Activity-based profiling of glycoconjugate processing enzymes
Witte, M.D.
Citation
Witte, M. D. (2009, December 22). Activity-based profiling of glycoconjugate processing enzymes. Retrieved from https://hdl.handle.net/1887/14551
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Introduction
Gaucher disease, the most frequent lysosomal storage disorder, is caused by the deficiency of acid beta-glucosidase.1 This lysosomal hydrolase is named glucocerebrosidase (GBA-1) based on its endogenous substrate, the glycosphingolipid glucocerebroside (glucosylceramide).2 A characteristic feature of Gaucher disease is the massive storage of glucosylceramide in lysosomes of tissue macrophages. Large amounts of lipid-laden macrophages, so-called Gaucher cells, progressively accumulate in tissues of Gaucher patients, particularly in the spleen, liver and bone marrow. These storage cells induce a variety of visceral symptoms, such as hepatosplenomegaly, leukopenia, thrombocytopenia and skeletal disease.3
Ultrasensitive visualization of active GBA-1
Activity-based labeling in vitro
and in situ
The life cycle of glucocerebrosidase differs considerably from that of other soluble lysosomal glycosidases. The ubiquitously expressed GBA-1 is initially synthesized as a 519 amino acid protein that co-translationally acquires four N-linked glycans. After removal of its 23 amino acid signal peptide, GBA-1 is thought to undergo no further posttranslational proteolytic modification, in contrast to other lysosomal glycosidases.1 The enzyme does not acquire mannose-6-phosphate moieties in the Golgi apparatus and is consequently sorted independently from mannose-6-phosphate receptors to its final lysosomal destination.4 Binding of GBA-1 in the Golgi-apparatus to the integral membrane protein LIMP-2 governs its routing to lysosomes.5 Given the normal lysosomal localization of GBA-1 in leukocytes of a LIMP-2 deficient individual, it is likely that an alternative sorting mechanism exists in such cells.6 For optimal hydrolysis of its lipid substrate GBA-1 requires saposin C, a small accessory protein which is proteolytically released from the precursor prosaponin in lysosomes. The relevance of saposin C is illustrated by individuals with symptoms of Gaucher disease as the result of an abnormal saposin C.1, 7
The manifestation of Gaucher disease in individuals with a defective glucocerebrosidase is remarkably heterogeneous with respect to onset, progression and nature of symptoms.1 Most patients develop visceral complications, but no lethal neurological manifestations.8 The clinical course of the common non-neuronopathic (type 1) variant of Gaucher disease varies remarkably among patients: some type 1 Gaucher patients may remain virtually asymptomatic, whilst others develop already during early childhood a rapidly progressive disease. Next to the type 1 variant, more severe manifestations of Gaucher disease occur with lethal neurological complications (type 2 and 3 variants) and in the most extreme cases with abnormalities in skin permeability (so-called collodion baby).1 The marked heterogeneity in phenotypic manifestation of Gaucher disease is partly explained by differences in underlying mutations in the GBA-1 gene and corresponding residual intralysosomal enzyme capacity. Most common among the abnormalities in GBA-1 in Gaucher patients are amino acid substitutions.1 The heteroallellic presence of N370S GBA- 1, the most frequent mutation in Caucasian individuals, protects against a neuronopathic manifestation, whilst homozygosity for L444P GBA-1 is associated with severe neurological symptoms. Several studies have indicated that the relationship between GBA-1 genotype and Gaucher phenotype is not very strict.9 Even phenotypic heterogeneity among identical twins has been reported, suggesting that additional factors influence the in situ residual activity of glucocerebrosidase.10
Gaucher disease has been the playground for development of new therapeutic approaches for lysosomal storage disorders. Two treatments for Gaucher disease are presently registered: enzyme replacement therapy and substrate reduction therapy. The pioneering work of Brady and co-workers resulted in enzyme replacement therapy which is based on chronic intravenous administration of recombinant glucocerebrosidase (Imiglucerase; Cerezyme) to Gaucher patients. The N-linked glycans of the therapeutic enzyme are modified to expose terminal mannose-moieties favouring the delivery to lysosomes of macrophages following endocytosis by the mannose receptor.11 Substrate
reduction therapy is based on chronic oral administration of N-butyldeoxynojirimycin, an inhibitor of the enzyme glucosylceramide synthase which catalyzes the formation of glucosylceramide.12 More recently an alternative approach received considerable attention, the so-called chaperone therapy. Common in Gaucher patients are mutant forms of glucocerebrosidase which show impaired folding and retention in the endoplasmic reticulum. This ultimately results in degradation of the mutant enzyme via the ubiquitin- proteasome system, a process known as ER-associated degradation (ERAD).13 Investigated are small compounds, designated as chemical chaperones, which are able to increase the amount of glucocerebrosidase by stabilizing and/or promoting folding of the enzyme. It has been demonstrated for cell lines that changing the environment of the endoplasmic reticulum stabilizes some of these mutant enzymes. Folding was noted to be enhanced by lowering temperature or by increasing ER calcium levels.14 A more specific intervention in this respect is nowadays also envisioned based on the observation that 1- deoxygalactonojirimycin, a competitive inhibitor of α-galactosidase A, is able to stabilize particular mutant forms of the enzyme.15 This finding has stimulated investigations on the use of inhibitors as so-called pharmacologic chaperones for glycosidases. It has been hypothesized that also pharmacologic chaperones may be selected which specifically interact with mutant glucocerebrosidase molecules and thus assist folding to a sufficient extent to ameliorate the clinical course of disease. Also in the case of glucocerebrosidase, compounds that act as competitive inhibitors have been considered as potential pharmacologic chaperones.16 Many compounds have meanwhile been screened on inhibitory potency and potential value as chaperone.17 One very well studied example is isofagomine, which interacts with the catalytic pocket of glucocerebrosidase and is a very potent inhibitor.18 Other competitive inhibitors commonly considered as potential pharmacologic chaperones are deoxynojirimycin-type structures.17,18 Beneficial effects on the amount and lysosomal localization of mutant GBA-1 forms in cultured cells have been reported for isofagomine and some other inhibitors, but to demonstrate increased degradative capacity quite artificial assays have been used in which cells are exposed at acidic pH to high concentrations of fluorogenic substrate.
It should be kept in mind that a priori pharmacologic chaperones will only exert a positive clinical effect at a particular dose range; their concentration should be sufficiently high to promote folding of the enzyme in the endoplasmic reticulum and transport to lysosomes, whereas concomitantly the concentration in lysosomes should be sufficiently low to prevent marked inhibition of catalytic activity. Key to the success of pharmacologic chaperone therapy is a drug-mediated increase of catalytic capacity rather than an increase of enzyme molecules per se. Hence, effective pharmacologic chaperones for Gaucher disease should result in an increment of detectable active GBA-1 molecules.
The present lack of a suitable method allowing specific visualization of active GBA-1 molecules in situ (available antibodies do not distinguish between active and inactive GBA- 1 molecules) forms a major limitation in research on pathogenesis as well as therapy of Gaucher disease. The activity-based glucosidase probes MDW933 and MDW941 described
in Chapter 5 (Figure 1) are both potent inhibitors that label recombinant glucocerebrosidase and glucocerebrosidases in cell homogenates selectively. Labeling was concentration dependent and abolished when the enzyme was denatured prior to incubation with the probes. Competition experiments with known inhibitors conduritol B epoxide19 and AMP-DNM20 demonstrated the active site dependency of labeling. The sensitivity of the probes was studied and it appeared that as little as 0.3 ng of GBA-1 could be visualized (Chapter 5). It was reasoned that these glucosidase probes MDW933 and MDW941 could be employed to visualize GBA-1 in situ. In this Chapter, the versatility of MDW933 and MDW941 is demonstrated by using these fluorescent probes to study features of glucocerebrosidase in cells and tissues as well as the effects of chaperones on the amount of active GBA-1.
Figure 1. Structures of MDW933 and MDW941.
Results and discussion
In vitro labeling of glucocerebrosidase with MDW933 and MDW941
GBA-1 is a very low abundant protein and constitutes < 10-5 of all cellular or tissue proteins and therefore the sensitivity and selectivity of the probe are of great importance. The labeling profile has partially been studied in Chapter 5. Here, the properties of MDW933 and MDW941 were further investigated. First the pH-dependency was examined by incubating the enzyme at varying pH in the presence and absence of the probes followed by determination of (residual) enzyme activity for 30 minutes. Inhibition of GBA-1 with probes exactly coincides with the pH profile of enzymatic activity towards 4- methylumbelliferyl-β-D-glucoside (Figure 2A). The sensitivity of detection of labeled GBA- 1 was determined by incubating 2 pmole GBA-1 with an excess of MDW933 (1 μM) for 1 hour at 37 °C, and subsequent titration of the amount applied on the gel (Figure 2B, upper panel). As little as 20 attomole GBA-1 was detectable by fluorescence scanning. Next, equal amounts of GBA-1 (2 pmole) were incubated for 30 minutes with decreasing amounts of MDW933, and all labeled protein was applied on gel. Incubation with as little as 20 attomole probe resulted in detectable GBA-1 on the slab gel (Figure 2B, lower panel).
Apparently, nearly all of the probe had covalently bound to recombinant GBA-1 (cerezyme), consistent with its very high potency (see Chapter 5). These experiments indicate that ultra-sensitive detection of GBA-1 is feasible on slab gels following in vitro labeling with the fluorescent probes.
OH HO OH N
O N N BN
F N F
OH HO OH N
O N N BN
F N F O
O
MDW941 MDW933
.
Figure 2. In vitro labeling of pure recombinant glucocerebrosidase (cerezyme). (A) Effect of pH on labeling and enzymatic activity. The activity of GBA-1 at various pHs was determined, normalized for the activity at pH 5.2 and plotted (dashed line/squares). Inhibition by MDW933 was examined at the same pH-ranges (solid line/circles).
The residual activity was determined by dividing the measured fluorescence by the fluorescence observed at the corresponding pH without the inhibitor. (B) Sensitivity of detection and labeling. Upper panel: cerezyme (2 pmole) was incubated with an excess of MDW933 and diluted. Lower panel: cerezyme (2 pmole) was incubated with a decreasing amount of MDW933.
Labeling of glucocerebrosidase in cell and tissue extracts
To determine the labeling specificity of both probes, homogenates of cultured cells and mouse tissues were incubated at pH 5.2 with 100 nM MDW933 for 30 minutes at 37°C. The preparations were subjected to SDS-PAGE and the wet gel-slabs scanned for fluorescence (Figure 3). In homogenates of cultured RAW cells, GBA-1 is labeled exclusively. The various GBA-1 forms with molecular mass ranging from 58-66 kDa due to glycan differences are visualized (Figure 3A). Similar results were obtained with several other cell types such as HepG2 cells, human fibroblasts, and mouse 3T3-L1 cells (data not shown). It is striking that no other cellular proteins are fluorescently labeled following incubation of cultured cell lysates with MDW933. The same high level of specificity was noted for MDW941 (data not shown). Very similar results, that is, highly specific labeling of GBA-1, were obtained using lysates of mouse tissues (see Figure 3B). The only exception was found for homogenates of mouse intestine. A high molecular weight protein was labeled which presumably is lactase and fragments thereof. Lactase (lactase-phloridzin hydrolase: LPH) is known to covalently bind CBE.21
Figure 3. Labeling of cell and tissue extracts. (a) Fluorescent labeling of GBA-1 in homogenates of RAW-cells. RAW- lysate (20 μg) was incubated with MDW933 (100 nM) for 30 min and resolved on a 7.5% SDS-PAGE gel. Left lane:
Labeled proteins were detected by fluorescence imaging.
Right lane: Coomassie brilliant blue (CBB) staining of the same gel. (b) Mouse tissues were exposed to MDW933 (100 nM) for 30 min followed by SDS-PAGE gel electrophoresis (10%). Examples of labeling of GBA-1 in different tissues.
Labeling of lactase was observed in the intestine (outermost left lane (B)). The arrow indicates GBA-1.
In situ labeling of glucocerebrosidase
To study whether labeling of GBA-1 in intact cells was also feasible, MDW933 and MDW941 were added to the culture medium at a concentration of 5 nM. At different time points the cells were harvested. Remaining glucocerebrosidase activity in the cell homogenates was determined with artificial substrate (Figure 4A). Inactivation of GBA-1 by the probes proceeds rapidly in intact cells. Furthermore, gel-electrophoresis of the homogenates revealed exclusive labeling of GBA-1. These results indicate that both probes can very easily enter the cellular compartments and when situated inside they selectively react with intracellular GBA-1. It is presently not clear how exactly the probes enter cellular compartments.
Figure 4. In situ labeling of glucocerebrosidase. (A) Inactivation of GBA-1 by MDW933 (squares) and MDW941 (circles) in situ. Fibroblasts were incubated with probes (5 nM) for the indicated time, after which the cells were homogenized and residual activity was determined with 4MU-β-glucoside. (B) FACS experiment. Cells were treated with 0 nM (red line), 2 nM (blue line) and 10 nM (green line) MDW933 for 300 min. As a control, cells were pretreated with CBE (0.3 mM) followed by incubation with 2 nM (brown line) and 10 nM (purple line) MDW933. (C) Representative micrographs of cells labeled with MDW933. 1) BODIPY-fluorescence of GBA-1, 2) Alexa594-fluorescence of GBA-1 visualized with monoclonal 8E4 antibodies, 3) nuclei stained with DAPI, 4) overlay of 1, 2 and 3. (D) Pulse-chase experiment. Cells were incubated overnight with 10 nM MDW941 (pulse, upper panel) followed by incubating with 10 nM MDW933 for the indicated time (chase, middle panel). Lower panel: overlay of the pulse and the chase.
Having assessed that MDW933 and MDW941 rapidly labeled GBA-1 in situ, the probes were applied in fluorescence activated cell sorting (FACS), fluorescence microscopy and pulse-chase experiments. FACS analysis nicely revealed dose-dependent fluorescent labeling of cells. The fluorescence intensity in cells incubated with an excess amount of the green fluorescent probe, MDW933, is larger than in those incubated with a sub-saturating concentration. To prove that the increase in fluorescence depended on the labeling of glucocerebrosidase, active GBA-1 was inhibited with the irreversible inhibitor CBE. Pre- incubation with an excess of CBE, indeed, reduced the fluorescent signal to background level (Figure 4B). Unfortunately, the red fluorescent probe, MDW941, could not be detected by FACS analysis.
For live-cell fluorescence microscopy, fibroblasts were cultured for 2 hours with MDW933 (5 nM) after which the labeled proteins were visualized. GBA-1 was also detected by indirect immunofluorescence using the specific anti-GBA-1 monoclonal antibody 8E4.
Figure 4C shows an illustrative example of the microscopy visualization of GBA-1. The intracellular pattern of labeling with the fluorescent probe showed an almost complete overlay with the detection by monoclonal antibody (Figure 4C). Additionally, similar results have been obtained with MDW941.
The life cycle of GBA-1 was visualized by means of a pulse-chase experiment using cultured cells. Previously conventional pulse-chase labeling experiments with radioactive methionine22 revealed that GBA-1 consists in three forms. In the ER, GBA-1 is formed as a 62.5-kDa precursor. Conversion of the high-mannose glycans into complex type glycans results in the formation of a 66-kDa intermediate form which eventually is transformed into 59-kDa mature GBA-1. Fibroblasts were incubated overnight with excess red fluorescent probe (10 nM). The cells were next incubated with the same concentration green fluorescent probe, and harvested at different time points (0-48 hours). Aliquots of cell homogenates were subjected to gel electrophoresis. Figure 4D shows the life cycle of GBA-1 as visualized in this manner. It should be noted that GBA-1 pulse-labeled with the red fluorescent probe, MDW941, disappeared gradually in time from the cells with an estimated half life of about 30 hours. This half life is entirely consistent with that earlier determined using conventional pulse-chase labeling. During the chase, a higher running form of GBA-1 became increasingly labeled with green fluorescent probe MDW933. Its molecular weight corresponds to that of the intermediate form of GBA-1, coinciding with the formation of new GBA-1 molecules (Figure 4D).
Analysis of GBA-1 in Gaucher fibroblasts and spleen samples
To study labeling of mutant glucocerebrosidase, cell-lysate of fibroblasts of Gaucher donors (a N370S GBA-1 homozygote, a L444P homozygote, and a collodion Gaucher patient which lack GBA-1 protein) was treated with MDW933 (10 nM) for one hour and subjected to SDS-PAGE. The labeling pattern was compared to the labeling pattern of a normal individual. This comparison revealed that the amount of GBA-1 is severely reduced in cell- lysate of the Gaucher donor homozygous for L444P GBA-1 (Figure 5A). It is indeed known that L444P GBA-1 undergoes largely premature degradation by ERAD as a result of
impaired folding. This phenomenon is far less striking in the case of N370S GBA-1.9b,23 The amount of labeled GBA-1 in cells from a Gaucher donor homozygous for N370S GBA-1 was also found to be reduced but to a lesser extent than in cells from a L444P homozygote.
As expected, cells from the collodion Gaucher did not show any labeled GBA-1 (Figure 5A).
It has earlier been noted that the irreversible inhibition by CBE of N370S GBA-1 is less avid compared to wild type GBA-1.9b Therefore inactivation curves of glucocerebrosidase activity were determined in cells of a normal subject and N370S GBA-1 homozygote (Figure 5B). Inactivation of glucocerebrosidase activity by MDW933 was found to be quite similar for wild-type and N370S GBA-1 cells. Treating spleen lysates with variable concentration of MDW933, resulted again in a lower amount of GBA-1 protein that was labeled in case of the patient’s spleen. However, the affinity of labeling was not dissimilar to that of GBA-1 in spleen from a normal individual (Figure 5C, D).
Figure 5. Labeling of mutant forms of glucocerebrosidase. (A) Detection of glucocerebrosidase in Gaucher fibroblasts. GBA-1 in wild type and homozygous N370S, L444P and collodion fibroblast were labeled with MDW933. As control recombinant glucocerebrosidase (cerezyme) was labeled with MDW933. (B) Inactivation curves of MDW933 (open symbol) and MDW941 (solid symbols) in N370S (triangles) and control fibroblasts (squares). (C) Labeling of GBA-1 in spleen homogenates. (D) The intensity of the fluorescent signal in control spleen (left panel) and N370S (right panel) was quantified and normalized for 100 nM MDW933 (100%).
The impact of isofagomine on N370S GBA-1 in cultured fibroblasts
It has been earlier reported for cells from N370S GBA-1 homozygotes that prolonged incubation with the reversible inhibitor isofagomine yields an increase in glucocerebrosidase activity as was determined by substrate based assays.18 The added chaperone may not only increase the amount of GBA-1, but also may partially inhibit GBA- 1. Since the assays used to examine the activity are rather artificial and require high concentrations, subtle differences in GBA-1 activity may not be detected. It was reasoned that MDW933 and MDW941 could be used to examine whether incubation of N370S GBA- 1 homozygous fibroblasts with isofagomine increases the amount of GBA-1 that can be
labeled with these probes. Cells were cultured for 3 days with different concentrations of isofagomine (0, 10, 30 and 300 nM) and next incubated for 2 hours with or without excess MDW933 in the presence of the original concentration isofagomine. The harvested cells were homogenized and glucocerebrosidase activity in homogenates of cells not treated with the probe was determined with 4-methylumbelliferyl-β-D-glucoside as substrate. As can be seen in Figure 6A (see page 120) a modest isofagomine dose-dependent increase in enzyme activity was noted. Aliquots from the homogenates of cells labeled with MDW933 were subjected to gel electrophoresis and the detected fluorescent GBA-1 was quantified. Again a modest dose-dependent increase was noted (Figure 6B), almost coinciding with the increase in enzyme activity in the homogenates. Analysis by fluorescence microscopy suggested an increase in probe-labeled GBA-1 in lysosomes of isofagomine treated cells (Figure 6C).
Apparently, the presence of 300 nM isofagomine did not compete significantly with MDW933 labeling of N370S GBA-1.
Conclusion
In conclusion, the fluorescent activity-based probes MDW933 (green fluorescent) and MDW941 (red fluorescent) are very versatile research tools to visualize active GBA-1, ultra- sensitively and specifically. The potential applications for the activity-based fluorescent probes MDW933 and MDW941 are enormous. They offer an alternative to antibodies which are species-specific and can not reach compartmental GBA-1 in intact cells.
Moreover, in contrast to antibodies these probes uniquely label active GBA-1 molecules.
The probes might contribute in the near future to get better insight in the in situ localization and activity of glucocerebrosidase in tissues, including the brain. Better understanding of the features of GBA-1 in the brain may increase insight in the neuropathology of Gaucher disease. It recently became apparent that carriership for a defective GBA gene is a major risk factor for Parkinson’s dissease.24 Therefore, the probes may also become useful to study Parkinsonism. Another area of application for these fluorescent probes may be found in the analysis of compounds on their possible inhibitory or chaperone effects. As demonstrated in this chapter, the beneficial effect of isofagomine on N370S GBA-1 in cultured fibroblasts could be confirmed with activity-based labeling.
This finding is of importance since it implies that at an optimal concentration of isofagomine, occupation of the catalytic centre by the competitive inhibitor is in situ sufficiently low to not impair activity towards the fluorescent probe. In other words, an appropriate concentration isofagomine indeed increases glucocerebrosidase levels and intralysosomal enzymatic capacity.
Figure 6. Impact of isofagomine on GBA-1-levels in N370S fibroblasts. (A) N370S cells were incubated with increasing concentrations of isofagomine for 3 days. The cells were harvested after which the activity was determined in a fluorescent substrate assay. (B) Cells incubated with isofagomine were treated with MDW933 prior to lysis. After resolving of the labeled proteins by SDS-PAGE, the amount of labeled GBA-1 was quantified.
(C) Microscopy analysis of GBA-1 labeled by MDW933 in non-treated N370S homozygous cells (left) and in N370S homozygous cells treated with isofagomine (right).
Experimental Section
General experimental
Chemicals were obtained from Sigma (St. Louis, USA) if not otherwise indicated. Recombinant glucocerebrosidase (Cerezyme) was obtained from Genzyme Corporation, Cambridge, USA.
Monoclonal anti-human glucocerebrosidase antibody 8E4 was produced from hybridoma cells as described earlier.25 Gaucher patients were diagnosed based on reduced glucocerebrosidase activity and demonstration of an abnormal genotype.26 Fibroblasts were obtained with consent from donors. Cell lines were cultured in HAMF12-DMEM medium (Invitrogen) supplied with 10% fetal calf serum.
Enzyme activity assays
Activity of glucocerebrosidase was measured at 37°C with 4-methylumbelliferyl-β-D-glucoside as substrate as reported previously.20 The incubation mixture contained 5 mM fluorogenic substrate, 0.2% (w/v) sodium taurocholate and 0.1% (v/v) Triton X-100 in 150 mM McIlvaine buffer pH 5.2.
After stopping the incubation with excess NaOH-glycine (pH 10.3) fluorescence was measured with a fluorimeter LS-30 (Perkin Elmer) using λex 366 nm and λem 445 nm.
Gel electrophoresis and fluorescence scanning
Electrophoresis in sodium dodecylsulfate containing 10% polyacrylamide gels was performed as earlier described.23 Wet slab gels were next scanned on fluorescence using the Typhoon Variable Mode Imager (Amersham Biosciences) using λex 488 nm and λem 520 nm (band pass 40) for MDW933, respectively λex 532 nm and λem 610 nM (band pass 30) for MDW941.
Fluorescence assisted cell sorting (FACS)
Fibroblasts were cultured in the presence or absence of 0.3 mM CBE overnight. Next cells were incubated with MDW933 (2 and 10 nM, for 300 min). Cells were suspended by trypsinization and analyzed by FACS using a FACS vantage (B.D. Bioscience, San José, U.S.A.), excitation 488 nm, emission 530 nm (bandpass filter 30 nm).
Fluorescence microscopy and indirect immunofluorescence
Fibroblasts were cultured on glass slides. Cells were incubated with 5 nM MDW933 for 2h. Next, cells were washed, fixed with 3% (w/v) paraformaldehyde in PBS for 15 min, washed and incubated with 0.05 % (w/v) saponine for 15 min, subsequently with 0.1 mM NH4Cl in PBS for 10 minutes, and with 3% (w/v) Bovine serum albumin in PBS for 1h. Next, the slides were and incubated with (1:500) anti- GBA-1 monoclonal antibody 8E4 in the presence of saponin. Bound murine monoclonal antibody was visualized with a secondary antibody conjugated with Alexa594. Nuclei were stained with DAPI. Cells
were examined using fluorescence microscopy (Leica DM5000B, Rijswijk, The Netherlands), applying A4 filter block for DAPI, L5 filter block for MDW933 (i.e. 480/40 nm BP excitation, 505 nm dichromatic mirror, 527/30 nm BP suppression), and TX2 filter block for TexasRed. Images were acquired using a Leica DFC500 camera.
Pulse-chase experiments
Fibroblast were cultured overnight in the presence of 10 nM MDW941. Next, cells were extensively washed and incubated with 10 nM MDW933. Cells were harvested at different time points, homogenates were prepared and subjected to SDS-PAGE. MDW933- and MDW941-labeled GBA-1 on the slab gel was separately visualized using the Typhoon Variable Mode Imager.
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