP-2 may be one of the factors that contribute to the phenotypic heterogeneity in patients with GD with the same genotype. Likewise, our data showed that this protein is involved in the transport of hrGCase to the lysosome where the enzyme must arrive to exert its function, and it may therefore be an important factor involved in the response of patients with GD to ERT with hrGCase. In conclusion, this work provides valuable new insights into the role of LIMP-2 in the trafficking of GCase in different human cell types. In addition, the neuronal model described in is a potential tool for the analysis of the molecular mechanism involved in neuronal pathology in both GD and AMRF syndrome. The authors thank Silvia Cattarossi for technical assistance and Federica Benvenuti for technical advice. This work was supported by Grant PRF 37/08 (clinical history and long-term cost-effectiveness of enzyme replacement therapy for Gaucher disease in Italy) from the Ministero della Salute. Some samples were obtained from the Cell Line and DNA Biobank from Patients Affected by Genetic Diseases (Giananni Gaslini Institute, Genoa, Italy), a Telethon Network of Genetic Biobanks (Project No. GTB12001A). The authors declare no conflicts of interest.

REFERENCES 1. Fujita, H., Ezaki, J., Noguchi, Y., Kono, A., Himeno, M., and Kato, K. (1991) Isolation and sequencing of a cDNA clone encoding 85kDa sialoglycoprotein in rat liver lysosomal membranes. Biochem. Biophys. Res. Commun. 178, 444–452 2. Febbraio, M., Hajjar, D. P., and Silverstein, R. L. (2001) CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J. Clin. Invest. 108, 785–791 3. Krieger, M. (2001) Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems. J. Clin. Invest. 108, 793–797 4. Reczek, D., Schwake, M., Schr¨oder, J., Hughes, H., Blanz, J., Jin, X., Brondyk, W., Van Patten, S., Edmunds, T., and Saftig, P. (2007) LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase. Cell 131, 770–783

13

5. Neculai, D., Schwake, M., Ravichandran, M., Zunke, F., Collins, R. F., Peters, J., Neculai, M., Plumb, J., Loppnau, P., Pizarro, J. C., Seitova, A., Trimble, W. S., Saftig, P., Grinstein, S., and Dhe-Paganon, S. (2013) Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36. Nature 504, 172–176 6. Zhao, Y., Ren, J., Padilla-Parra, S., Fry, E. E., and Stuart, D. I. (2014) Lysosome sorting of b-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor. Nat. Commun. 5, 4321 7. Liou, B., Haffey, W. D., Greis, K. D., and Grabowski, G. A. (2014) The LIMP-2/SCARB2 binding motif on acid b-glucosidase: basic and applied implications for Gaucher disease and associated neurodegenerative diseases. J. Biol. Chem. 289, 30063–30074 8. Badhwar, A., Berkovic, S. F., Dowling, J. P., Gonzales, M., Narayanan, S., Brodtmann, A., Berzen, L., Caviness, J., Trenkwalder, C., Winkelmann, J., Rivest, J., Lambert, M., Hernandez-Cossio, O., Carpenter, S., Andermann, F., and Andermann, E. (2004) Action myoclonus-renal failure syndrome: characterization of a unique cerebro-renal disorder. Brain 127, 2173–2182 9. Dardis, A., Filocamo, M., Grossi, S., Ciana, G., Franceschetti, S., Dominissini, S., Rubboli, G., Di Rocco, M., and Bembi, B. (2009) Biochemical and molecular findings in a patient with myoclonic epilepsy due to a mistarget of the beta-glucosidase enzyme. Mol. Genet. Metab. 97, 309–311 10. Dibbens, L. M., Michelucci, R., Gambardella, A., Andermann, F., Rubboli, G., Bayly, M. A., Joensuu, T., Vears, D. F., Franceschetti, S., Canafoglia, L., Wallace, R., Bassuk, A. G., Power, D. A., Tassinari, C. A., Andermann, E., Lehesjoki, A. E., and Berkovic, S. F. (2009) SCARB2 mutations in progressive myoclonus epilepsy (PME) without renal failure. Ann. Neurol. 66, 532–536 11. Dibbens, L. M., Karakis, I., Bayly, M. A., Costello, D. J., Cole, A. J., and Berkovic, S. F. (2011) Mutation of SCARB2 in a patient with progressive myoclonus epilepsy and demyelinating peripheral neuropathy. Arch. Neurol. 68, 812–813 12. Rubboli, G., Franceschetti, S., Berkovic, S. F., Canafoglia, L., Gambardella, A., Dibbens, L. M., Riguzzi, P., Campieri, C., Magaudda, A., Tassinari, C. A., and Michelucci, R. (2011) Clinical and neurophysiologic features of progressive myoclonus epilepsy without renal failure caused by SCARB2 mutations. Epilepsia 52, 2356–2363 13. Guerrero-L´opez, R., Garc´ıa-Ruiz, P. J., Gir´aldez, B. G., Dur´anHerrera, C., Querol-Pascual, M. R., Ram´ırez-Moreno, J. M., M´as, S., and Serratosa, J. M. (2012) A new SCARB2 mutation in a patient with progressive myoclonus ataxia without renal failure. Mov. Disord. 27, 1827–1828 14. Higashiyama, Y., Doi, H., Wakabayashi, M., Tsurusaki, Y., Miyake, N., Saitsu, H., Ohba, C., Fukai, R., Miyatake, S., Joki, H., Koyano, S., Suzuki, Y., Tanaka, F., Kuroiwa, Y., and Matsumoto, N. (2013) A novel SCARB2 mutation causing late-onset progressive myoclonus epilepsy. Mov. Disord. 28, 552–553 15. Beutler, E., and Grabowski, G. A. (2001) Gaucher disease. In The Metabolic and Molecular Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Kinzler, K. W., and and Vogelstein, B., eds), Vol. 3. pp. 3635–3668, McGraw-Hill, New York, USA 16. Balreira, A., Gaspar, P., Caiola, D., Chaves, J., Beirão, I., Lima, J. L., Azevedo, J. E., and Miranda, M. C. (2008) A nonsense mutation in the LIMP-2 gene associated with progressive myoclonic epilepsy and nephrotic syndrome. Hum. Mol. Genet. 17, 2238–2243 17. Chaves, J., Beirão, I., Balreira, A., Gaspar, P., Caiola, D., S´a-Miranda, M. C., and Lima, J. L. (2011) Progressive myoclonus epilepsy with nephropathy C1q due to SCARB2/LIMP-2 deficiency: clinical report of two siblings. Seizure 20, 738–740 18. Cox, T. M. (2010) Gaucher disease: clinical profile and therapeutic developments. Biologics 4, 299–313 19. Gaspar, P., Kallemeijn, W. W., Strijland, A., Scheij, S., Van Eijk, M., Aten, J., Overkleeft, H. S., Balreira, A., Zunke, F., Schwake, M., S´a Miranda, C., and Aerts, J. M. (2014) Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis. J. Lipid Res. 55, 138–145

14

Vol. 29 September 2015

20. Park, J. K., Orvisky, E., Tayebi, N., Kaneski, C., Lamarca, M. E., Stubblefield, B. K., Martin, B. M., Schiffmann, R., and Sidransky, E. (2003) Myoclonic epilepsy in Gaucher disease: genotype-phenotype insights from a rare patient subgroup. Pediatr. Res. 53, 387–395 21. Beltrami, A. P., Cesselli, D., Bergamin, N., Marcon, P., Rigo, S., Puppato, E., D’Aurizio, F., Verardo, R., Piazza, S., Pignatelli, A., Poz, A., Baccarani, U., Damiani, D., Fanin, R., Mariuzzi, L., Finato, N., Masolini, P., Burelli, S., Belluzzi, O., Schneider, C., and Beltrami, C. A. (2007) Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow). Blood 110, 3438–3446 22. Cesselli, D., Beltrami, A. P., Rigo, S., Bergamin, N., D’Aurizio, F., Verardo, R., Piazza, S., Klaric, E., Fanin, R., Toffoletto, B., Marzinotto, S., Mariuzzi, L., Finato, N., Pandolfi, M., Leri, A., Schneider, C., Beltrami, C. A., and Anversa, P. (2009) Multipotent progenitor cells are present in human peripheral blood. Circ. Res. 104, 1225–1234 23. Bergamin, N., Dardis, A., Beltrami, A., Cesselli, D., Rigo, S., Zampieri, S., Domenis, R., Bembi, B., and Beltrami, C. A. (2013) A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient’s skin. Orphanet J. Rare Dis. 8, 34 24. Van de Wetering, M., Oving, I., Muncan, V., Pon Fong, M. T., Brantjes, H., van Leenen, D., Holstege, F. C., Brummelkamp, T. R., Agami, R., and Clevers, H. (2003) Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615 25. Schr¨oter, C. J., Braun, M., Englert, J., Beck, H., Schmid, H., and Kalbacher, H. (1999) A rapid method to separate endosomes from lysosomal contents using differential centrifugation and hypotonic lysis of lysosomes. J. Immunol. Methods 227, 161–168 26. Mioˇci´c, S., Filocamo, M., Dominissini, S., Montalvo, A. L., Vlahovicek, K., Deganuto, M., Mazzotti, R., Cariati, R., Bembi, B., and Pittis, M. G. (2005) Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1. Hum. Mutat. 25, 100 27. Blanz, J., Groth, J., Zachos, C., Wehling, C., Saftig, P., and Schwake, M. (2010) Disease-causing mutations within the lysosomal integral membrane protein type 2 (LIMP-2) reveal the nature of binding to its ligand beta-glucocerebrosidase. Hum. Mol. Genet. 19, 563–572 28. Maley, F., Trimble, R. B., Tarentino, A. L., and Plummer, T. H., Jr. (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal. Biochem. 180, 195–204 29. Trimble, R. B., and Tarentino, A. L. (1991) Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1, endo F2, and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. J. Biol. Chem. 266, 1646–1651 30. Van Weely, S., Aerts, J. M., Van Leeuwen, M. B., Heikoop, J. C., Donker-Koopman, W. E., Barranger, J. A., Tager, J. M., and Schram, A. W. (1990) Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase. Eur. J. Biochem. 191, 669–677 31. Su´arez-Quian, C. A. (1987) The distribution of four lysosomal integral membrane proteins (LIMPs) in rat basophilic leukemia cells. Tissue Cell 19, 495–504 32. Okazaki, I., Himeno, M., Ezaki, J., Ishikawa, T., and Kato, K. (1992) Purification and characterization of an 85 kDa sialoglycoprotein in rat liver lysosomal membranes. J. Biochem. 111, 763–769 (Tokyo) 33. Mistry, P. K., Wraight, E. P., and Cox, T. M. (1996) Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher’s disease. Lancet 348, 1555–1559 34. Friedman, B., Vaddi, K., Preston, C., Mahon, E., Cataldo, J. R., and McPherson, J. M. (1999) A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placentalderived b-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease. Blood 93, 2807–2816

The FASEB Journal x www.fasebj.org

Received for publication February 3, 2015. Accepted for publication May 11, 2015.

MALIN ET AL.