Human glucocerebrosidase: heterologous expression of active site ...

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Jean-Paul Mornon2 and Pierre Lehn1. INSERM U 458, Hôpital Robert Debré, 48 Bd Sérurier, .... Aerts et al., 1986). It is generally agreed that the relationships.
Glycobiology vol. 10 no. 11 pp. 1217–1224, 2000

Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells

Sylvie Fabrega, Patrick Durand2, Patrice Codogno3, Chantal Bauvy3, Claudine Delomenie, Bernard Henrissat4, Brian M.Martin5, Cindy McKinney5, Edward I.Ginns5, Jean-Paul Mornon2 and Pierre Lehn1 INSERM U 458, Hôpital Robert Debré, 48 Bd Sérurier, 75019 Paris, France, 2Systèmes Moléculaires et Biologie Structurale, Laboratoire de MinéralogieCristallographie, CNRS UMR 7590, Universités Paris VI-Paris VII, T16, case 115, 4 place Jussieu, F-75252 Paris Cedex 5, France, 3INSERM U 504, 16 Av Paul Vaillant-Couturier, 94807 Villejuif Cedex, France, 4Architecture et Fonctions des Macromolécules Biologiques, CNRS UPR 9039, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France, and 5Clinical Neuroscience Branch, National Institute of Mental Health, Building 49, 49 Convent Drive, MSC 4405, Bethesda, MD 20892, USA Received on April 18, 2000; revised on June 23, 2000; accepted on June 26, 2000

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/ base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase. Key words: active site residues/catalytic machinery/Gaucher disease/glycoside hydrolases/human glucocerebrosidase/ lysosomal enzymes/site-directed mutagenesis

1To

whom correspondence should be addressed

© 2000 Oxford University Press

Introduction Glucocerebrosidase (acid β-glucosidase, glucosylceramidase, EC.3.2.1.45) is a lysosomal glycoside hydrolase which cleaves the β-glucosidic linkage of glucosylceramide, a normal intermediate in glycolipid catabolism (Barranger and Ginns, 1989; Beutler and Grabowski, 1995). The mature human protein is a membrane associated glycoprotein which is composed of 497 amino acids and four N-linked oligosaccharide chains which are mainly of the “complex-type,” mature human placental glucocerebrosidase retaining at least one highmannose glycan (Barranger and Ginns, 1989; Takasaki et al., 1984). The crucial role of glucocerebrosidase (GCase) has been established by targeted disruption of the GCase gene in mice (Tybulewicz et al., 1992). Deficiency of GCase activity in man is responsible for Gaucher disease (Barranger and Ginns, 1989). The majority of gene mutations in Gaucher disease are missense mutations that result in the synthesis of GCases with decreased catalytic function and/or stability. As Gaucher disease is the most prevalent lysosomal storage disorder, it has been a model for the development of experimental therapies such as enzyme replacement and gene therapy (Barton et al., 1991; Dunbar et al., 1998). Glycoside hydrolases (EC.3.2.1–3.2.3) are a widespread group of enzymes which function by using one of two general mechanisms leading to either overall retention or inversion of the anomeric configuration at the hydrolysis site (Davies and Henrissat, 1995; Withers and Aebersold, 1995). In both mechanisms, acid/base catalysis requires a pair of carboxylic acid groups and therefore involves two critical amino acid residues. “Retaining” enzymes, such as GCase, function through a two-step mechanism involving a covalent glycosideenzyme intermediate (Withers and Aebersold, 1995). In this double-displacement reaction, a critical active site residue functions as a nucleophile to form the intermediate, whereas the other (acid/base catalyst) acts as a general acid catalyst during glycosylation and then as a general base during deglycosylation. These two critical amino acid residues are situated on the two opposites sides of the glycosidic bond and are separated by a distance of ∼5.5 Å. Various strategies, including sequence alignment, 3D structure analysis, and labeling techniques, have been used for the identification of the critical residues in the active site of glycosidases. Interestingly, active site labeling with mechanismbased inhibitors (2-deoxy-2-fluoro glycosides) has enabled Withers and co-workers to identify the catalytic nucleophile in several glycosidases (Withers and Aebersold, 1995). In the case of human GCase, the nucleophilic amino acid residue was conclusively identified as Glu 340 (Miao et al., 1994). 1217

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Sequence alignment strategies have permitted classification of glycoside hydrolases into families on the basis of amino acid sequence similarities and mechanistic considerations (Henrissat, 1991). In a recent update, a group of families has been named clan GH-A; this group is currently composed of families 1, 2, 5, 10, 17, 26, 30, 35, 39, 42, 51, and 53 (Henrissat and Bairoch, 1996). Clan GH-A enzymes hydrolyze the glycosidic bond with net retention of the anomeric configuration. Human GCase is a member of clan GH-A as it does belong to family 30 of glycoside hydrolases. In order to detect folding similarities, we recently used a bioinformatics approach to analyze the protein sequences of clan GH-A available in databanks (Henrissat et al., 1995; Durand et al., 1997, 2000). Basically, we collected information concerning the known 3D structures from families of clan GH-A and used the 2D Hydrophobic Cluster Analysis (HCA) method to investigate whether the common features observed in the 3D structures were conserved for the entire clan GH-A (Callebaut et al., 1997). Our results showed that all the proteins of clan GH-A likely share a similar catalytic domain consisting of a (β/α)8 barrel with the catalytic acid/base and nucleophile residues located at the C-terminal ends of strands β4 and β7, respectively. In the case of human GCase, Glu 235 (amino acid numbering as in the mature protein) was predicted to be the putative acid/base catalyst; in addition, our analysis located the nucleophile at Glu 340, in agreement with the previous study of Miao et al. (1994). In the present work, we performed site-directed mutagenesis to obtain experimental evidence supporting our computerbased predictions concerning the role of the aforementioned glutamic acid residues in the catalytic site of human GCase. Thus, Glu235Ala (E235A) and Glu340Ala (E340A) mutants were constructed and expressed in GCase-deficient murine null cells to allow us to examine the effect on enzyme activity, processing and sorting to lysosomes, of replacing the presumed critical glutamic acid residues with alanine residues whose methyl side chains are unable to participate in the enzymatic reaction. The results reported herein clearly indicate that Glu235 plays a crucial role in the catalytic machinery of human GCase (and may indeed be the acid/base catalyst) and provide additional support to the identification of Glu340 as the active site nucleophile. Results and discussion Expression of wild-type and mutant human GCases in murine GCase-deficient GSK cells Heterologous expression of human wild-type (wt) and mutant GCases in murine GCase-deficient GSK cells provided a convenient system to study the consequences of replacing the presumed critical active site glutamic acid residues 235 and 340 with alanine residues. The GSK cells were derived from brain tissue explants of null (GCase knock-out) mice (Tybulewicz et al., 1992) and are probably of fibroblast origin as they express vimentin but not the glial fibrillary acidic protein (data not shown). We used recombinant retroviruses to transfer the normal and mutant human GCase cDNAs into GSK cells. GCase cDNA mutants were generated and recombinant retroviruses were produced as indicated in Materials and methods. Figure 1 1218

Fig. 1. Structure of retroviral vectors. The mutant human GCase cDNAs were inserted between the NcoI and BamHI sites of the MFG backbone. MFG vectors, which retain the viral splice donor (SD) and splice acceptor (SA) sites, use the Moloney murine leukemia virus 5′ long terminal repeat (5′ LTR) to generate a spliced mRNA responsible for expression of the inserted sequence. Codon substitutions corresponding to E235A and E340A mutants are indicated.

shows a diagram of the MFG proviruses with the codon substitution corresponding to the E235A and E340A mutants. We used retroviral vectors as the GSK cells were relatively resistant to lipofection (data not shown). In addition, retroviruses have already been used to transfer the human GCase cDNA into Gaucher fibroblasts and hematopoietic cells (Choudary et al., 1986; Dunbar et al., 1998). MFG recombinant retroviruses can yield stable and high-level expression of the transgene via a molecular mechanism involving augmented levels of spliced RNA (Krall et al., 1996). Of note is that the use of retroviral transfer permitted normalization of the level of immunoreactive human GCase protein in GSK cells transduced with the different constructs by adjusting the number of incubations of the cells with the respective viral stocks. Expression of wt and mutant proteins in GSK cells was evaluated by Western blotting using the R386 rabbit polyclonal anti-human GCase antibody (Figure 2). Cross-reacting immunological material (CRIM) was detected in transduced GSK cells (Figure 2, lanes 1–3) whereas no immunoreactive material was observed in untransduced cells (Figure 2, lane 4). Most importantly, the same three-banded immunoblot pattern was observed in GSK cells transduced with the different retroviruses (Figure 2, lanes 1–3) indicating that the same steadystate molecular species (as judged by their apparent molecular mass) were produced in all cases. In addition, the apparent molecular mass values of the three CRIM forms were in good agreement with those previously reported (59 kDa, 63 kDa, and 66 kDa) from Western blotting and pulse chase analyses of human cells (Ginns et al., 1982; Erickson et al., 1985; Fabbro et al., 1987; Jonsson et al., 1987; Willemsen et al., 1987); this is also supported by the immunoblot pattern observed with normal human fibroblasts (Figure 2, lane 5). Of note, a typical three-banded pattern was also observed when using different extraction conditions (data not shown); thus, there was no differential extraction of the three individual CRIM forms. Moreover, this three-banded pattern was not modified when analyzing lysates of GSK cells that had been grown for up to 72 h in the presence of leupeptin (data not shown), indicating

Active site residues of human glucocerebrosidase

Table I. Glucocerebrosidase activity in murine GCase-deficient GSK cells expressing wild-type, E235A, and E340A mutant human GCases Units/mg protein of GCase activitya Controlb

Wild-type

E235A

E340A

4.72 ± 1.1

274.35 ± 4.55

4.18 ± 1.22

4.63 ± 1

Cell lysates from GSK cells transduced with the corresponding recombinant retroviruses were analyzed for GCase activity using the artificial substrate 4-methylumbelliferyl-β-D-glucopyranoside as described in the text. aGCase activity is expressed as units/mg total protein, one unit being the amount of activity that releases 1 nmol of 4-methylumbelliferone/h (mean ± SD, n = 6). bUntransduced GCase-deficient GSK cells. Fig. 2. Western blot analysis of normal and mutant human GCase expressed in murine GCase-deficient GSK cells. Lysates from GSK cells transduced with recombinant retroviruses encoding wild-type (lane 1), E235A mutant (lane2), or E340A mutant (lane 3) human GCase were subjected to 10% SDS/PAGE electrophoresis, transblotted and incubated with the R386 rabbit polyclonal anti-human GCase antibody. The immunoblot pattern was visualized using the ECL chemiluminescence system. Lane 4, control uninfected GSK cells. Lane 5, normal human fibroblasts. The apparent molecular weights of the three typical human CRIM species are indicated (arrows).

that the immunoblot pattern was not the result of intracellular protein degradation. It should also be stressed that the relative intensity of the three individual CRIM bands was similar in all cases, suggesting that similar levels of correctly processed immunoreactive material and hence the different molecular species were produced in the GSK cells transduced with the different retroviral constructs. Indeed, it has been reported that the three different CRIM forms correspond to biosynthetic species whose molecular mass values vary due to differential glycosylation (Takasaki et al., 1984; Erickson et al., 1985; Aerts et al., 1986). It is generally agreed that the relationships of these forms are as follows: (1) after cleavage of the signal sequence, the unglycosylated 56 kDa polypeptide is core glycosylated yielding a “high mannose” precursor of 63 kDa; (2) this first molecular form is then converted in the Golgi into a 66 kDa intermediate form characterized by a mixed pattern of glycosylation (carbohydrate chains which are intermediates between polymannose and complex type); (3) finally, this intermediate form is further processed by exoglycosidases in the lysosome to the mature form with a molecular mass of 59 kDa and with oligosaccharide chains not terminated by mannose residues (Barranger and Ginns, 1989). Taken together, our results indicate that both wt and mutant human GCases can be stably produced in GSK cells and they also suggest that processing of the mutant proteins is identical to that of the wt protein. Thus, we next undertook to: (1) study the enzymatic activity of the different recombinant proteins expressed in GSK cells and (2) further investigate the glycosylation status and the intracellular sorting of the mutant proteins versus the wt enzyme. Activity of the wild-type enzyme and mutant proteins in GSK cells Activity of the wt and mutant human GCase proteins expressed in GSK cells was evaluated at pH 5.9 using the artificial substrate 4-methylumbelliferyl-β-D-glucopyranoside (4 MUGP). As shown in Table I, there was insignificant activity in

untransduced GSK cells, in good agreement with that previously observed in null mice from which the GSK cells were established (Tybulewicz et al., 1992). Importantly, GCase activity observed in GSK cells expressing either of the mutant proteins (E235A or E340A), was similar to that measured in untransduced GSK cells, whereas a high level of activity was detected in GSK cells expressing the wt protein. The wt human GCase expressed in GSK cells had a Km value of approximately 2 mM, close to that reported for human GCase expressed in Spodoptera frugiperda (Sf9) cells (Km = 3.6 mM) and to that reported for human spleen glucocerebrosidase (Km = 8 mM) (Basu et al., 1984; Martin et al., 1988; Grabowski et al., 1989). In contrast, Km values could not be obtained for the mutant proteins as the activities measured with increasing concentrations of substrate remained similar to the background level found in control GSK cells (data not shown). Taken together, these results indicate that active human GCase can be expressed in murine GSK cells and that both mutant proteins are catalytically inactive. With respect to the E340A mutant, our results are in good agreement with those reported by Miao et al., who showed that the CRIM specific activity of a E340G mutant was 103-fold reduced compared to normal GCase (Miao et al., 1994). These authors also suggested that the small amount of residual activity was likely due to translational misreading. As concerns the E235A mutant, our results indicate that Glu 235 plays a key role in the catalytic activity of human GCase and suggest that Glu 235 may indeed be the acid/base catalyst predicted by our bioinformatics analysis (Henrissat et al., 1995; Durand et al., 1997, 2000). Glycosylation pattern of wild-type and mutant GCase proteins expressed in GSK cells In order to verify that catalytic inactivity of the mutant proteins was not the consequence of incorrect processing, we investigated the glycosylation pattern of the recombinant proteins produced in GSK cells. Indeed, after cleavage of the signal peptide, maturation of human GC involves only carbohydrate modifications (Barranger and Ginns, 1989). Thus, GSK lysates were treated with N-glycanase F or endoglycosidase-H and the resulting changes in the CRIM pattern were examined by Western blotting (Figure 3). N-Glycanase F was used to investigate the presence of N-linked oligosaccharide chains and the size of the protein backbone, as human GCase has four N-linked carbohydrate chains. Identical results were observed for the wt and the 1219

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Subcellular localization of recombinant wt and mutant proteins in GSK cells

Fig. 3. Immunoblot pattern of normal and mutant human GCase after digestion with glycosidases. Lysates from GSK cells expressing wild-type (lanes 2 and 5), E235A mutant (lanes 3 and 6) or E340A mutant (lanes 4 and 7) human GCase protein were subjected to digestion with endoglycosidase-H or N-glycanase and subsequently analyzed by Western blotting. The immunoblot patterns from endoglycosidase-H and N-glycanase digested samples are shown in lanes 2–4 and 5–7, respectively. Lane 1, control undigested lysate from GSK cells expressing wt human GCase.

mutant proteins as shown in Figure 3. After complete deglycosylation with N-glycanase F, all the CRIM forms detected in undigested lysates from transduced GSK cells (Figure 3, lane 1) were reduced to a single CRIM form of 56 kDa, in good agreement with the calculated molecular mass of the nascent protein (Figure 3, lanes 5–7). These data are consistent with the results reported for human GCase extracted from placenta or expressed in Sf9 cells (Erickson et al., 1985; Martin et al., 1988; Grabowski et al., 1989). Deglycosylation studies with endoglycosidase-H also demonstrated similar results for the wt and the mutant proteins. As shown in Figure 3 (lanes 2–4), the immature (63 kDa and 66 kDa) CRIM forms were sensitive to endoglycosidase-H yielding a molecular species of approximately 56 kDa, a molecular mass similar to that of the nascent polypeptide. In contrast, the mature 59 kDa form was not clearly affected by endoglycosidase-H treatment. These results are consistent with previous analyses showing the progressive loss of endoglycosidase-H sensitivity during maturation of human GCase, as there is conversion of “high mannose” oligosaccharides into “complex type” carbohydrate chains; accordingly, endoglycosidase-H sensitivity of the 66 kDa species indicates that high-mannosyl oligosaccharides are still clearly present in this intermediate form (Barranger and Ginns, 1989). Importantly, this also demonstrates that both the wt and the mutant GCase precursors do migrate from the endoplasmic reticulum to the Golgi apparatus where “complex type” glycosylation occurs and strongly suggests that they are properly folded. In summary, these results indicate that processing of wt human GCase in GSK cells is similar to that in human tissues. The data also show that both E235A and E340A mutant proteins are correctly processed in GSK cells and because their folding and glycosylation are likely to be correct, their catalytic inactivity is unlikely the result of incorrect folding/processing. 1220

Since the aforementioned biosynthetic glycosylation steps occur at different locations in the cell (Barranger and Ginns, 1989), we also studied the intracellular localization of the recombinant proteins in order to verify that they were indeed correctly sorted. Western blotting was performed on subcellular components obtained by fractionation of post-nuclear supernatants on a Percoll density gradient; of note, a similar fractionation procedure has previously allowed us to show that differentiation-dependent autophagy controls the fate of newly synthesized N-linked glycoproteins in colon cancer cells (Houri et al., 1995). Eighteen fractions were collected from the top to the bottom of the gradient. A typical gradient density profile is shown in Figure 4A. It is generally agreed that β-1,4-galactosyltransferase and β-hexosaminidase enzyme activities are markers of endoplasmic reticulum/Golgi and lysosomal fractions, respectively. As shown in Figure 4B, a high level of β-hexosaminidase activity was found in the high-density fractions which typically have very low β-1,4-galactosyltransferase activity, a combination of features identifying these fractions as lysosomal. Western blot results for fractions 11–18 are shown in Figures 4C–E and, again, the data are identical for the wt and mutant proteins. Interestingly, although the immature (63 kDa and 66 kDa) CRIM forms were detected in all these fractions, the mature 59 kDa molecular species was only detected in the last fractions of the gradient; quantification of the 59 kDa signal by phosphorimager analysis showed that fractions 14–18 contained 97%, 92%, and 85% of the 59 kDa mature molecular species of the wt, E235A mutant and E340A mutant protein, respectively. These data clearly indicate that, in GSK cells, the mature form of wt and mutant proteins is correctly sorted to the lysosome. On the other hand, as concerns the two CRIM species characterized by molecular weights lower than 59 kDa which were occasionally detected in GSK cells expressing wt human GCase, we observed that they did not specifically accumulate in the lysosomal fractions, a finding further suggesting that they were degraded forms (data not shown). Next, we confirmed that correct lysosomal targeting occurs for the wt and mutant proteins by confocal immunofluorescence microscopy. The lamp-1 protein was used as a lysosomal marker and human GCase protein was detected using the 8E4 mouse monoclonal antibody (Barneveld et al., 1983). As shown in Figure 5, a similar vesicular pattern was observed for wt and mutant proteins and all three recombinant proteins were found to accumulate in the same intracellular compartments as the lamp-1 protein. Negative controls were performed by omitting the respective primary antibodies (data not shown). Taken together, these results demonstrate that the mutant as well as the wt proteins are correctly sorted in GSK cells. Conclusions and perspectives In summary, active, correctly processed and sorted human GCase can be expressed in murine GSK cells established from GCase knock-out mice representing an animal model of type 2 Gaucher disease. Our results also demonstrate that E235A and E340A mutants can be expressed in GSK cells, but that both mutants are catalytically inactive although they are correctly processed and sorted to the lysosomes, a finding suggesting

Active site residues of human glucocerebrosidase

With respect to the catalytic role of Glu 340, our results are consistent with those of Miao et al. (1994) obtained via active site labeling. In addition, we demonstrate that the inactive E340A mutant protein is correctly processed and sorted. Additional studies will permit further insights into the role of Glu 235. This studies may involve detailed kinetic analysis of purified mutants as has been done for other glycoside hydrolases (MacLeod et al., 1994; Wang et al., 1995; Viladot et al., 1998). It may also be possible to use a direct labeling approach analogous to that recently described by Howard and Withers in the case of α-glucosidase from Saccharomyces cerevisiae (Howard and Withers, 1998). Ultimately, 3D structure determination would reveal the full details of the catalytic machinery of human GCase. Interestingly, no mutations involving Glu 235 or Glu 340 have so far been described in Gaucher patients. According to our results, it is indeed possible that mutations of these highly critical active site residues would lead to a clinical status which is incompatible with life, a hypothesis also in good agreement with the observation that (1) GCase knock-out mice die within 24 h after birth and (2) homozygosity for GCase null mutation results in prenatal lethality in humans (Tybulewicz et al., 1992; Tayebi et al., 1997). In a broader perspective, it should be stressed that similar approaches may be successfully applied to other glycoside hydrolases, especially the other human lysosomal enzymes for which we have previously also identified active-site motifs. In the case of human β-glucuronidase, recent studies of active site mutants support our predictions based on HCA implicating Glu 451 and Glu 540 as the acid-base/nucleophile pair (Islam et al., 1999). Finally, our work illustrates the usefulness of bioinformatics analyses for the identification of critical amino acid residues in the context of a rapidly increasing number of coding sequences available through genome sequencing. Materials and methods Materials

Fig. 4. Subcellular localization of normal and mutant human GCase proteins in GSK cells. Postnuclear supernatants from GSK cells transduced with the different recombinant retroviruses were prepared and subfractionated by Percoll gradient centrifugation. Eighteen fractions were collected from the top to the bottom of the gradient. (A) shows a typical gradient density profile. Representative β-hexosaminidase (squares) and β-1,4-galactosyltransferase (diamonds) profiles are indicated in panel B. Proteins from each gradient fraction were precipitated and the samples were subjected to Western blotting. (C–E) show the immunoblot patterns for fractions 11–18 collected when analyzing postnuclear supernatants from GSK cells expressing wild-type, E235A, and E340A mutant human GCase, respectively.

that their catalytic inactivity is not related to incorrect folding/ processing. Thus, these studies clearly indicate that Glu 235 and Glu 340 play essential roles in the active site of human GCase and are in good agreement with our previous HCA-based predictions identifying Glu 235 as the putative acid/base catalyst and Glu 340 as the key nucleophilic residue (Henrissat et al., 1995; Durand et al., 1997, 2000).

The wild-type (wt) human GCase cDNA has been previously described (Ginns et al., 1984; Tsuji et al., 1986). Plasmids pGEM-5Zf(+) and pCI-Neo were from Promega. Oligonucleotides for mutagenesis were purchased from Genosys. The T7 sequencing kit was from Pharmacia. Nitrocellulose Hybond ECL membrane and ECL chemiluminescence system were from Amersham. The artificial substrates 4-methylumbelliferyl-β-D-glucopyranoside (4MUGP) and p-nitrophenyl N-acetyl-β-D-glucosaminide were obtained from Sigma. The bicinchoninic acid (BCA) protein assay kit was from Pierce. Endoglycosidase H and N-glycanase were purchased from Oxford GlycoSciences. Fetal calf serum was from ICN, whereas newborn calf serum was from Hyclone. Cationic liposomes BGTC/DOPE were generously supplied by J.P.Vigneron (Collège de France, Paris). G418 was obtained from Gibco and polybrene was from Sigma. The R386 rabbit polyclonal and 8E4 mouse monoclonal anti-human GCase antibodies have been previously described (Barneveld et al., 1983). Polyclonal antibody 126 directed against lamp-1 protein was a gift from S.Meresse (CIML, Marseilles, France). Fluorescent secondary antibodies were purchased from Jackson Immuno Research Laboratories. 1221

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Fig. 5. Immunofluorescence staining of GSK cells expressing wild-type (wt), E235A mutant and E340A mutant human GCase. GSK cells transduced with the corresponding recombinant retroviral vectors were fixed and double stained with an anti-human GCase antibody and an anti-lamp-1 antibody; secondary antibodies conjugated to fluorescein and Texas red were used to reveal the GCase and lamp-1 staining patterns, respectively. Finally, cells were observed by confocal fluorescence microscopy. Anti-Gcase, GCase staining. Anti-lamp 1, lamp-1 staining. Merge, double staining showing intracellular colocalization of the different recombinant human GCase proteins and lamp-1, a lysosomal marker.

Cells and culture conditions GSK cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml Penicillin. CRIP packaging cells (Danos and Mulligan, 1988) were grown under similar conditions, except that fetal calf serum was replaced by newborn calf serum. Normal human fibroblasts were obtained from C.Caillaud (ICGM, Paris, France). Site-directed mutagenesis A 987 bp NcoI–NsiI fragment of wt human GCase cDNA encompassing the codons for E235 and E340 was subcloned into pGEM-5Zf(+) and the resulting plasmid was used for the generation of the mutants by the Kunkel’s method (Kunkel, 1985). The mutant oligonucleotides (sens) were as follows: (1) 5′-GCTGAAAATGCGCCTTCTGCTGG-3′ for construction of the E235A mutant by an A to C substitution, (2) 5′-CTTTGCCTCAGCTGCCTGTGTGGG-3′ for construction of the E340A mutant by modifying the GAG triplet coding for Glu 340 into a GCT triplet coding for Ala (in order to create a PvuII restriction site facilitating screening). Both mutant fragments were verified to exclude undesired mutations by DNA sequencing by the dideoxy chain termination method using the T7 sequencing kit. Retroviral vectors MFG retroviral vectors carrying the mutant cDNAs were constructed using standard molecular biology techniques. The MFG backbone and the MGC vector which contains the wt human GCase cDNA inserted between the NcoI and BamHI cloning sites of the MFG backbone have been previously 1222

described (Dranoff et al., 1993; Krall et al., 1996). The same strategy was followed for construction of the MFG vectors expressing the E235A and E340A mutants. Briefly, following coupling of a 481 bp NcoI fragment corresponding to the 5′ end of the wt cDNA to the mutant NcoI–NsiI fragment in pGEM-5Zf(+), a 1331 bp HindIII–NsiI fragment was excised and swapped with the wt fragment in the MGC vector. In addition, we also used a retroviral construct (kindly provided by J.M.Heard, Institut Pasteur, Paris) where the wt human GCase cDNA is driven by an internal phosphoglycerate kinase promoter. Recombinant retroviruses were generated by transfecting the respective retroviral construct into CRIP packaging cells using BGTC/ DOPE cationic liposomes as previously described (Vigneron et al., 1996). Because a pCI-Neo plasmid was co-transfected into the packaging cells, G418 could be used for the selection of individual producer clones. High titer producer clones were identified via a single infection of GCase-deficient GSK cells (in the presence of polybrene at 8 µg/ml) with the corresponding supernatants and estimation of the total amount of human GCase protein expressed by Western blotting as described below. Transduction of GSK cells Viral stocks obtained from high titer producer clones were used for transduction of the different cDNAs into GSK cells. Normalization of the level of expression of immunoreactive human GCase protein in GSK cells transduced with the different plasmids was obtained by adjusting the number of incubations of the cells with the respective viral stocks. Cell lysis and protein determination Confluent GSK cells were harvested and resuspended in extraction buffer (60 mM potassium phosphate, 0.1% Triton

Active site residues of human glucocerebrosidase

X-100). The cell suspension was sonicated at 50W for 30 s on ice (Bioblock apparatus). Following centrifugation (20,000 × g, 5 min, 4°C), the supernatants were recovered for analysis. Total protein concentration was determined by the BCA assay. Glucocerebrosidase activity GCase activity in cell extracts was assayed at pH 5.9 using the fluorogenic synthetic substrate 4MUGP in 50 mM citrate phosphate buffer containing 0.15% Triton X-100 and 0.125% sodium taurocholate (Ginns et al., 1982). Samples of 20 µg total protein containing similar levels of immunoreactive human GCase protein were studied. The amount of 4-methylumbelliferone generated by enzymatic cleavage of 4-MUGP was determined fluorimetrically with a Bioblock spectrometer TD 700. GCase activity was expressed as units/mg total protein, one unit being the amount of activity that releases 1 nmol of 4methylumbelliferone/h.

Immunofluorescence staining Transduced GSK cells grown on glass coverslips were fixed for 15 min in 2% paraformaldehyde, incubated for 10 min with 50 mM ammonium chloride and permeabilized by treatment with 0.075% saponin for 20 min. The cells were then incubated with the 8E4 monoclonal antibody (Barneveld et al., 1983) (used at a 1:100 dilution) and the rabbit polyclonal antibody 126 directed against interspecies-conserved amino acid residues of lamp-1 protein (used at a 1:500 dilution). Cells were next incubated with FITC-conjugated secondary anti-mouse antibody to reveal human GCase protein and with a Texas red– conjugated secondary anti-rabbit antibody to detect the lamp-1 protein. Negative controls were performed by omitting the primary antibodies. Finally, samples were examined by confocal microscopy using a LEICA TCS equipped with DMR inverted microscope and E63/1.4 objective.

Western blot analysis

Acknowledgments

Human GCase proteins expressed in GSK cells transduced with the different retroviral constructs were analyzed by immunoblotting as described previously (Ginns et al., 1982). Briefly, cell extracts (∼10 µg total protein per sample) were subjected to 10% sodium dodecyl sulfate/polyacrylamide (SDS–PAGE) gel electrophoresis under reducing conditions and transblotted to nitrocellulose Hybond ECL membranes. Following a blocking step, the blots were incubated with the R386 rabbit polyclonal antibody used at a 1:1000 dilution. Finally, the blots were incubated with peroxidase-labeled antirabbit antibody and the immunoblot pattern was visualized using the ECL chemiluminescence system (Amersham).

This work was supported by grants from the Association Vaincre les Maladies Lysosomales (VML, Evry, France). Institut Fédératif de Recherche 02 (Cellules Epithéliales) is acknowledged for confocal microscope facilities.

Deglycosylation studies

References

GSK lysates were subjected to endoglycosidase-H and N-glycanase digestions according to the manufacturer’s instructions (Oxford GlycoSciences). Digestions were for 20 h at 37°C. Following endoglycosidase-H and N-glycanase digestions, the samples were analyzed by Western blotting as described above. Subcellular fractionation of transduced GSK cells Transduced GSK cells were submitted to subcellular fractionation on Percoll gradients by a modification of the method described by Rijnboutt (Houri et al., 1995; Rijnboutt et al., 1992). Confluent cells were harvested by scraping, resuspended in 5 ml of isotonic homogenization buffer (250 mM sucrose, 1 mM EDTA, 20mM HEPES, 1 mM PMSF, and 1 µg/ml of leupeptin) and disrupted on ice with a glass/Teflon homogenizer (15 strokes; Thomas Scientific Type A, Polylabo, Paris, France). Intact cells and nuclei were removed by centrifugation at 300 × g for 10 min, and the postnuclear supernatant was subfractionated on a 30% Percoll, self-generating gradient at 68,000 × g for 35 min in a Beckman Ti-50 rotor. Eighteen fractions were collected from the top to the bottom of the gradient. Determination of β-hexosaminidase activity was as described by Opheim (Opheim and Touster, 1978). Analysis of β-1,4-galactosyltransferase activity was performed radiometrically by the method of Barker et al. (1972). Western blotting of total protein from each gradient fraction was performed as described above.

Abbreviations GCase, glucocerebrosidase; HCA, hydrophobic cluster analysis; wt, wild-type; CRIM, cross-reacting immunological material; 4MUGP, 4-methylumbelliferyl β-D-glucopyranoside.

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