Use of fluorescent substrates for characterization of Gaucher disease mutations

Blood Cells, Molecules, and Diseases 35 (2005) 57 – 65 www.elsevier.com/locate/ybcmd

Use of fluorescent substrates for characterization of Gaucher disease mutations Idit Rona, Arie Daganb, Shimon Gattb, Metzada Pasmanik-Chora, Mia Horowitza,* b

a Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, 69978, Israel The Department of Biochemistry, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel

Submitted 21 March 2005 Available online 23 May 2005 (Communicated by E. Beutler, M.D., 24 March 2005)

Abstract Gaucher disease results from impaired activity of the lysosomal enzyme h-glucocerebrosidase. More than 200 mutations within the glucocerebrosidase gene have been associated with this disease. In this study we tested the effect of several mutations (K157Q, D140H, E326K, D140H+E326K, V394L and R463C) on RNA stability, protein stability and activity toward four different fluorescent substrates (LR-12-GC, Bodipy-12-GC, LR-0-PAP-glucose and 4-MUG), using the vaccinia-derived expression system. The results indicated that the K157Q mutation leads to RNA instability, causing low protein levels and a concomitant reduction in hglucocerebrosidase activity. All other tested mutations led to production of glucocerebrosidase RNA and protein with stabilities comparable to those of the normal counterpart. The D140H variant exhibited a high activity toward the tested substrates while the variant enzymes containing either the E326K or D140H and E326k mutations together expressed low h-glucocerebrosidase activity. The V394L variant exhibited low activity toward the tested substrates, while a higher activity was presented by the R463C containing glucocerebrosidase variant. Our results strongly indicated that the LR-12-GC substrate distinguishes between severities of different mutant glucocerebrosidase variants overexpressed in a heterologous system. D 2005 Elsevier Inc. All rights reserved. Keywords: Fluorescent substrate; Gaucher disease; h-glucocerebrosidase

Introduction Gaucher disease, characterized by accumulation of glucosylceramide mainly in cells of the reticuloendothelial system [1,2], results from impaired activity of the lysosomal enzyme h-glucocerebrosidase [2 –4]. On the basis of age of

Abbreviations: Bodipy-12-GC, Bodipy dodecanoyl h glucosylceramide; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GCase, glucocerebrosidase; LR-12-GC, lissamine rhodamine dodecanoyl h glucosylceramide; LR-0-PAP, lissamine rhodamine-p-aminophenyl h-glucose; 4-MUG, 4-methyl-umbeliferyl-glucopyranoside; 4MU, 4-methylumbeliferone; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; TLC, thin layer chromatography. * Corresponding author. Fax: +972 3 642 2046. E-mail address: [email protected] (M. Horowitz). 1079-9796/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.03.006

onset, clinical signs and involvement of neurological symptoms, the disease has been sub-divided into three clinical categories. Type 1 (MIM# 230800, adult type, chronic, non-neuronopathic) is the most common form, characterized by the lack of central nervous system involvement. It is very heterogeneous in its clinical features [3,5] and is known as the most prevalent genetic disease among Ashkenazi Jews, with a carrier frequency of 1:17 in the Israeli Ashkenazi Jewish population [6]. Type 2 (MIM# 230900, infantile, acute neuronopathic) is a rare and lethal form of the disease. It is characterized by early appearance of visceral signs, central nervous system involvement and death a few months after birth. Type 3 (MIM# 231000, juvenile, neuronopathic) is characterized by early onset of visceral impairment and a later appearance of central nervous system symptoms [3,5]. Over 200 mutations,

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identified thus far in the glucocerebrosidase gene, have been associated with Gaucher disease (www.hgmd.org) [7]. The mutations include mostly point mutations, but also some frame shift mutations and deletions. Complex alleles that contain more than one point mutation or point mutations and a deletion were also described [8]. Most of them exist normally in the glucocerebrosidase pseudogene that occupies the same locus as the normal counterpart, on chromosome 1q21 [9]. Aiming at understanding the biochemical basis for the different mutations, several heterologous systems for glucocerebrosidase expression were developed. We have used and already described the vaccinia-derived expression system [10 – 12]. The recombinant vaccinia virus vTF7-3 [11] provides an expression of the bacteriophage T7 RNA polymerase, a prokaryotic enzyme, which does not contain a nuclear localization signal. The prokaryotic RNA polymerase can transcribe any T7 promoter-containing DNA, which is introduced into the cytoplasm of the infected cell. The target gene is coupled to the T7 polymerase promoter, followed by the 5V encephalomyocarditis virus-5V-untranslated region (ECMV-5V-UTR). In the presence of T7 RNA polymerase, high transcription levels are obtained and the EMCV-5V-UTR ensures highly efficient cap-independent translation from the transcribed mRNA [10]. In the present study we used the vaccinia-derived system to express glucocerebrosidase variants that contained the mutations: K157Q, D140H and E326K, R463C, V394L [13 –16] along with the already reported: N370S [17], L444P [18] and P415R mutations [19,20] for RNA stability, protein levels as well as their activities toward different fluorescent substrates, tested under in vitro conditions. The N370S mutation [17], associated with a mild form of the disease, is the most prevalent one among Ashkenazi Jews [6], whereas the L444P mutation, a severe form associated with neuronopathic disease at homozygosity [18], is the most prevalent mutation among non-Jews. The P415R is a very severe mutation, associated with type 2 Gaucher disease. The D140H/E326K and the K157Q mutations were initially identified in a Gaucher disease family, with two affected brothers [13]. One was severely affected and died at the age of 28 of what was initially described as type 3 Gaucher disease while the other brother is mildly affected [13]. While the D140H and the K157Q mutations are unique, the E326K mutation was reported in seven different families, always in conjunction with another mutation on the same allele [13,21 –25]. The fact that this mutation never appeared alone raised the question whether it is a disease-producing mutation or merely non-diseaseproducing polymorphism [26]. The R463C substitution has been reported in homozygosity among patients with severe type I Gaucher disease [27], implying that this mutation, like the N370S mutation, is not associated with the neuronopathic forms of Gaucher disease. The V394L mutation has been documented among patients with Gaucher types 1, 2 and 3. The mutation has not been found

in homozygosity, suggesting that homozygosity for this mutation may be lethal [28]. Usually, glucocerebrosidase activity is tested in vitro with 4-MUG as a substrate. It has been shown [28] that activity of overexpressed mutant glucocerebrosidase variants toward this substrate varied between <0.2% for severe mutations, such as D409H and P415R, to 13% for mild mutations such as N370S and R463C. We chose to test few other fluorescently labeled substrates, namely, LR-12-GC, Bodipy-12-GC and LR-0-PAP-h-glucose. Our results strongly indicate that all the tested base pair changes are disease-producing mutations and that the fluorescent substrate LR-12 GC is the most reliable in predicting severity of all the tested mutations, using heterologous expression.

Materials and methods Materials The following antibodies were used in this study: mouse monoclonal anti-glucocerebrosidase 2C7, kindly provided by Dr. H. Aerts (E. C. Slater Institute for Biochemical Research, University of Amsterdam, the Netherlands). Rabbit anti-Erk (C16 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Mouse monoclonal anti-myc (9B11 Cell Signaling Technology. Beverly, MA, USA). The secondary antibodies: goat anti-rabbit and goat anti-mouse conjugated to horseradish peroxidase were purchased from Jackson Laboratories Inc., PA, USA. Restriction enzymes were purchased from several companies and employed according to the manufacturers’ recommendations. TLC plates (TLC, Silica gel 60A) were purchased from Whatman International Ltd. England. Western blotting luminal reagents were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Cells and viruses HeLa, CV-1 and BSC-1 cells were grown in DMEM supplemented with 10% FCS. Thymidine kinase-deficient 143B cells (TK-cells) were grown in DMEM supplemented with 10% FCS and 25 Ag/ml Brdu (Sigma-Aldrich, Israel). All cells were grown at 37-C in the presence of 5% CO2. Wild-type vaccinia virus (v-WR), vTF7-3 (expressing the T7 RNA polymerase) and recombinant viruses were propagated in HeLa cells. Titers were determined using BSC-1 cells as described elsewhere [29]. Stocks were stored at 80-C. Plasmid construction Construction of the plasmid containing the human glucocerebrosidase cDNAs was described elsewhere [30]. The mutations D140H, K157Q, E326K, V394L and R463C were introduced into the glucocerebrosidase cDNA within the vaccinia vector. Each one of the base pair changes was

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introduced into M13 by the in vitro mutagenesis technique as described elsewhere [29]. Double-stranded DNA was prepared from the M13 clones and was digested with the restriction enzyme EcoNI and SacI. The resulting fragments were ligated to pTM1-glu digested with the same enzymes, as described [29]. To create the vaccinia-derived vector carrying the D140H and the E326K mutations, the pTM1-glu (D140H) and the pTM1-glu (E326K) vectors were digested with SacI. The 3.1-kb fragment of the pTM-glu (D140H) was ligated to the 3.9-kb fragment of the pTM-glu (E326K). To create recombinant myc-tagged glucocerebrosidase, the cDNA encoding the human glucocerebrosidase containing its 38 amino acid leader, was cloned into the EcoRI and XhoI sites of the pcDNA4 myc-his-plasmid (Invitrogen Life-Technologies, Carlsbad, CA, USA). This construct was mutated using the Quick Change site directed mutagenesis kit (Invitrogen Life-Technologies, Carlsbad, CA, USA) to create variant forms with the mutations: N370S and L444P. Transfections Transfection was performed using Fugene transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions. Immunoprecipitation Cells were transiently transfected with plasmids expressing myc-tagged glucocerebrosidase. Forty-eight hours later, cells, washed three times with ice-cold PBS, were lysed at 4-C in 1 ml of lysis buffer (10 mM Hepes pH 8, 100 mM NaCl, 1 mM MgCl2 and 0.5% NP40), containing 10 mg/ml aprotinin, 0.1 mM PMSF and 10 mg/ml leupeptin (SigmaAldrich, Israel). Following incubation on ice for 30 min, lysates were centrifuged at 10,000  g for 15 min at 4-C, and the supernatant was precleared for 1 h at 4-C with protein A Agarose (Hoffmann-La Roche Ltd, Basel, Switzerland). Samples were centrifuged at 15,000  g for 1 min at 4-C, and the supernatant was incubated overnight at 4-C with monoclonal anti-myc antibody immobilized on protein G Sepharose (Sigma-Aldrich, Israel). Immune complexes were precipitated, followed by four washes with 1 ml of lysis buffer. Immunoprecipitated proteins were eluted for 10 min at 100-C with 5  loading buffer, electrophoresed through 10% SDS-PAGE and blotted. The corresponding blot was interacted with anti-myc antibody. Generation of recombinant vaccinia viruses Generation of recombinant vaccinia viruses was essentially as described elsewhere [29]. RNA preparation and Northern blot analysis RNA preparation and Northern blot analysis was as described elsewhere [30].

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Enzymatic activity Subconfluent monolayers of HeLa cells (¨5  106 cells) were infected with 10 pfu/cell of vTF7-3, a vaccinia virus expressing the T7 polymerase from a vaccinia early gene and any one of the recombinant viruses. 20 h later, cells were washed twice with PBS, collected in 1 ml sterile water and frozen at 80-C. Samples containing the same amount of protein, determined by the Bradford technique [31], were assayed for h-glucocerebrosidase activity with different fluorescent substrates. (A) With 4-MUG (Genzyme Corp., Boston, MA, USA): The enzymatic reaction was performed in 0.2 ml of 100 mM potassium phosphate buffer, pH 4.5, 0.15% Triton X-100 (v/v, Sigma, Israel) and 0.125% taurocholate (w/v, Calbiochem, grade A, Merck, Darmstadt, Germany) in the presence of 3 mM 4-MUG, for 60 min at 37-C. The reaction was stopped by addition of 1 ml 0.1 M glycine in 0.1 M NaOH pH 10. The amount of 4-MU was quantified using a Perkin Elmer Luminescence Spectrometer LS 50 (excitation length: 340 nm; emission: 448 nm). (B) With LR-12-GC, Bodipy-12-GC and LR-0-PAPglucose: The enzymatic reaction was performed in 0.2 ml as above, in the presence of 2.5 nM substrate. At the end of the reaction, lipids were separated by TLC in chloroformbutanol-ethyl acetate-KCl (0.25%) – methanol (25:25:25: 9:16). The TLC plates were visualized under a UV lamp. The intensity of the substrates and products was scanned using FLA2000 FUJIFILM fluorimeter (Fuji Co., Tokyo, Japan) (excitation wavelength: 505 nm; emission: 511 nm for Bodipy and excitation wavelength: 583 nm; emission: 568 nm for lissamine rhodamine) and quantified using Tina 2.10 program. SDS – PAGE and Western blotting Subconfluent monolayers (¨5  106 cells) of HeLa cells were infected with 10 pfu/cell of vTF7-3 and any one of the recombinant viruses, or transfected with a plasmid expressing different myc-tagged glucocerebrosidase variants. 20 h after infection or 48 h after transfection, cell lysates were prepared by washing the monolayers 3 times with ice-cold PBS and addition of 400 Al of lysis buffer (10 mM Hepes pH 8, 100 mM NaCl, 1 mM MgCl2 and 1% Triton X-100) containing 10 Ag/ml aprotinin, 0.1 mM PMSF and 10 Ag/ml leupeptin (Sigma Aldrich, Israel), at 4-C. Following 30 min on ice, the lysates were centrifuged at 10,000  g for 15 min at 4-C and the precipitates were discarded. Samples containing the same amount of protein were electrophoresed through 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane. Membranes were blocked in 5% skim milk and 0.1% Tween 20 in TBS for 1 h at room temperature, and incubated with the primary antibodies for 1 h at RT. The membranes were then washed three times in 0.1% Tween 20 in TBS and incubated with the appropriate secondary antibody for 1 h at room

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temperature. After washing, membranes were incubated with Western blotting Luminol reagent (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) and analyzed by luminescent image analyzer (LAS-1000; Fujifilm, Tokyo, Japan). Random-primed labeling Kit was purchased from Fermentas (Fermentas Inc. Hanover, MD, USA) and labeling was performed according to the manufacturer’s recommendations. Synthesis of LR-PAP-glucose Solid lissamine rhodamine chloride (Molecular Probes, Eugene, OR, USA) (20 Amol) was added to a 10-ml solution of p-aminophenyl-h-d-glucopyranoside (30 Amol), dissolved in isopropanol: 5% potassium carbonate (in water), 4:1. The mixture was stirred for 24 h, and the solvents were evaporated to dryness using a rotavapor. The dried material was dissolved in a small amount of dichloromethane – methanol (1:2) and purified by thin-layer chromatography (TLC) on preparative silica gel plates, which were developed in chloroform – methanol – water (50:50:8). The two red spots (normal and pH-sensitive) were scraped individually and eluted with a mixture of chloroform –methanol – water (1:2:1, by volume).

Results Stability of the mutated RNAs In order to compare RNA stability of the normal and mutated glucocerebrosidase variants, RNA prepared from HeLa cells, co-transfected with VTF-3 and the wild-type or different mutated glucocerebrosidase variants, was subjected to Northern blot analysis. The blot was hybridized with the glucocerebrosidase cDNA as a probe and with rRNA probe as a control for total RNA levels. As evident

from the results presented in Fig. 1, a major 1.8-kb RNA species was detected. This RNA was highly overexpressed, as exemplified by the difference between VTF7-3-infected cells and cells co-infected with vTF7-3 and each of the recombinant viruses. Under the exposure conditions used in this experiment, the endogenous glucocerebrosidase RNA level in cells infected only with vTF7-3 was invisible. Since the same promoter was used in all cases, the changes in the amount of steady-state RNA levels reflect changes in stability of the different RNA molecules. The results strongly indicate that the K157Q allele directed synthesis of an unstable RNA. No significant difference in RNA levels were detected in any of the other tested mutated variants. Levels of the mutant glucocerebrosidase variants To test whether different mutations (D140H, K157Q, E326K D140H+E326K, V394L, N370S, P415R, L444P and R463C) affect glucocerebrosidase protein levels, samples of lysates, prepared from HeLa cells co-transfected with the different mutants or wild-type glucocerebrosidase variants and the VTF-3, containing the same amount of protein, were subjected to Western blot analysis. The blots were reacted with monoclonal anti-glucocerebrosidase antibody and polyclonal anti-Erk antibodies as a control for protein levels. The results strongly indicate (Fig. 2) that there are low levels of the K157Q variant, most probably due to its low RNA levels. All the other tested mutants accumulated in infected cells in levels comparable to those of normal glucocerebrosidase. As described before, the 2C7 monoclonal antibody did not recognize the L444P substitution, although this mutant protein is expressed [30]. Therefore the corresponding protein is not visible on the blot. To confirm this observation, we expressed the myctagged L444P glucocerebrosidase variant and followed its presence by immunoprecipitation or Western blotting, using an anti-myc antibody. As evident from the results presented in Figs. 2B and C, myc-tagged L444P glucocerebrosidase accumulated in transfected cells in levels

Fig. 1. Stability of human-mutated glucocerebrosidase RNA variants. (A) HeLa cells were co-infected with vTF7-3 and the different viruses harboring the normal or mutated glucocerebrosidase cDNAs. 18 h after infection, RNA was extracted and electrophoresed through a formaldehyde-agarose gel, blotted and hybridized with 32P-labeled human glucocerebrosidase cDNA. (B) To quantify glucocerebrosidase RNA in the different samples, blots were stripped and rehybridized to human 32P-labeled rRNA cDNA.

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Fig. 2. Recombinant-mutated glucocerebrosidase variant levels. (A) HeLa cells were co-infected with vTF7-3 and the different viruses harboring the normal or mutated glucocerebrosidase cDNAs. 20 h after infection, lysates were prepared. Samples containing the same amounts of protein were electrophoresed through 10% SDS – PAGE and blotted. The corresponding blot was interacted with anti-glucocerebrosidase and anti-erk antibodies. Detection was carried out using horseradish peroxidase conjugated to a specific secondary antibody followed by enhanced chemiluminescence reaction. (B) 48 h after transfection of HeLa cells with normal or mutated myc-tagged glucocerebrosidase variants, lysates were prepared, electrophoresed through 10% SDS – PAGE and blotted. Recombinant glucocerebrosidase level was detected by interacting the blot with anti-myc antibody (C) 48 h after transfection of HeLa cells with wild-type or mutated myc-tagged glucocerebrosidase, cell lysates were prepared and immunoprecipitated with anti-myc antibody. The precipitates were electrophoresed through 10% SDS – PAGE and blotted. The corresponding blot was interacted with anti-myc antibody.

comparable to the N370S or the normal glucocerebrosidase forms. Enzymatic activity of recombinant glucocerebrosidase variants toward four different fluorescent substrates Aiming at characterizing the activity of the different mutant glucocerebrosidase variants and in an attempt to find a substrate that distinguishes between the severity of mutations, we tested their activities toward four fluorescent substrates: 4-MUG, LR-12-GC, Bodipy-12-GC and LR-0PAP-h-glucose (Fig. 3). LR-12-GC and Bodipy-12-GC were synthesized as described elsewhere [32]. The results presented in Fig. 4 clearly indicate that the K157Q protein has a very low activity, of about 10% of normal toward all the substrates. The E326K mutant had reduced activity toward all the substrates (about 25% of normal activity), indicating that this base pair change is a disease-producing mutation. The D140H protein presented a high activity toward all the tested substrates (70 – 80% of normal). Thus, the D140H base pair change is unique in presenting a high enzymatic activity, yet lower than that presented by the normal glucocerebrosidase. The protein carrying the D140H and the E326K mutations showed an activity similar to that presented by the E326K-carrying protein (about 30% of normal). It is worth noting that the activities of the recombinant variants carrying the K157Q, D140H, E326K and the D140H+E326K mutations toward 4-MUG are in accordance with published results by Grace et al. [21].

The enzymatic activity of the R463C mutant protein was moderate (30 –50% of the normal) indicating that, at least under the experimental conditions used, this mutation has a residual activity that may account for its association with type 1 Gaucher disease. The V394L-mutated protein presented a very low enzymatic activity toward all substrates (less than 10% of normal activity), indicating that this is a severe mutation. As expected, the P415R mutation revealed negligible levels of activity toward all substrates, approving that this mutation is very severe. The L444P mutation demonstrated a very low activity toward all tested substrates (about 10% of the normal), showing that this mutation is a severe one, while the N370S mutation had somewhat higher activities of 20– 70% of normal depending on the specific substrate. While its activity toward the non-natural substrates 4-MUG as well as LR-0-PAP-h-glucose was about 20% of normal, the activity toward the 2 other natural substrates (LR-12-GC and Bodipy12-GC) was significantly higher (35% toward Bodipy-12-GC and 71% toward LR-12-GC). Gaucher disease mutations have been divided to lethal (null), severe and mild, according to the disease they produce [33]. It seems that in vitro activity of recombinant glucocerebrosidase variants toward natural fluorescent substrates, particularly LR-12-GC, allows biochemical division of mild mutation to moderate and mild. According to the results presented in Table 1, mutated alleles presenting 25 –50% of normal activity are moderate (like the E326K and the R463C) and mutations that express 50– 75% activity toward the substrate are mild (like the D140H and the

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Fig. 3. Structure of the fluorescent substrates. Chemical composition of the fluorescent substrates used in this study is shown.

Fig. 4. In vitro enzymatic activity of recombinant glucocerebrosidase variants. HeLa cells were co-infected with VTF7-3 and the different recombinant viruses. 20 h later, cell lysates were prepared and samples containing 2 and 4 Ag protein were tested for glucocerebrosidase activity toward 2.5 nmol of the substrates: LR12-GC, LR-0-PAP-glucose and Bodipy-12-GC. Samples containing 10 and 20 Ag of protein were tested for glucocerebrosidase activity toward 1.5 mM of the artificial substrate 4-MUG. After 1 h at 37-C, fluorescent ceramides were separated from the fluorescent substrates by TLC and their fluorescence was quantified using FLA2000 FUJIFILM fluorimeter (Fuji Film Co., Tokyo, Japan). In the case of 4-MUG, after 1 h incubation at 37-C the amount of fluorescent 4-MU was measured. The results represent the mean + SEM as percentage of the activity of normal protein, measured in 3 experiments with 2 repetitions for each one.

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Table 1 Disease severity and In vitro glucocerebrosidase activity of the tested mutations Mutation

cDNA location

Mutation in Gaucher type

Known mutation severity*

Predicted mutation severity**

Activity toward 4-MUG (percentage of normal)

K157Q D140H E326K D140H+ E326K R463C V394L N370S L444P P415R

c586 AYG c535 GYC c1093 GYA c535 GYC + c1093 GYA c1504 CYT c1297 GYT c1226 AYG c1448 TYC c1361 CYG

Types Types Types Types

Unknown Unknown Unknown Unknown

Severe Mild Moderate Moderate

9.7 73.4 25.1 24.6

T T T T

1.8 17.2 7.2 6

6.48 67.25 26.46 29.34

4.1 13.9 5.3 7.7

10.6 71.3 27.4 28.4

T T T T

9.4 11.8 4.9 5.9

11.8 T 60.3 T 21.4 T 24.3 T

Mild/severe Severe Mild Severe Severe

Moderate Severe Mild Severe Severe

30.8 3.7 21.5 9.2 2.1

T T T T T

12 0.2 12.1 1.5 0.5

32.11 T 5.5 1.52 T 0.8 71.08 T 22.5 13.5 T 1.8 0T0

48.5 4 23.5 12 4.6

T T T T T

7.1 1.4 10.6 11.1 3.6

52.4 7.2 37.5 9.3 2.8

1 1 1 1

and and and and

3 3 3 3

Types 1, 2 and 3 Types 1, 2 and 3 Type 1 Types 1, 2 and 3 Types 2 and 3

Activity toward LR-12-GC (percentage of normal) T T T T

Activity toward Bodipy-12-GC (percentage of normal)

Activity toward LR-0-PAP (percentage of normal)

T T T T T

1.8 14.9 4.8 4.6 20 7 9.3 0.2 3.6

The table summarizes the known and/or predicted severities of each of the tested mutations as well as their activities toward LR-12-GC, LR-0-PAP, Bodipy-12GC and the artificial substrate 4-MUG. The results represent the mean T SEM, as percentage of activity of normal protein, of 3 experiments with 2 repetitions for each one. Concerning the K157Q and the D140H+E326K mutations, they appeared in two Gaucher disease brothers, one is type 1 patient while the other one died at the age of 28 of a non-Gaucher-related neurological disease [13]. Therefore, it was difficult to judge the severity of the mutations according to the phenotypes they are associated with. The D140H mutation alone has not been described in Gaucher patients. The E326K appeared with other base pair changes on the same allele, as discussed in the text. * Severities of the mutations were defined according to Beutler et al. [33]. Homozygosity for the R463C mutation has been described in two nonneuronopathic patients [27,38] and one patient with type 3 Gaucher disease [34]. It also appeared in compound heterozygotes presenting type 2 and type 3 Gaucher disease [34 – 37]. Therefore it is a mild/severe mutation. V394L allele appeared in type 3 patient with the RecTL (severe) allele; therefore, V394L is a severe mutation [16]. P415R is a severe mutation that appeared in a type 2 patient with the L444P allele [19]. ** Severity according to the present study.

N370S mutations). Mutant alleles with less than 25% activity toward LR-12-GC (like the K157Q, P415R, L444P and the V394L) are severe.

Discussion In this study we aimed at evaluating different mutated alleles of the glucocerebrosidase gene for their defective biochemical properties such as RNA stability, protein level and in vitro enzymatic activity toward various substrates using the vaccinia-derived expression system. We also searched for a fluorescent substrate that will enable us to correlate between the activity and the severity of the different mutations. Our data indicated that the K157Q amino acid substitution is a severe mutation, leading to RNA instability, resulting in a low protein level and therefore, a low enzymatic activity, as measured in vitro using four different substrates. The D140H base pair change, however, exhibited normal levels of protein and RNA stability and a high activity toward all four tested substrates. Its activity was even higher than that presented by the mild N370S mutation. This finding led us to conclude that the D140H is a mild mutation. The E326K mutation did not affect RNA stability or protein level but led to a decreased activity of about 21 – 27% of normal, as measured in vitro with any of the tested substrates. However another publication [26] argued that the E326K base change might present a polymorphism and not a

disease-producing mutation. This argument was based on several observations: (a) The E326K mutation was never found as an only mutation on a Gaucher associated allele. (b) Of 310 alleles screened from patients with Gaucher disease the E326K mutation was detected in four alleles (1.3%). (c) Screening for the E326K mutation among normal controls from a random population revealed that three alleles among 316 screened (0.9%) carried the E326K base pair change. In contrast to this observation, Grace et al. [21] demonstrated that the E326K substitution is a disease-producing mutation. It was found as a biallelic change in conjunction with the L444P mutation. When expressed in sf-9 cells using baculovirus as a vector, the mutant protein carrying the E326K substitution had 31% of normal activity toward 4MUG. The occurrence of the E326K mutation in combination with another mutation on the same allele in Gaucher patients is still an unresolved puzzle. The protein carrying the D140H and the E326K mutations presented some activity toward all the tested substrates (about 25% of normal) in a very similar pattern to the E326K mutation, implying that the E326K lowers the activity of the D140H carrying protein and therefore is a disease-producing mutation. The R463C mutation did not change RNA stability or protein level and presented an activity of about 30 –50% of normal protein toward the tested substrates. Patients homozygous for this mutation presented severe type I Gaucher disease [27,38], arguing that this mutation, like the N370S mutation, does not lead to development of neurological signs. However, there was a homozygous patient with type 3 Gaucher disease [34]. Also, it was found in

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compound heterozygotes with type 2 and type 3 Gaucher disease [34 –37]. The V394L mutation showed a significant decrease in the activity toward all the tested substrates, which was less than 10% of normal. This mutation has been documented among patients with types 1, 2 and 3 Gaucher disease. This mutation has not been found in homozygosity, proposing the possibility that it is homozygous lethal. A previous report documented a large decrease in catalytic activity and instability of this protein in the lysosomal pH as well [28]. It seems that the activity of the different mutated glucocerebrosidase variants toward the artificial substrate 4-MUG permit one to divide them into two groups: (1) Classical mutations that present a very low activity toward the artificial substrate, including mild mutations, like the 370S. (2) Base pair changes that modify the normal activity but are not classical mutations and present at least 50% of normal, wild-type, activity. On the other hand, the in vitro activity of different recombinant glucocerebrosidase variants toward the natural fluorescent substrates, particularly the LR-12-GC substrate, showed some variability. It seems that the mutations defined as mild by Beutler et al. [33] can be subdivided into moderate and mild on the basis of the in vitro activity of the recombinant proteins toward the substrate LR-12-GC. Alleles presenting 25 –50% of normal activity are moderate. Mutations that present 50 – 75% activity toward LR-12-GC are mild mutations. To summarize, we characterized several Gaucher-associated mutations. We also showed that LR-12-GC is a reliable substrate for differentiating between severe, moderate and mild recombinant mutant glucocerebrosidase variants. Acknowledgments This work was supported by a grant from Genzyme Corp. USA to M.H. and the Israel Science Foundation (Grant no. 648/02, to M.H and Grant no 607/02 to S.G. and A.D). References [1] R.O. Brady, J.N. Kafner, D. Shapiro, Metabolism of glucocerebrosidase II. Evidence of an enzymatic deficiency in Gaucher’s disease, Biochem. Biophys. Res. Commun. 18 (2) (1965) 221 – 225. [2] E. Beutler, G.A. Grabowski, Gaucher disease, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Diseases, vol. 2, McGraw-Hill, New York, 1995, pp. 2641 – 2663. [3] E. Beutler, Gaucher disease, a paradigm for single gene defects, Experientia 51 (1995) 196 – 197. [4] G.A. Grabowski, S. Gatt, M. Horowitz, Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease, Crit. Rev. Biochem. Mol. Biol. 25 (1990) 385 – 414. [5] E. Beutler, T. Gelbart, Glucocerebrosidase (Gaucher disease), Hum. Mut. 8 (1996) 207 – 213.

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