The cytosolic β-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation

Blood Cells, Molecules, and Diseases 46 (2011) 19–26

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Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d

The cytosolic β-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation Nick Dekker a, Tineke Voorn-Brouwer a, Marri Verhoek a, Tom Wennekes c, Ravi S. Narayan a, Dave Speijer a, Carla E.M. Hollak b, Hermen S. Overkleeft c, Rolf G. Boot a, Johannes M.F.G. Aerts a,⁎ a b c

Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands

a r t i c l e

i n f o

Article history: Submitted 7 July 2010 Available online 21 August 2010 (Communicated by A. Zimran, M.D., 7 July 2010)

a b s t r a c t GBA3, also known as cytosolic β-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a β-glucosidase degrading glucosylceramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and influence the clinical manifestation of Gaucher disease. Recombinant GBA3 was found to hydrolyze artificial substrates such as 4-methylumbelliferyl-β-D-glucoside and C6-NBD-glucosylceramide, but hydrolysis of naturally occurring lipids like glucosylceramide and glucosylsphingosine was hardly detected. Consistent with this, inhibition of GBA3 in cultured cells using a novel inhibitor (alpha-1-C-nonyl-DIX) did not result in an additional increase in glucosylceramide as compared to GBA inhibition alone. Examination of the GBA3 gene led to the identification of a common substitution in its open reading frame (1368T→A), resulting in a truncated GBA3 protein missing the last α-helix of its (β/α)8 barrel. Both recombinant 1368A GBA3 and 1368A enzyme from spleen of a homozygous individual were found to be inactive. Amongst nonneuronopathic (type 1) Gaucher disease patients, we subsequently identified individuals being wild-type, heterozygous, or homozygous for the GBA3 1368T→A mutation. No correlation was observed between GBA3 1368A/T haplotypes and severity of type 1 Gaucher disease manifestation. In conclusion, GBA3 does not seem to modify type 1 Gaucher disease manifestation. © 2010 Elsevier Inc. All rights reserved.

Introduction The presence in mammalian tissues of a cytosolic β-glucosidase with broad substrate specificity (GBA3; EC 3.2.1.21) was first documented several decades ago. Seminal work by Glew and collaborators led to the molecular characterization of GBA3 (formerly referred to as non-specific β-glucosidase, broad-specificity β-glycosidase, or cytosolic β-glucosidase, and more recently as klotho-related protein) [1–4,6]. GBA3 is able to hydrolyze a variety of substrates with a β-glucose, β-galactose, β-xylose, or α-arabinose moiety linked to a hydrophobic group [2]. GBA3 is resistant to inhibition by conduritolβ-epoxide (CBE), but can be inhibited by β-D-glucosylsphingosine [2,5]. The GBA3 gene is located in locus 4p15,31 and its cDNA was independently cloned by two research groups [4,7]. The cDNA encodes a protein of 469 amino acid residues, possessing neither a signal peptide for secretion nor a transmembrane domain. GBA3 is most closely related to mammalian lactase phlorizin hydrolase and is

⁎ Corresponding author. Fax: + 31 20 691 5519. E-mail address: [email protected] (J.M.F.G. Aerts). 1079-9796/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2010.07.009

classified as belonging to family 1 of β-glycosidases [8]. The crystal structure of GBA3 was recently solved and crucial structural features for substrate (aglycone form) recognition were identified [6,9]. GBA3 is particularly expressed in liver, kidney, and intestine and found in the cytosol of the cell [6]. Since guinea pig liver cytosolic β-glycosidase is able to hydrolyze plant-derived phenolic (e.g. arbutin and salicin) and cyanogenic glucosides (e.g. prunasin), it has been suggested that human GBA3 may play an important role in the biotransformation of glycosylated xenobiotics by enterocytes in the intestine [3,10,11]. Another β-glucosidase, the lysosomal glucocerebrosidase (GBA) is crucial for cellular metabolism. Deficiency in GBA (EC.3.2.1.45), results in Gaucher disease, a recessively inherited lysosomal storage disorder [12]. Gaucher disease is characterized by massive accumulation of glucosylceramide in tissue macrophages. The phagocytic cells, designated as Gaucher cells, secrete various hydrolases, cytokines and chemokines, driving visceral pathophysiology [13–15]. Three variants of Gaucher disease are generally distinguished. Type I, the most common variant, does not involve prominent neurological manifestations. Patients with the more rare type 2 disease develop lethal neuropathology at childhood, whereas in type 3 Gaucher patients these complications occur later in life. The findings that neither the GBA genotype nor the residual β-glucosidase activity in cultured fibroblasts

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of Gaucher patients strictly predicts the severity of Gaucher disease manifestation, prompted investigations on other β-glucosidases involved in glucosylceramide metabolism which could act as disease modifiers. Indeed, a membrane-bound, non-lysosomal glucosylceramidase was discovered [16]. Recently its identity as GBA2 was elucidated [17,18]. So far, no indications that genetic heterogeneity in GBA2 influences the clinical outcome of GBA deficiency were found. Attention was then drawn to GBA3 as a possible Gaucher disease modifier. It has been reported that in some severely affected neuronopathic Gaucher patients, soluble β-glucosidase activity was impaired [2,19]. In addition, marked variations in cytosolic β-glucosidase activity in leukocytes have been reported [20]. This information prompted Beutler and coworkers to study the relationship of four GBA3 gene polymorphisms with severity of Gaucher disease manifestation [21]. They could not find any correlation between the GBA3 haplotypes and Gaucher disease severity. It was concluded that GBA3 was not likely to modulate the course of Gaucher disease. Recently, this view has been disputed by the report of Hayashi and coworkers [6]. These researchers provided convincing evidence that recombinant GBA3 was able to efficiently hydrolyze C6NBD-glucosylceramide. A much less striking activity towards -naturalglucosylceramide was reported, leading again to the speculation that GBA3 may modify Gaucher disease manifestation [6]. Theoretically, degradation of glucosylceramide by GBA3 in the cytosol might indeed occur. Glucosylceramide is present in significant amounts at the cytosolic leaflet of the Golgi apparatus where it is synthesized and transported to the luminal side of the endoplasmic reticulum. The possibility that, in contrast to earlier reports, GBA3 might metabolize glucosylceramide in the cytosol, prompted us to re-investigate the enzymatic features of GBA3. In our hands, GBA3 was found to be hardly active towards natural glucosylceramide. Moreover, we show that specific inhibition of GBA3 does not significantly alter overall cellular glucosylceramide levels when GBA is impaired and that a loss of function mutation in GBA3 does not correlate with type 1 Gaucher disease severity. Materials and methods Patients Fifty-nine patients with Gaucher disease type I were included in the study. Diagnosis of Gaucher disease was confirmed by glucocerebrosidase activity measurements in leukocytes or fibroblasts [22] and genotyping [23]. SSI scoring of disease severity was performed at baseline according to Zimran et al. [24,25]. Approval was obtained from the Ethics Committee. Informed consent was provided according to the Declaration of Helsinki. Sequencing patient material DNA from EDTA plasma was isolated using the QIAamp DNA Blood Kit, according to the manufacturer's protocol (Qiagen). Sequencing was performed using the BigDye® Terminator Cycle Sequencing Kit, according to the manufacturer's protocol (Applied Biosystems). Cloning Frozen Gaucher disease spleen tissue (stored at −80 °C) was homogenized in RNase free sterile water using a mortar. Subsequently, total RNA was isolated using RNAzol according to the manufacturer's protocol (RNA-Bee™, TEL-TEST). First strand cDNA synthesis was performed on 10 μg of total RNA using SuperScript® II Reverse Transcriptase (Invitrogen) and oligo(dT). The complete open reading frame from GBA3 (NM_001128432) was amplified from cDNA, using a forward primer containing the EcoR1 restriction site and an extended Kozak sequence in front of the start codon and a reverse primer containing the Not1 restriction site. For overexpression, full-length

cDNA was cloned into the pCDNA3.1-myc/His expression vector (A+, Invitrogen). For detection of GBA3 on Western blot, the open reading frame was cloned in pCDNA3.1-myc/His, using a forward primer containing an extended Kozak sequence followed by a haemagglutinin-tag coding sequence. The reverse oligos contained either a thymidine at position 1368 to clone the entire open reading frame or an adenine to express the truncated form of GBA3. Transient transfections Transient transfection experiments in COS-7 cells were performed using Fugene-6 transfection reagent (Roche Applied Science) according to the manufacturer's protocol. Western blotting Protein concentrations were measured using the Bradford method with BSA as a standard. COS-7 cell lysates were resolved at 72.5 μg of protein per lane on a SDS/8% polyacrylamide gel under reducing conditions, blotted onto nitrocellulose membranes (0.45 μm; Schleicher & Schuell, Keene, NH), and probed with an anti-HA monoclonal antibody (Cell Signaling Technology) and an anti-α-tubulin antibody (Sigma-Aldrich). Blots were incubated with Cy3-conjugated goatanti-mouse IgG (Jackson ImmunoResearch) and scanned on an Odyssey image scanner (GE Healthcare). Substrates and inhibitors 4-Methylumbelliferyl-β-glucoside (4-MU-β-glucoside), glucosylceramide, glucosylsphingosine, sphingosine, octylglucoside, octylgalactoside, and conduritol-β-epoxide (CBE) were purchased from Sigma-Aldrich. Ceramide was purchased from Avanti Polar Lipids. BSA-complexed C6-NBD-glucosylceramide and N-(5 adamantane-1yl-methoxy)pentyl-deoxynojirimycin (AMP-DNM) were synthesized according to Overkleeft et al. [26]. Alpha-1-C-nonyl-DIX (anDIX), reported earlier [27], was synthesized according to Wennekes et al. [28]. To inhibit GBA and GBA2 activity, CBE and AMP-DNM were used at final concentrations of 1 mM and 20 nM, respectively. To inhibit GBA3 activity, anDIX was used at a final concentration of 1 μM. Cell culture COS-7, Hek-293, Meb-4, and HuH-7 cells were grown to confluency, lysed in Hepes buffer (100 mM, pH 7.0), and incubated with CBE and/or anDIX for 48 h at final concentrations of 1 mM and 1 μM, respectively. Lipids were extracted, separated by HPLC, and quantified using C17-sphinganine as internal standard, according to Groener et al. [29]. Enzyme assays All enzyme measurements were performed in triplicate. 4-MU-βglucoside, NBD-C6-glucosylceramide, glucosylsphingosine, or glucosylceramide was used as substrate to measure GBA and GBA3 activity. As source for GBA3, either COS-7 cells (overexpressing wild-type GBA3), or homogenized donor spleen tissue from a Gaucher type I patient (mutation N370S) were used. COS-7 lysates were generated by sonication on ice for 3 × 10 s at 10 μm (MSE Soniprep 150) in 100 mM Hepes buffer pH 7.0. Frozen spleen tissue (0.5 g, stored at − 80 °C) was homogenized on ice in 1.5 ml of sterile water (4 °C) using an Ultra-Turrax and sonication followed by 2 subsequent centrifugation steps for 10 min at 15.000 × g. The supernatant was used as spleen extract. GBA3 activity was measured for 45 min at 37 °C in Hepes buffer (100 mM Hepes, pH 7.0) containing 3.7 mM 4MU-β-glucoside as substrate after pre-incubation for 20 min on ice with CBE to inhibit GBA activity. For pH optimum determination,

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activity assays were performed for 30 min in the indicated buffers at a concentration of 100 mM. For fractionation of GBA3, COS-7 lysates were subjected to 3 subsequent centrifugation steps at 4 °C; 5 min at 1000 × g, 5 min at 16.000 × g, and 1 h at 100.000 × g, and activity was measured towards 4-MU-β-glucoside. Half maximal inhibitory concentrations (IC50) of anDIX and other inhibitors were determined by pre-incubation with GBA3 for 20 min on ice. IC50 values of anDIX for GBA, GBA2, and GBA3 were 0.001, 250, and 0.01 μM, respectively. Residual activity on 4-MU-β-glucoside was measured for 20 min at 37 °C. For measurement of endogenous cellular β-glucosidase activity, the immortalized cell lines COS-7, Hek-293, Meb-4, and HuH-7 were grown to confluency, lysed, and pre-incubated with CBE (final assay concentration 1 mM) for 20 min on ice to eliminate GBA activity. β-glucosidase activity was measured towards 4-MU-βglucoside for 1 h at 37 °C in 100 mM Hepes, pH 7.0. As source for GBA, a commercially available preparation of purified recombinant GBA was used (Cerezyme, Genzyme). GBA activity was measured towards 4-MU-β-glucoside as substrate (final concentration 3.7 mM) for 20 min at 37 °C in McIlvain buffer (0.1 mM citrate and 0.2 mM phosphate buffer, pH 5.2) containing 0.25% w/v sodium taurocholate and 0.1% v/v Triton X-100. Assays were stopped by the addition of glycine/NaOH (pH 10.6). The amount of liberated 4-MU was determined with an LS30 fluorometer (PerkinElmer Life Sciences).

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with the report by Hayashi and coworkers, GBA3 was found to hydrolyze C6-NBD-GlcCer [6]. However, the ratio of activity (relative turnover C6-NBD-GlcCer/4-MU-β-glucoside) was only 0.01. The ratio of activity towards GlcSph as compared with 4-MU-β-glucoside was 0.0013, whereas the ratio of activity of towards GlcCer as compared with 4-MU-β-glucoside was even lower: only 0.0004. For comparison, we also determined the activity of recombinant GBA (Cerezyme) towards 4-MU-β-glucoside, C6-NBD-GlcCer, GlcCer and GlcSph at optimal conditions for this enzyme (Fig. 2B). The specific activity of GBA towards 4-MU-β-glucoside under these conditions is 1.2 mmol substrate hydrolysis/mg protein/h. The ratio of activity of GBA towards C6-NBD-GlcCer, GlcCer and GlcSph relative to the activity towards 4-MU-β-glucoside was 0.23, 0.07 and 0.0007, respectively (Fig. 2B). At optimal conditions for 4-MU-β-glucoside hydrolysis, GBA3 hydrolyzed C6-NBD-GlcCer, GlcCer and GlcSph much less efficient than GBA, about 100-, 230- and 8-fold, respectively.

Activity quantification Quantification of GBA and GBA3 activity towards NBD-C6-glucosylceramide, glucosylsphingosine, and glucosylceramide was achieved by using high performance liquid chromatography. The lipids (400 pmol) were incubated with purified recombinant GBA (Cerezyme) or GBA3 overexpressing lysates (amounts normalized by using their activity towards the artificial substrate 4-MU-β-glucoside). The lipids were dissolved in McIlvain buffer (pH 5.2) containing 0.25% w/v sodium taurocholate and 0.1% v/v Triton X-100, to measure GBA activity, or in 100 mM Hepes buffer (pH 7.0) containing 0.25% w/v sodium taurocholate to measure GBA3 activity. To improve glucosylceramide solubility, 0.1% v/v Triton X-100 was added. After incubation for 30 and 60 min at 37 °C, the lipids were extracted using the Bligh and Dyer procedure and separated by HPLC according to Groener et al. [29]. Results Enzymological characterization of GBA3 GBA3 was overexpressed in COS-7 cells and its features in cell lysates were examined using the artificial substrate 4-methylumbelliferyl-β-glucoside (4-MU-β-glucoside). As a negative control, lysates were made of comparably MOCK-transfected cells. Differential centrifugation revealed that, in accordance with earlier reports, GBA3 activity was exclusively present in the soluble fraction [2,6,30] (Fig. 1A). Next, we studied the enzymatic features of GBA3 using lysates of GBA3 overexpressing COS-7 cells. To eliminate substrate hydrolysis by the lysosomal GBA, lysates were pre-incubated with 1 mM conduritol-β-epoxide (CBE), an irreversible inhibitor of GBA [16]. GBA3 activity in the lysate of transfected COS-7 cells was found to be optimal around pH 6.0, in accordance with previous reports [2,6] (Fig. 1B). A specific activity of purified GBA3 towards 4-MU-βglucoside of 0.28 mmol substrate hydrolysis/mg protein/h was previously determined and again confirmed [30]. Subsequently, we studied the substrate preferences of GBA3 (Fig. 2A). Hence GBA3 activity in cell lysates, pre-treated with CBE, towards 4-MU-βglucoside was determined. In addition, lysates were incubated with 0.5 μM albumin-complexed C6-NBD-glucosylceramide (C6-NBDGlcCer), glucosylceramide (GlcCer), or glucosylsphingosine (GlcSph), and substrate hydrolysis was quantified by HPLC (Fig. 2A). Consistent

Fig. 1. GBA3 characteristics. (A) GBA3 is a soluble enzyme. Lysates of COS-7 cells overexpressing GBA3 were subjected to differential centrifugation as described in Materials and methods. Activity towards artificial 4-MU-β-glucoside was measured in the presence of an excess of CBE to selectively inhibit GBA activity. Activity is presented as percentage of activity compared to that of the total cell lysate. (B) pH optimum for GBA3, using 4-MU-β-glucoside as substrate. Enzymatic activity was measured in COS-7 cell lysates in the presence of an excess of CBE. Enzymatic activity is represented as percentage of maximum activity.

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N. Dekker et al. / Blood Cells, Molecules, and Diseases 46 (2011) 19–26 Table 1 Enzyme inhibition by various lipids.

Glucosylceramide Glucosylsphingosine Ceramide Sphingosine Octylglucoside Octylgalactoside

GBA

GBA3

N 25 2.5 N 25 20 N100 N100

N25 1 N25 4 3 6

IC50 values are shown in μM. Activities towards 4-MU-β-glucoside were measured as described in Materials and methods.

Impact of GBA3 inhibition in cultured cells To better assess the possible contribution of GBA3 to glucosylceramide metabolism, we searched for a potent inhibitor of GBA3. To this end a library of aza-C-glycosides was generated and screened [28]. Compound alpha-1-C-nonyl-DIX (AnDIX) (Fig. 3A) was found to potently inhibit GBA3 (IC50 ~100 nM) as well as GBA (IC50 ~1 nM). To study the impact of GBA3 inhibition in GlcCer metabolism we made use of a human hepatoma cell line, HuH-7, which, in comparison with the immortalized cell lines COS-7, Hek-293, and Meb-4, expresses a relative large amount of GBA3 (Fig. 3B). Cells were incubated with 1.0 μM anDIX, 2 mM CBE, or a combination of both inhibitors for 2 days and cellular glucosylceramide and ceramide were determined (Fig. 3C and D). Ceramide levels did not change upon GBA3 and/or GBA inhibition (Fig. 3D). Inhibition of GBA alone with CBE led to a marked increase in cellular glucosylceramide. Concomitant inhibition of GBA3 by 1.0 μM anDIX did not result in an additional accumulation of the lipid, suggesting that GBA3 does not significantly contribute to cellular degradation of glucosylceramide (Fig. 3C). A common 1368T→A substitution leads to truncated, non-functional GBA3

Fig. 2. Substrate hydrolysis by GBA3 and GBA. Lysates of cells overexpressing GBA3 (A), or recombinant GBA (B), were incubated with C6-NBD-glucosylceramide, natural glucosylceramide, or natural glucosylsphingosine. Quantification of substrate hydrolysis was performed by HPLC. Enzyme activity from 3 independent experiments is presented as the mean ± SD of the ratio of hydrolysis of the indicated substrate relative to that of 4-MU-β-glucoside.

To further characterize GBA3, we studied the inhibitory effects of GlcCer, GlcSph, ceramide, sphingosine, octylglucoside and octylgalactoside on GBA3 activity using 4-MU-β-glucoside as substrate in the absence of detergents (Table 1). Compared to GlcCer and ceramide, the more water-soluble, smaller amphiphilic GlcSph, sphingosine, octylglucoside, and octylgalactoside were more potent inhibitors of GBA3 (Table 1). This implies that amphiphilic structures compete better with 4-MU-β-glucoside in the catalytic pocket, suggesting that GBA3 might prefer small amphiphilic structures as substrates.

A number of GBA3 gene polymorphisms have been reported previously by Beutler and colleagues [21]. Of particular interest is one of the single-nucleotide substitutions, with a relatively high frequency (on average 12%). This single-nucleotide change in the GBA3 gene results in the conversion of the tyrosine codon at position 456 into a stop codon. As a consequence, the truncated protein lacks most of the carboxy-terminal α-helix (helix number 8) from the classical (β/α)8 TIM barrel, the catalytic core of the enzyme (Fig. 4A). We hypothesized that this truncation would inevitably lead to a catalytically inactive GBA3 enzyme due to a perturbation of the catalytic centre or overall arrangement of the TIM barrel structure. To investigate this, we cloned the full-length cDNA and the cDNA carrying the T→A substitution at position 1368. Subsequently, we transiently expressed the cDNAs encoding in addition an N-terminal haemagglutinin (HA) epitope tag. Transfected COS-7 cells produced similar amounts of the recombinant tagged GBA3 proteins (Fig. 4B). Expression of the cDNA containing the 1368T→A substitution gave a protein with a molecular mass of ~50 kDa as compared to ~ 52 kDa for wild-type GBA3, which is in good agreement with their calculated molecular masses of 52.1 and 53.7 kDa, respectively, indicating absence of posttranslational modifications (Fig. 4B). Next, we measured β-glucosidase activity in COS-7 lysates overexpressing either HA-tagged wild-type or 1368A GBA3. To irreversibly inhibit GBA, an excess of CBE was used. In sharp contrast to HA-tagged wildtype GBA3, 1368A GBA3 was completely inactive towards the artificial substrate 4-MU-β-D-glucoside (Fig. 4C). The relative high frequency of the 1368T→A mutation in GBA3 allowed us to identify type 1 Gaucher patients homozygous or heterozygous for the mutation. To investigate the impact of GBA3 in vivo, available spleen tissue samples from two Gaucher patients were studied more closely. One patient expressed wild-type GBA3, the

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Fig. 3. Impact of GBA3 inhibition in cultured cells. (A) Structure of the aza-C-glucoside inhibitor alpha-1-C-nonyl-DIX used in this study. (B) Endogenous β-glucosidase activity measured in lysates of COS-7, Hek-293, Meb-4, and HuH-7 cells in the presence of an excess of CBE to selectively inhibit GBA activity. β-Glucosidase activity towards artificial 4-MUβ-glucoside was measured in triplicate (mean ± SD). Effect of the inhibition of GBA (CBE), or GBA and GBA3 concomitantly (AnDIX) on glucosylceramide (C) and ceramide (D) levels in HuH-7 cells. Lipid quantification was performed by HPLC and the means ± SD from 3 independent experiments is shown.

other was homozygous for the 1368T→A mutation. Both patients were heterozygous for the N370S mutation in the GBA gene. The βglucosidase activity in the two spleens was studied using 4-MU-βglucoside as substrate, employing specific inhibitors for GBA (1 mM CBE) and GBA2 (20 nM AMP-DNM). In addition the effect of anDIX was examined. Total β-glucosidase activity was 60% lower in the spleen extract from the Gaucher patient expressing mutant GBA3 as compared to the patient expressing wild-type GBA3 (2.4 versus 0.94 nmol/mg/h, respectively) (Fig. 4D). This loss in activity is probably caused by the loss of GBA3 activity due to the homoallelic GBA3 1368T→A mutation. Indeed, in the spleen extract from the Gaucher patient homozygous for 1368T→A virtually no GBA3 activity, i.e. activity inhibitable by anDIX, but insensitive to CBE or AMP-DNM, could be detected (Fig. 4D). In conclusion, in complete agreement with the findings with recombinant 1368A GBA3, the 1368T→A mutation in GBA3 also rendered the GBA3 enzyme inactive in spleen tissue from a Gaucher patient. The 1368T→A mutation in GBA3 does not correlate with type I Gaucher disease severity In a cohort of 59 patients with Gaucher disease type I we determined the 1368T/A GBA3 haplotypes and correlated this to the clinical severity of disease. Disease severity is generally quantified by the scoring index SSI (composite severity scoring index that scores various clinical manifestations of Gaucher disease). As shown in Fig. 5,

the SSI scores of type I Gaucher disease patients were unaffected by the presence of the GBA3 1368T→A mutation. Discussion The marked heterogeneity in clinical manifestation of Gaucher disease has stimulated speculations on the existence of additional βglucosidases that might modulate the outcome of a deficiency in the lysosomal glucocerebrosidase GBA. One such candidate is GBA3, also designated as the cytosolic β-glucosidase. The recent report by Hayashi and colleagues showing that GBA3 is able to hydrolyze glucosylceramide [6], prompted us to study the enzyme and its gene in more detail. Our investigation confirms that GBA3 is able to hydrolyze the artificial substrate C6-NBD-glucosylceramide, albeit far less efficiently than lysosomal GBA. Since this non-physiological substrate lacks a large portion of the hydrophobic acyl chain present in natural glucosylceramide, activity towards the artificial substrate may not reflect activity towards natural lipid substrates. Thus we tested the activity of GBA3 to natural glucosylceramide and glucosylsphingosine directly. The activity of GBA3 towards both natural lipids was found to be extremely low. At each enzyme's optimal conditions for 4-MU-βglucoside hydrolysis, GBA3 hydrolyzed glucosylceramide 230-fold less efficient than GBA. This observation is in agreement with the findings of Daniels et al., who detected no glucosylceramidase activity for a cytosolic β-glucosidase isolated from human liver [2]. Very

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Fig. 4. Substitution 1368T→A in the GBA3 gene renders GBA3 inactive. (A) A ribbon representation of the (β/α)8 barrel structure of GBA3. A 1368T→A substitution in GBA3 leads to a truncation of the carboxy-terminal α-helix of the (β/α)8 catalytic core of the enzyme (α-helices in red; β-sheets in yellow; part of the α-helix missing in 1368A GBA3 in blue). (B) Expression of amino-terminally haemagglutinin (HA) tagged GBA3 in COS-7 cells. Total cell lysates expressing either wild-type or mutant GBA3 were resolved by 8% SDS-PAGE under reducing conditions and visualized by immunoblot analysis using an anti-HA mAb. α-Tubulin was used as a loading control. (C) β-Glucosidase activity in lysates of COS-7 cells expressing wild-type GBA3 (circles) or 1368A GBA3 (triangles). β-Glucosidase activity was measured in triplicate using 4-MU-β-glucoside as a substrate and liberated 4-MU (pmol) is presented by the mean ± SD (D) β-Glucosidase activity in spleen extracts from two Gaucher patients (N370S mutation in GBA) expressing wild-type GBA3 (black bars) or 1368A GBA3 (grey bars). Specific GBA3 activity towards artificial 4-MU-β-glucoside was measured by simultaneous inhibition of GBA and GBA2 using an excess of CBE (1 mM) and AMPDNM (20 nM), respectively. Inhibition of GBA3 activity was achieved using an excess (1 μM) of anDIX. Note that in spleen extract expressing 1368A GBA3 no extra activity can be inhibited with anDIX as compared to the combined inhibition by CBE and AMP-DNM alone (compare wildtype). Enzymatic activity is represented in arbitrary units (arbs) per μg of total protein.

recently, the crystal structure of GBA3 was solved, revealing details regarding substrate glycone and aglycone specificity [6,9]. GBA3 contains a (β/α)8-barrel fold similar to that of other enzymes from family 1 of glycoside hydrolases [8], and hydrolyzes O-linked glycosides from the non-reducing end, with retention of anomeric configuration [6]. The catalytic centre of GBA3 comprises a compact glycone binding motif, and a hydrophobic aglycone binding motif. In contrast to the GBA motif, the aglycon binding motif of GBA3 displays an oblong, narrowly shaped entrance [9]. Our observation that a large molecule such as glucosylceramide is not hydrolyzed by GBA3 is consistent with the observation that the catalytic pocket of the enzyme displays such a narrow entrance. This is corroborated by the fact that the smaller amphiphilic compounds, such as glucosyl-

sphingosine, octylglucoside, and octylgalactoside are efficient inhibitors of GBA3. Our investigation of GBA3 activity in living cells did also not point to a role for the enzyme in glucosylceramide metabolism. Concomitant inhibition of GBA3 and GBA activity with alpha-1-C-nonyl-DIX did not further increase glucosylceramide levels as compared to those found with GBA inhibition alone. To confirm our findings on the role of GBA3 in glucosylceramide metabolism, we investigated the GBA3 gene. A common 1368T→A substitution in the GBA3 gene has been described previously. This polymorphic GBA3 allele is carried by 10–20% of individuals [21]. Interestingly, the 1368T→A substitution results in a change of a tyrosinecodon to a stopcodon, leading to the premature termination

N. Dekker et al. / Blood Cells, Molecules, and Diseases 46 (2011) 19–26

Fig. 5. Mutation 1368T→A in GBA3 does not correlate with clinical manifestation of type I Gaucher disease severity. DNA of type I Gaucher disease patients was isolated from plasma, screened for the 1368T→A mutation in GBA3, and correlated with disease severity. SSI scoring of disease severity was performed at baseline according Zimran et al. [25]. Lines represent mean values in each group. P-values (using Welch’s t-test) between all groups were higher than 0.05.

of the GBA3 protein. We demonstrate for the first time that this truncation of GBA3 disrupts its activity. In spleen tissue of a homozygote for the 1368T→A mutation, no GBA3 activity could be detected. Absolutely consistent with this, COS-7 cells expressing 1368A-GBA3 showed no activity, whereas COS-7 cells expressing GBA3 displayed clear activity. This, in turn, prompted us to examine the correlation between the GBA3 1368T→A haplotype and disease severity in a cohort of type 1 Gaucher disease patients. Disease severity, as measured with a commonly employed composite score, did not correlate with the GBA3 1368A haplotype of the type 1 Gaucher disease patients examined. Our findings support earlier observations by Beutler and colleagues and substantiate their conclusion that GBA3 does not influence clinical severity of type I Gaucher disease [21]. The common 1368T→A mutation in the GBA3 gene may contribute to the wide range of cytosolic β-glucosidase activity measured in human leukocytes [20]. The lack of residues at the carboxy terminus forming the last α-helix might disrupt correct folding of the (β/α)8 barrel and positioning of the residues comprising the catalytic pocket. One such residue is phenylalanine in loop 8 at position 433 that together with other hydrophobic residues forms the wall of the catalytic pocket of the aglycone motif possibly regulating substrate specificity [9]. A priori, our study does not exclude that GBA3 plays a modulating role in very severely affected, neuronopathic type 2 and 3 Gaucher disease patients. Compared to GBA, GBA3 shows an approximate 8fold lower ability to hydrolyze glucosylsphingosine. It has been hypothesized that glucosylsphingosine may contribute to neurological manifestations in Gaucher disease [31,32]. It is conceivable that a combined deficiency in GBA and GBA3 further favours glucosylsphingosine accumulation (see below). At present the physiological function of GBA3 remains enigmatic. Detailed analyses of exogenously expressed GBA3 have shown that, in strong contrast to other glycosidases from glycoside hydrolase family 1, it has very broad substrate specificity in terms of substrate glycone and aglycone moiety. Although it is generally assumed that most of the dietary glycosidated xenobiotics are hydrolyzed by glycosidases produced by the colon microflora, endogenous glycosidases expressed by the small intestine, such as GBA3 and lactase phlorizin hydrolase, may still play an important role in xenobiotics metabolism. Indeed, glycosidated forms of xenobiotics, such as phytoestrogens, flavonoids, simple phenolics, and cyanogens are hydrolyzed efficiently by GBA3 [3,10]. This might find its importance in the rapid uptake of some xenobiotics by the intestine for further metabolism and hence may

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reduce toxicity of some of these compounds, prior to reaching the large intestine and colon [10]. The large amount of individuals deficient in GBA3, such as homozygotes for the 1368T→A mutation, offers opportunities for research on the impact of GBA3 deficiency on metabolism of dietary glycosidated xenobiotics. The marked expression of GBA3 in the intestine, liver and kidney also indicates a role in biotransformation of xenobiotics. In conclusion, our study finally lays the discussion regarding a possible modifier role for GBA3 in the clinical manifestation of type 1 Gaucher disease to rest. GBA3 is not involved, as: 1. (Over) expressed GBA3 did not show significant glucosylceramidase activity when using natural, instead of artificial ‘lipid’ substrates. 2. Inhibition of GBA3 (against a background of total inhibition of GBA) in living cells did not lead to any further glucosylceramide accumulation. 3. The common 1368T→A substitution in the GBA3 gene results in a truncated and completely inactive protein, but the presence or absence of the 1368A haplotype shows no correlation whatsoever with the severity of disease manifestation in type 1 Gaucher disease patients (Fig. 5). The remaining question whether GBA3 does have a modifier role in type 2 and/or type 3 Gaucher disease, has to be established by studies of large cohorts of neuronopathic Gaucher disease patients. Acknowledgments We are grateful to the Netherlands Gaucher Society and their patient members for cooperation and support. We would like to acknowledge Wilma Donker-Koopman and Anneke Strijland for technical assistance, and Boris Bleijlevens for helpful discussions. Dedicated to the memory of Prof. Ernest Beutler (2008†) who momentuously contributed over many decades to the knowledge of Gaucher disease. References [1] R.H. Glew, S.P. Peters, A.R. Christopher, Isolation and characterization of βglucosidase from the cytosol of rat kidney cortex, Biochim. Biophys. Acta 422 (1976) 179–1992. [2] L.B. Daniels, P.J. Coyle, Y.B. Chiao, R.H. Glew, R.S. Labow, Purification and characterization of a cytosolic broad specificity β-glucosidase from human liver, J. Biol. Chem. 256 (1981) 13004–13013. [3] V. Gopalan, A. Pastuszyn, W.R. Galey Jr., R.H. Glew, Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic β-glucosidase, J. Biol. Chem. 267 (1992) 14027–14032. [4] K. Yahata, K. Mori, H. Arai, S. Koide, Y. Ogawa, M. Mukoyama, A. Sugawara, S. Ozaki, I. Tanaka, Y. Nabeshima, K. Nakao, Molecular cloning and expression of a novel klotho-related protein, J. Mol. Med. 78 (2000) 389–394. [5] V. Gopalan, L.B. Daniels, R.H. Glew, M. Claeyssens, Kinetic analysis of the interaction of alkyl glycosides with two human β-glucosidases, Biochem. J. 262 (1989) 541–548. [6] Y. Hayashi, N. Okino, Y. Kakuta, T. Shikanai, M. Tani, H. Narimatsu, M. Ito, Klothorelated protein is a novel cytosolic neutral β-glycosylceramidase, J. Biol. Chem. 282 (2007) 30889–30900. [7] M. de Graaf, I.C. van Veen, I.H. van der Meulen-Muileman, W.R. Gerritsen, H.M. Pinedo, H.J. Haisma, Cloning and characterization of human liver cytosolic βglycosidase, Biochem. J. 356 (2001) 907–910. [8] B. Henrissat, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280 (Pt 2) (1991) 309–316. [9] S. Tribolo, J.G. Berrin, P.A. Kroon, M. Czjzek, N. Juge, The crystal structure of human cytosolic β-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase, J. Mol. Biol. 370 (2007) 964–975. [10] K. Németh, G.W. Plumb, J.G. Berrin, N. Juge, R. Jacob, H.Y. Naim, G. Williamson, D. M. Swallow, P.A. Kroon, Deglycosylation by small intestinal epithelial cell βglucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans, Eur. J. Nutr. 42 (2003) 29–42. [11] C. Henry-Vitrac, A. Desmouliere, D. Girard, J.M. Merillon, S. Krisa, Transport, deglycosylation, and metabolism of trans-piceid by small intestinal epithelial cells, Eur. J. Nutr. 45 (2006) 376–382. [12] J.A. Barranger, E.I. Ginns, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The metabolic basis of inherited disease, 2nd Ed., McGraw-Hill, New York, 1989, pp. 1677–1698. [13] C.E. Hollak, S. van Weely, M.H. van Oers, J.M. Aerts, Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease, J. Clin. Invest. 93 (1994) 1288–1292. [14] R.G. Boot, M. Verhoek, M. de Fost, C.E. Hollak, M. Maas, B. Bleijlevens, M.J. van Breemen, M. van Meurs, L.A. Boven, J.D. Laman, M.T. Moran, T.M. Cox, J.M. Aerts,

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