INSERM U 458, Hôpital Robert Debré, 48 Bd Sérurier, 75019 Paris, France, 2Systèmes Moléculaires et Biologie Structurale, Laboratoire de Minéralogie-Cristallographie, 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.
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Abstract |
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Key words: active site residues/catalytic machinery/Gaucher disease/glycoside hydrolases/human glucocerebrosidase/lysosomal enzymes/site-directed mutagenesis
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Introduction |
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Glycoside hydrolases (EC.3.2.13.2.3) 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 glycoside-enzyme 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 mechanism-based 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
).
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 (ß/
)8 barrel with the catalytic acid/base and nucleophile residues located at the C-terminal ends of strands ß4 and ß7, 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 computer-based 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.
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Results and discussion |
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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 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.
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Deglycosylation studies with endoglycosidase-H also demonstrated similar results for the wt and the mutant proteins. As shown in Figure 3 (lanes 24), 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.
Subcellular localization of recombinant wt and mutant proteins in GSK cells
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 ß-1,4-galactosyltransferase and ß-hexosaminidase enzyme activities are markers of endoplasmic reticulum/Golgi and lysosomal fractions, respectively. As shown in Figure 4B, a high level of ß-hexosaminidase activity was found in the high-density fractions which typically have very low ß-1,4-galactosyltransferase activity, a combination of features identifying these fractions as lysosomal. Western blot results for fractions 1118 are shown in Figures 4CE 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 1418 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).
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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
-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 ß-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.
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Materials and methods |
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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.
Cells and culture conditions
GSK cells were grown in Dulbeccos Modified Eagles 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 NcoINsiI 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 Kunkels 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 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 NcoINsiI fragment in pGEM-5Zf(+), a 1331 bp HindIIINsiI 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 X-100). The cell suspension was sonicated at 50W for 30 s on ice (Bioblock apparatus). Following centrifugation (20,000 x 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 4-methylumbelliferone/h.
Western blot analysis
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 (SDSPAGE) 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 anti-rabbit antibody and the immunoblot pattern was visualized using the ECL chemiluminescence system (Amersham).
Deglycosylation studies
GSK lysates were subjected to endoglycosidase-H and N-glycanase digestions according to the manufacturers 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 x g for 10 min, and the postnuclear supernatant was subfractionated on a 30% Percoll, self-generating gradient at 68,000 x 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 ß-hexosaminidase activity was as described by Opheim (Opheim and Touster, 1978
). Analysis of ß-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.
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 redconjugated 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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