3Lysosomal Diseases Research Unit, Department of Chemical Pathology, Womens and Childrens Hospital, King William Road, North Adelaide, SA 5006, Australia; 4INSERM U458, Hopital Robert Debré, 48 Bd Serurier, 75019 Paris, France; 5Systèmes Moléculaires et Biologie Structurale, Laboratoire de Minéralogie-Cristallographie, CNRS UMR 7590, Universités Paris VI-Paris VII, T16, case 115, 4 place Jussieu, 75252 Paris Cedex 5, France; 6Federal University of Rio Grande do Sul, Porto Alegre, Brazil; and 7Architecture et Fonction des Macromolécules Biologiques, CNRS UMR 6098, Universités dAix-Marseille I and II, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Received on March 1, 2001; revised on May 30, 2001; accepted on June 1, 2001..
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Abstract |
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Key words: active site residues/catalytic machinery/ -L-iduronidase/Hurler syndrome/mucopolysaccharidosis I
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Introduction |
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MPS I patients present with a wide spectrum of clinical phenotypes, ranging from very severe for Hurler syndrome to nearly normal in Scheie syndrome (Neufeld and Muenzer, 1995). About 57 IDUA gene mutations have been reported (Scott et al., 1995
; Bunge et al., 1995
; Yamagishi et al., 1996
; Krawczak and Cooper, 1997
). The most common mutant alleles in Caucasians, involve truncation of the
-L-iduronidase protein and include the W402X and Q70X "null alleles," which produce no detectable
-L-iduronidase protein (Scott et al., 1992a
,b; Ashton et al., 1992
). Together, these nonsense mutations account for approximately 60% of disease alleles in Caucasians and in the homozygous condition are associated with the most severe form of MPS I patient clinical phenotype. R89Q (Scott et al., 1993
) is one of the most common mutant alleles in Japanese MPS I patients (Yamagishi et al., 1996
) and is associated with a clinical phenotype of intermediate severity (Scott et al., 1993
, 1995). To date, no active site mutations have been reported for
-L-iduronidase, although one mutation (D349N) has been shown to result in a high level of catalytically inactive protein (Brooks et al., 1992
).
Glycoside hydrolases (EC. 3.2.13.2.3) are a large group of enzymes that function by two distinct catalytic mechanisms, involving either overall retention or inversion of configuration at the anomeric carbon of the substrate (Koshland, 1953; Sinnott, 1990
; McCarter and Withers, 1994
; Davies and Henrissat, 1995
). In both mechanisms, catalysis requires a pair of carboxylic acid groups, typically provided by either aspartic acid or glutamic acid residues. In the "retaining" enzymes like
-L-iduronidase, the critical residues are organized on either side of the glycosidic bond and are separated by a distance of
5.5Å (McCarter and Withers, 1994
). These two critical amino acid residues are involved in a two-step catalysis. In the first step, one of the residues performs a nucleophilic attack at the sugar anomeric carbon, while the other residue functions as an acid/base and assists aglycon departure by protonation of the glycosidic oxygen. The result of this first step is the formation of a covalent glycosyl-enzyme intermediate. In the second step, the deprotonated acid/base residue abstracts a proton from a water molecule, which attacks the glycosyl-enzyme to release a sugar with a stereochemistry identical to that of the substrate (Koshland, 1953
; Sinnott, 1990
; McCarter and Withers, 1994
; Davies and Henrissat, 1995
).
A number of strategies, including sequence alignment, 3D structure analysis, labeling techniques and combined bioinformatics approaches have been used to identify active site residues in glycosidases (Withers and Aebersold, 1995; Henrissat et al., 1995
; Durand et al., 1997
, 2000; Callebaut et al., 1997
). The Cellulomonas fimi ß-1,4-glycanase Cex was the first retaining glycoside hydrolase for which the 3D structure of a catalytically competent glycosyl-enzyme intermediate was determined (White et al., 1996
). Based on amino acid similarities, glycoside hydrolases have been classified into a number of families (these families can be found on a continuously updated server at http://afmb.cnrs-mrs.fr/
pedro/CAZY/db.html). A higher hierarchical level of classification was introduced, the "clans," which groups families displaying the same fold and catalytic machinery (Henrissat and Bairoch, 1996
; Henrissat and Davies, 1997
). The largest of these clans, clan GH-A, contains many of the glycosidases responsible for lysosomal storage disorders, including ß-glucuronidase (Sly syndrome), ß-glucocerebrosidase (Gaucher disease), ß-galactosidase (GM-1 gangliosidosis, Morquio type B syndrome), ß-mannosidase (mannosidosis), and
-L-iduronidase (Hurler and Scheie syndromes) (Durand et al., 2000
). All clan GH-A members share the same retaining mechanism and a common 3D structure, a (ß/
)8 barrel, in which the acid/base and the nucleophilic residues are located at the C-terminal end of strands ß4 and ß7, respectively. Sequence comparisons and computer modeling have previously allowed us to predict that E182 and E299 are the likely acid/base and nucleophilic residues of
-L-iduronidase (Henrissat et al., 1995
; Durand et al., 1997
, 2000). In addition, Arg 89 was shown to be located at the C-terminal end of strand ß2 and was hypothesized to play a role in activation of the nucleophile, a prediction in agreement with the involvement of a R89Q mutation in MPS I disease (Durand et al., 1997
).
The aim of the present work was to generate experimental data supporting that residues E182 and E299 play a critical role in the catalytic machinery of human -L-iduronidase. Thus, we have constructed the E182A and E299A mutants (as a Glu to Ala mutation is isosteric) as well as an E182K mutant (as a patient with a severe MPS I clinical phenotype has been identified with this allele). In these mutants, the putative catalytic residues have been replaced by amino acid residues whose side chains are unable to participate in the enzymatic reaction. We herein report the results of expression studies of these mutant human
-L-iduronidase proteins in Chinese hamster ovary (CHO)K1 cells, which confirm our bioinformatics predictions.
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Results and discussion |
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To characterize the expression of wild-type and mutant -L-iduronidase protein from CHO-K1 cells, cell extracts and media were immunoquantified using a polyclonal capture step and an ID1A monoclonal antibody detection step (Table I). In this assay, no detectable
-L-iduronidase reactivity was observed for untransfected CHO-K1 cell extracts, demonstrating that ID1A had no immunoreactivity with endogenous CHO-K1
-L-iduronidase. In contrast, the CHO-K1 cell line transfected with the wild-type construct had human
-L-iduronidase protein both in the cell extract and secreted into the culture medium. The E182A and E299A mutants had similar levels of human
-L-iduronidase protein detected in CHO-K1 cell extracts, when compared to the wild-type control. Both E182A and E299A cells also showed human
-L-iduronidase protein in the cell culture media. There was an increase in the level of
-L-iduronidase protein secreted by the E182A cells (and also E182K cells), compared to the wild-type cells (Table I), indicating a possible minor alteration in the efficacy of mannose-6-phosphorylation. Clearly, the ability of the CHO-K1 cell lines transfected with the mutant constructs to produce high amounts of human
-L-iduronidase protein and their capacity to also secrete
-L-iduronidase protein strongly suggested that the mutant proteins could correctly fold and pass the ER quality control system. Of note, despite having the highest level of intracellular
-L-iduronidase protein, the E299A cell line had the lowest level of
-L-iduronidase protein in the cell culture medium.
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Characterization of an MPS I patient with an E182K mutation
An MPS I patient with a severe clinical phenotype was identified as having the genotype W402X/E182K and was selected for further analysis. W402X has previously been identified as a null allele, because no -L-iduronidase protein was detected in MPS I patients with a W402X/W402X genotype (Scott et al., 1992a
; Ashton et al., 1992
). Although a W402X mutant protein may possibly contain the (ß/
)8-barrel catalytic domain (although strand ß8 is yet to be identified), the truncated protein was presumably unstable and failed to fold correctly, being retained and degraded by the ER quality control system. Here, expression studies with the E182K mutant were undertaken to better understanding how this mutation contributes to a severe clinical phenotype. From a mechanistic point of view, the E182K protein should be catalytically inactive, because a lysine residue cannot perform the acid/base catalysis required for glycosidase activity. This was confirmed by the production of a catalytically inactive E182K
-L-iduronidase protein in CHO-K1 cells (Table I), supporting the hypothesis that the E182K mutation would contribute to a severe clinical outcome. Interestingly, the mutation of the glutamic acid to a lysine at position 182 would introduce a net positive charge into the active site of human
-L-iduronidase, but this was not sufficient to reduce the intracellular level of the mutant protein. Most other
-L-iduronidase mutations appear to cause problems with protein folding, evoking ER recognition/degradation of the mutant protein and resulting in low levels of intracellular
-L-iduronidase protein (reviewed in Brooks, 1997
).
Identification of the molecular forms of wild-type and mutant human -L-iduronidase produced in CHO-K1 cells
Cell extracts from the wild-type, E182A, E182K, and E299A human -L-iduronidase-expressing CHO-K1 cells were immunoblotted to define the
-L-iduronidase molecular species produced at steady state (Figure 3). Cell extract from CHO-K1 cells expressing wild-type enzyme demonstrated molecular species of 74 (minor), 69 (major), and 65 (major) kDa by immunoblot analysis (plus molecular forms of 49 and 44 kDa, which were barely visible), while in the culture medium an 81-kDa molecular species was detected. These processed forms were consistent with the extensive processing of
-L-iduronidase, which has been previously reported in human skin fibroblasts (Taylor et al., 1991
; Scott et al., 1991
). Despite not being proteolytically processed, the 81 kDa wild type
-L-iduronidase detected in the culture medium was active (see Table I), indicating that proteolytic processing was not a necessary prerequisite for
-L-iduronidase enzyme activity. Importantly, a similar pattern of processed forms was observed for E182A, E182K, and wild-type
-L-iduronidase. In contrast, the E299A immunoblot pattern was characterized by a major 74-kDa species and only a minor 69 kDa molecular form, a finding that suggests delayed traffic or altered proteolytic processing. Delayed processing/traffic of E299A
-L-iduronidase was also supported by the immunoquantification data above, which showed a high level of intracellular compared to secreted E299A
-L-iduronidase (at steady state). Whereas the traffic and processing of the E299A mutant appeared to be delayed or altered, its culture medium contained the same 81-kDa human
-L-iduronidase species detected in both of the E182 mutants and wild-type culture media. This indicated that the E299A mutant protein eventually reached the culture medium following processing in the Golgi, but was still catalytically inactive (Table I).
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The E299A mutation, which involves the residue proposed to act as the nucleophile in the catalytic mechanism of -L-iduronidase, was also investigated. E299A
-L-iduronidase protein was synthesized and secreted, with a molecular mass similar to that of the wild-type and the two E182 mutants described above. This indicated that the E299A mutant protein could pass the ER/Golgi quality control process. The E299A mutant protein was catalytically inactive, a finding again providing support for our bioinformatics prediction that E299 may be the nucleophilic residue in the catalytic site of
-L-iduronidase (Henrissat et al., 1995
; Durand et al., 1997
, 2000). However, there was evidence of a delayed rate of traffic and secretion, and the E299A mutant protein that reached the lysosomes was not proteolytically processed to the 69/65 species. This may indicate that, unlike other glycosidases, the glutamic acid to alanine change in
-L-iduronidase at position 299 has some minor structural effect on the mutant protein. This suggested that despite folding and subsequent traffic to the correct destination, this minor protein structure modification could impede interaction with processing enzymes.
The glutamic acid residues studied here in human -L-iduronidase are also present in dog and mouse
-L-iduronidase (Stoltzfus et al., 1992
; Clarke et al., 1994
). There are regions of absolute sequence identity around these critical glutamic acid residues for both dog and mouse, compared to human
-L-iduronidase, and despite an altered positional location within the overall sequence, the two residues are exactly the same number of residues apart within all three sequences. This suggests that the location of the two glutamic acid residues is important and is consistent with the organization of these glutamic acid residues in other glycosidases, on either side of the glycosidic bond, with a precise spatial separation (McCarter and Withers, 1994
). Additional studies are planned to investigate the interaction of substrate with the wild-type, E182A, and E299A
-L-iduronidase to confirm the role of these glutamic acid residues. Studies with E182D and E299D mutants, where the key glutamic acid residues are replaced by aspartic acid residues whose side chains also bear a carboxylic acid group, should allow further insight into the structural constraints existing in the catalytic site of human
-L-iduronidase. Finally, it will be important to confirm these conclusions by direct active site labelling (Withers and Aebersold, 1995
) and ultimately by 3D structural analysis.
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Materials and methods |
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Polyvinylchloride plates (96-well, enzyme-linked immunosorbent assay [ELISA] plates) were obtained from Costar. Nitrocellulose membrane and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate were purchased from Bio-Rad. Pansorbin cells (507861), and the -L-iduronidase substrate MUI were from Calbiochem. Ovalbumin and bovine serum albumin (BSA) were from Sigma. G418 was purchased from either Gibco or Sigma. Sheep anti-mouse immunoglobulin, horseradish peroxidaseconjugated sheep anti-mouse immunoglobulin (SAM-Ig) and horseradish peroxidaseconjugated sheep anti-rabbit immunoglobulin were purchased from Silenus Laboratories (a subsidiary of Chemicon).
-L-iduronidase peptide pins were synthesized by Chiron Mimitopes. Fetal calf serum (FCS) for tissue culture, L-glutamine, penicillin, streptomycin, and Dulbeccos Modified Eagles Medium/Hams Nutrient Mixture F-12 Coons Modified were from Life Technologies/Gibco BRL. 35S-methionine radiolabel was purchased from NEN.
Site-directed mutagenesis for construction of mutants E182A and E299A
The wild-type human IDUA cDNA was isolated from plasmid pBS-II-KS+-IDUA by EcoRI digestion, subcloned into pCI-Neo and the resulting plasmid pCI-wt-Neo was used for the generation of mutants, by Kunkels method (Kunkel, 1985). The mutant oligonucleotides (antisense) were as follows: (1) 5'-GTCAAAGTCGTGGTGGTCTGGTGCATTCCACGTC-3' for construction of the E182A mutant by modifying the GAG triplet coding for Glu182 into a GCA triplet coding for Ala to create a BsmI restriction site facilitating screening; (2) 5'-CGGGTCCGCGGCGTCGTTGTAAATG-3' for construction of the E299A mutant by replacing the GAG triplet coding for Glu299 with a GCC triplet coding for Ala to create a SacII restriction site facilitating screening. Both mutant cDNAs were verified to exclude undesired mutations by DNA sequencing by the dideoxy chain termination method.
Generation of CHO-K1 clones expressing wild-type, E182A, and E299A human IDUA proteins
Plasmids pCI-wt-Neo, pCI-E182A-Neo, and pCI-E299A-Neo (where the wild-type and E182A and E299A mutant human IDUA cDNAs, respectively, are under the transcriptional control of the cytomeglovirus promoter) were transfected into CHO cells using BGTC/DOPE cationic liposomes as previously described (Vigneron et al., 1996). Because these plasmids also contain a Neo expression cassette, stably transfected CHO-K1 clones could be generated via G418 selection. CHO-K1 clones characterized by a high copy number of the different plasmid DNAs were identified by Southern blotting and used for analysis of the corresponding human IDUA proteins expressed. The control cell line expressing wild-type human IDUA was also selected via determination of the level of enzyme activity by a MUI assay.
Generation of E182K expression cell line
The E182K missense mutation was engineered into the wild-type cDNA using a quick change site-directed mutagenesis kit (Stratagene). A clone containing the mutagenized IDUA cDNA construct was identified by hybridization with an allele-specific oligonucleatide, sequenced to ensure that no changes other than E182K were introduced and then subcloned into the expression vector pEFNeo (Unger et al., 1994). Large-scale plasmid stocks were prepared using a BRESApure plasmid kit (Bresatec). Ten micrograms of pEFNeoE182K was electroporated into CHO-K1 cells as previously described (Anson et al., 1992
) and selected with 0.75 mg/ml G418. G418-resistant mass-cultures were maintained in medium containing 0.5 mg/ml G418 for at least 2 weeks.
Cell culture
CHO-K1 cells were grown in either Dulbeccos Modified Eagles Medium or Hams Nutrient Mixture F-12 Coons Modified supplemented with 10% FCS and antibiotics. The culture media from confluent 75 cm2 flasks were collected, clarified by centrifugation (200 x g for 10 min at 4°C) and stored sterile at 4°C. For harvesting, the cell layers were washed twice with 10 ml of Dulbeccos phosphate buffered saline (PBS) and the cells released from the culture surface by incubation with 10% (v/v) trypsin versene solution for 2 min at 37°C. The harvested cells were centrifuged at 200 x g for 5 min and the cell pellet washed with an additional 10 ml of PBS and then recentrifuged. The supernatant was removed and the cell pellet used for either organelle fractionation or cell extracts prepared for immunoquantification, immunobinding assay, and western blotting.
Monoclonal antibody epitope mapping
Peptide pin technology (Chiron Mimitopes) was used to determine the epitope reactivity of the monoclonal antibody ID1A (Ashton et al., 1992) with linear peptide sequences of
-L-iduronidase, as previously described with another lysosomal protein (Turner et al., 1999
). Briefly, individual 13 amino acid peptides were synthesized onto polyethylene pins as previously described (Geyson et al., 1984
). A six-amino-acid overlap for consecutive peptides ensured linear sequence epitopes were not split between peptides. The array of peptide pins (96-well format) were subject to ELISA to quantify reactive linear sequence epitopes. The peptide pins were slotted into a 96-well plate with each well containing 200 µl of block buffer (1% [w/v] ovalbumin and 0.1% [v/v] Tween 20, in 1 x PBS, pH 7.2), for 1 h at 20°C to reduce nonspecific background reactivity with the peptide pins. All incubations were at 20°C and aided by the use of a plate shaker (Milenia, Micromix 4). The peptide pins were washed by submerging in PBS, pH 7.2, for 10 min at 20°C on an orbital shaker to remove unbound protein. The monoclonal antibody was diluted 1:3 in block buffer (1% [w/v] ovalbumin and 0.1% (v/v) Tween 20 in PBS, pH 7.2) and 200 µl added to each well of an ELISA plate. Peptide pins were submerged into each well and incubated at 20°C for 1 h, prior to an overnight incubation at 4°C. Plate pins were sequentially washed three times in PBS, pH 7.2, to remove unbound monoclonal antibody. A volume of 200 µl of a 1:1000 dilution of horseradish peroxidaseconjugated SAM-Ig in block buffer, was added to each well of a fresh 96-well plate. Peptide pins were submerged into each well and incubated at 20°C for 1 h, then washed (as above) to remove unbound antibody. A volume of 200 µl of ABTS peroxidase substrate solution was added to each well of a fresh 96-well plate and the peptide pins submerged into the wells. Color reaction was recorded after a 20-min incubation at 20°C, by determining the optical density of each well at 414 nm (Ceres 900). All results were compared to positive and negative control pins, and a second antibody-only control with each individual peptide pin (i.e., performed as a separate assay on the same peptide pin plate).
Preparation of cell extracts and cell protein assays
Confluent CHO cells were harvested as described above and a cell pellet produced. For immunoquantification, immunobinding assay and western blot analysis, each cell pellet was resuspended in 200 µl of 20 mM TrisHCl pH 7.0 containing 0.5 M NaCl, then freeze/thawed six times. The samples were then centrifuged at 10,000 x g for 5 min and the supernatant collected and stored at 20°C. The amount of protein in the media and each cell lysate was determined by the method of Bradford (1976).
Immunoquantification
-L-iduronidase polypeptide levels were measured with an immunoquantification assay, as previously described (Ashton et al., 1992
), using a monospecific polyclonal antibody to capture the protein and then ID1A monoclonal antibody and a peroxidase-labeled second antibody to detect and quantify the bound protein.
Immunobinding assay
An immune capture method was developed to specifically bind human -L-iduronidase protein and followed by a MUI assay to determine
-L-iduronidase enzyme activity. Briefly, 100 µl of affinity purified SAM-Ig (20 µg/ml in 0.1 M NaHCO3, pH 8.5) was attached to each well of a 96-well polyvinylchloride plate (ELISA plate) by incubation at 37°C for 2 h, then overnight at 4°C. The plate was then washed to remove unbound antibody (cycle involving three successive washes on a plate washer using 0.02 M TrisHCl, pH 7.0, containing 0.25 M NaCl). Each well on the plate was then incubated with 200 µl of 0.02 M TrisHCl, pH 7.0, containing 0.25 M NaCl and 1% (w/v) ovalbumin to reduce nonspecific binding and block any remaining reactive sites on the ELISA plate. The monoclonal antibody ID1A was then adsorbed to the SAM-Ig on the plate, by incubation of each well with 100 µl of ID1A monoclonal antibody culture supernatant, for 4 h at 20°C. The plate was then rewashed, as above, to remove unbound antibody. Dilutions of the
-L-iduronidase to be assayed were then incubated (50 µl/well) in each well of the plate (e.g., standard curve of known enzyme activity plus dilutions of cell extract containing unknown levels of
-L-iduronidase) for 16 h at 4°C. The plate was then rewashed, as above, to remove unbound sample and non-
-L-iduronidase protein. Wells were then assayed for
-L-iduronidase activity by incubating 30 µl of MUI substrate (0.5 mM), containing 0.05 M sodium dimethylglutarate, pH 5.7, and 3.5 mg/ml BSA in 0.9% (w/v) NaCl, for 4 h at 37°C. Reactions were stopped by placing the plate on ice, then recovering the substrate and adding 2 ml of 0.05 M glycine-carbonate buffer, pH 10.7. Enzyme activity was estimated by the release of 4-methylumbelliferone using a Perkin Elmer spectrofluorimeter and an excitation wavelength of 366 nm and an emission wavelength of 446 nm. Results were interpolated through a standard curve and enzyme activity expressed as nmol/min/ml of sample.
Western blot analysis
Cell lysates (60 µg) and media samples (110 µg) were electrophoresed on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) using standard conditions (Laemmli, 1970) and transferred to nitrocellulose and immunoblotted as previously described (Clements et al., 1989
). Briefly, the immunoblot was blocked with 5% (w/v) BSA in 0.25 M NaCl/20 mM Tris, pH 7.0, and then incubated in the primary antibody, which was a monospecific polyclonal against human
-L-iduronidase (1:1000 dilution in 1% [w/v] ovalbumin in 0.25 M NaCl/20 mM Tris, pH 7.0). Horseradish peroxidaseconjugated sheep anti-rabbit immunoglobulin was used as a second antibody (1:1000 dilution) and followed by detection using 4-chloro-1-naphthol and H2O2 in 0.02 M TrisHCl, pH 7.0, containing 0.25 M NaCl.
Pulse chase labeling
Confluent 75-cm2 tissue culture flasks of CHO-K1 cells were preincubated for 45 min in cystine/methionine free Dulbeccos Modified Eagles Medium containing 10% (v/v) FCS and 2 mM glutamine, before labeling for 30 min at 37°C with 0.49 mCi of 35S-methionine in 5 ml of the same culture medium. The culture medium containing the radiolabel was then aspirated, and the tissue culture flasks washed twice with 10 ml of PBS before either harvesting (pulse label) or adding 8 ml of fresh culture media (for chase, which was in Hams-F12 containing 10% [v/v] FCS and 2 mM glutamine). The pulse label was followed by either 4 h or 24 h chase, then the culture medium was collected from each flask and centrifuged at 200 x g for 5 min and stored sterile at 4°C prior to immunoprecipitation. The CHO-K1 cell layers were harvested at appropriate time points, as described above. The cell pellet was resuspended in 10 ml of solubilization buffer (PBS containing 1% [w/v] Na deoxycholate, 0.1% [w/v] SDS, 0.5% [v/v] Triton X100). The samples were left at 4°C for a minimum of 24 h before immunoprecipitation.
Immunoprecipitation
Radiolabeled cell extracts were immunoprecipitated using antibody attached to pansorbin cells (i.e., source of protein A). The Pansorbin cells were equilibrated by washing four times with solubilization buffer, using centrifugation at 10,000 x g for 2 min at 4°C. Cell extracts and culture media were precleared by incubation first, with 60 µl of equilibrated Pansorbin cells for 2 h at 4°C, with constant mixing (i.e., minus antibody). Second, 60 µl of SAM-Ig bound Pansorbin cells (15 µg of SAM-Ig per 60 µl of Pansorbin cells) was incubated for 2 h at 4°C. Subsequently, 5 ml of Id1A monoclonal culture supernatant (approximately 10 µg/ml monoclonal antibody) was bound to an additional 500 µl of Pansorbin-SAM-Ig (by incubation at 4°C for 2 h, with mixing), then 60 µl of this Pansorbin mix incubated with each cell extract/culture medium, overnight at 4°C with mixing. The Pansorbin complex was pelleted by centrifugation at 10,000 x g for 5 min, prior to three 1-ml washes with solubilization buffer and finally one wash with 0.5 ml water. The Pansorbin complex was then resuspended in SDSPAGE sample buffer, reduced using ß-mercaptoethanol, boiled for 5 min to release the -L-iduronidase protein then centrifuged at 10,000 g for 5 min. The supernatant was loaded onto a 10% polyacrylamide gel, and electophoresed as described above. The gel was fixed in 40% (v/v) methanol:10% (v/v) acetic acid for 1 h, prior to placing in Amplify solution overnight at 20°C and subsequent autoradiographic detection of radiolabeled
-L-iduronidase.
Organelle fractionation of CHO-K1 cells
CHO-K1 cells were harvested and a granular fraction prepared for organelle subfractionation, on an 18% (v/v) Percoll gradient, as previously described (Brooks et al., 1997). Briefly, cells were harvested as described above and the cell pellet resuspended in 10 mM HEPES, pH 7.0, containing 0.25 M sucrose, 1 mM ethylenediamine tetraacetic acid, and protease inhibitors. The cell suspension was drawn through a 23-gauge needle and then subjected to hypobaric shock, prior to centrifugation at 200 x g for 10 min at 4°C to remove cellular debris. The postnuclear supernatant (whole granular fraction) produced was then subfractionated on an 18% (v/v) Percoll gradient, by centrifugation at 31,400 x g (average g) for 1 h at 4°C. Fractions were collected from the top of the gradient, as 10 2-ml samples. Each fraction was assayed for ß-hexosaminidase and acid phosphatase activity as previously described (Brooks et al., 1992
), using the fluorogenic substrates 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide and 4-methylumbelliferyl-phosphate respectively (Leaback and Walker, 1961
; Kolodny and Mumford, 1976
). The salt concentration of each fraction was adjusted to 0.25 M and then freeze/thawed six times before centrifuging at 100,000 x g for 1 h at 4°C to remove the Percoll and cell membranes. The supernatant of each fraction (20 µl of the total) was run on a 10% SDSPAGE and immunoblotted as described above.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: Hybrigenics S.A., 180 avenue Daumesnil, 75012 Paris, France
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References |
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