From the Department of Biophysics, Boston University
School of Medicine, Boston, Massachusetts 02118-2526 and the
New England Biolabs, Beverly, Massachusetts 01915-5599
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ABSTRACT |
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Glycosylasparaginase (GA) is a member of a novel
family of N-terminal nucleophile hydrolases that catalytically use an
N-terminal residue as both a polarizing base and a nucleophile. These
enzymes are activated from a single chain precursor by intramolecular autoproteolysis to yield the N-terminal nucleophile. A deficiency of GA
results in the human genetic disorder known as aspartylglycosaminuria. In this study, we report the crystal structure of recombinant GA from
Flavobacterium meningosepticum. Similar to the human
structure, the bacterial GA forms an sandwich. However,
some significant differences are observed between the
Flavobacterium and human structures. The active site of
Flavobacterium glycosylasparaginase is in an open
conformation when compared with the human structure. We also describe
the structure of a mutant wherein the N-terminal nucleophile
Thr152 is substituted by a cysteine. In the bacterial GA
crystals, we observe a heterotetrameric structure similar to that found
in the human structure, as well as that observed in solution for eukaryotic glycosylasparaginases. The results confirm the suitability of the bacterial enzyme as a model to study the consequences of mutations in aspartylglycosaminuria patients. They also suggest that
further studies are necessary to understand the detail mechanism of
this enzyme. The presence of the heterotetrameric structure in the
crystals is significant because dimerization of precursors has been
suggested in the human enzyme to be a prerequisite to trigger
autoproteolysis.
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INTRODUCTION |
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Eukaryotic glycosylasparaginase (glycoasparaginase,
N4(-N-acetyl-D-glucosaminyl)-L-asparaginase,
1-aspartamido-
-N-acetylglucosamine amidohydrolase, aspartylglycosylaminase, aspartylglucosaminidase, EC
3.5.1.26) is a well known lysosomal enzyme that cleaves the amide bond
of asparagine-linked glycoproteins (1). It is widely distributed in
vertebrate tissues (2) and insect cells (3) and is also found in
bacteria (4). Substrate preferences for this enzyme include free
-amino and
-carboxyl groups on the asparagine, and that position
6 of N-acetylglucosamine does not contain fucose. However, a recent
study suggests that the
-amino and
-carboxyl groups on the
asparagine part of the substrate may not strictly be required for
hydrolysis (5).
A deficiency of glycosylasparaginase (GA)1 results in accumulation of glycoasparagines in tissue lysosomes and leads to severe clinical symptoms, known as aspartylglycosaminuria (AGU). AGU is the most common disorder of glycoprotein degradation. It severely involves the central nervous system and causes skeletal abnormalities and connective tissue lesions. Among children in eastern Finland, AGU was found to be the leading genetic cause for mental retardation after trisomy 21 and fragile X syndrome (1).
Glycosylasparaginase has been biochemically characterized from
different species and is composed of two nonidentical subunits of
approximately 24 and 20 kDa, associated by noncovalent forces. These
respective subunits are referred to as the a- and b-subunits (or heavy
and light subunits). The enzyme is encoded by a single gene, and
post-translational cleavage of the nascent polypeptide into a mature
a/b heterodimer is required for activation. Neither the single chain
precursor (6, 7) nor the isolated subunits (8) are enzymatically active
by themselves. Expression of the a- and b-subunits of GA on separate
DNA constructs showed that independently folded subunits lack enzyme
activity, and even when co-expressed in vitro they fail to
produce an active heterodimer (9). A common feature of GA from
different species is a new N-terminal threonine of the C-terminal
product (the b-subunit) resulting from the autoproteolytic activation
(10). A study demonstrated that an irreversible inhibitor specifically
reacts with the N-terminal threonine on the b-subunit of the human
leukocyte enzyme via an -ketone ether linkage with the hydroxyl side
chain (8), indicating that this N-terminal threonine acts as a
nucleophile during substrate hydrolysis. The crystal structure of human
GA shows a topology similar to other N-terminal nucleophile hydrolases (11, 12) and reveals interactions between the N-terminal threonine and
aspartate, one of the reaction products (13).
GA from Flavobacterium meningosepticum is the only prokaryotic homolog characterized so far. It differs from the human counterpart in several aspects: (i) sequence alignment of these two enzymes reveals only about 30% sequence identity and shows a difference in one gap/insertion of 31 residues (3, 4); (ii) part of the 31-residue insertion in the human enzyme is removed from the new C terminus of the a-subunit in the lysosome (6); no trimming occurs in the bacterial enzyme; (iii) the human enzyme contains N-linked glycans on both the a- and b-subunits (Asn15 and Asn285) (14), whereas the bacterial enzyme is nonglycosylated (4); (iv) according to previous sequence alignments (3, 4), neither the position nor the pattern of disulfide bridges is conserved between these two enzymes. The disulfide bonds have been shown to be essential for initial protein folding and activation of human GA (15). Together, these differences suggest the possibility that the structure of Flavobacterium GA may be significantly different from the human enzyme. Here we report the crystal structures of recombinant GA from F. meningosepticum. Structures of the wild type GA and a single amino acid mutant in their mature forms have been determined at 2.2 and 2.1 Å, respectively.
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MATERIALS AND METHODS |
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Crystallization and Crystal Preparation-- Protein expression and purification will be described elsewhere.2 Crystals were grown in hanging drops equilibrated by vapor diffusion against well solutions of 15% PEG 3300, 100 mM HEPES, pH 7.5, 0.1% sodium azide. Microseeding was routinely used to improve crystal quality. Crystals were harvested into a modified well solution containing 20% PEG 3300. Shortly before data collection, crystals were placed in dialysis buttons and dialyzed stepwise against harvest buffer supplemented with a progressively higher concentrations of glycerol. The final glycerol concentration was 20%, and the transfer steps were between 5 and 10% glycerol and varied from 3 h to a couple of days for each step. No correlation was observed between transfer procedure and the quality of the diffraction data. Heavy atom derivatives were obtained by soaking the native crystals in the harvest buffer supplemented with 10% glycerol and 0.1-10 mM of heavy atom compound, from 15 h to a few days, before step up to the final glycerol concentration.
Data Collection and Processing-- Oscillation data were collected from crystals frozen at 100 K, mounted in a thin film of harvest buffer plus 20% glycerol, and supported by a loop made of dental floss. Diffraction data were collected using an R-AXIS IIC image plate detector mounted on a Rigaku RU300 rotating anode generator. All intensity data were processed and scaled using the programs DENZO and SCALEPACK (16) and converted to structure factors using TRUNCATE from the CCP4 software package (17). The space group was determined by examination of the differences in intensities of potential pairs of reflections across putative mirror planes within the data and confirmed by examining the electron density maps.
Experimental Phases-- Heavy atom positions were initially obtained from isomorphous Patterson maps calculated in XTALVIEW (18). The heavy atom parameters were then refined by MLPHARE from the CCP4 package (17) and were confirmed by the cross Fourier. At this stage, the figure of merit was 0.55 (0.71 calculated by XTALVIEW). The MIR phases were then extended to the resolution range 12-2.5 Å and were improved by noncrystallographic symmetry averaging, solvent flatterning, and histogram matching using the program DM (17) with a figure of merit of 0.785.
Model Building and Refinement--
The first map was calculated
at 2.5 Å resolution (see Fig. 3) and skeletonized to build a C
trace in the program O (19). The first 244 of 275 residues were built
based on the DM-modified MIR map. Automated refinement included rigid
body, overall temperature factor, positional, and restrained atomic
temperature factor refinement, as well as simulated annealing using a
slow cooling protocol in X-PLOR (20). After the first round of manual
rebuilding, without the N-terminal nucleophile (amino acid 152), the
structure, after rigid body fit by AMORE (17), was used for refinement
of both the wild type and the T152C mutant. SIGMAA (17) was used in the
early cycles of refinement and manual rebuilding to combine model
phases with experimental phases. Initially, strict noncrystallographic symmetry constraints were applied, and in later stages of refinement, tight noncrystallographic symmetry restraints were applied, exclusive of residues that were involved in crystal contacts. After a few rounds
of model rebuilding, stepwise resolution extension, and automated
refinement, clear electron density could be seen for all residues in
the final model. Refinement protocols were aimed at decreasing the
Rfree (21) rather than the conventional
Rcryst to avoid errors introduced by overfitting
of the data. When the Rfree appeared to have
reached a minimum at the final resolution, water molecules were added
and subjected to another round of automated refinement and manual
rebuilding. The statistics of the final structures are shown in Table
II, with a root mean square (r.m.s.) deviation of 0.20-0.23 Å for
main chain atoms between crystallographically independent
molecules.
Structural Comparisons-- All superimpositions of different structures were performed using LSQKAB (17). For Fig. 4, all atoms of residues contacting the reaction product, aspartate, in the human structure are superimposed (atoms equivalent to those in bacterial Thr152, Thr170, Arg180, Asp183, Thr203, and Gly204).
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RESULTS |
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Description of the Structure--
The enzymes crystallized in
space group P21 with unit cell constants a = 46.2Å, b = 97.3Å, c = 61.8Å, and
= 90.3°. The initial phases were obtained by MIR method with four
heavy atom derivatives (Table I). The
wild type structure has been determined at 2.2 Å and refined to an
Rfree (21) of 29.70% and an
Rcryst of 24.65% with all reflections (Table
II). There are two a/b heterodimers per
asymmetric unit. In the final model, each heterodimer comprises 136 residues (3-138) of the a-subunit and 139 residues (152-290) of the
b-subunit. No electron density is observed for the 13 residues spanning
the segment (139-151) that connects the a- and b-subunits in the
precursor protein. In the crystal, this linker segment appears to face
into the solvent channels. 93% of the nonglycine residues fall in the
most favored regions of Ramachandran plot, as defined in PROCHECK (22),
and no residues are in the disallowed regions.
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Structural Differences between Flavobacterium and Human Structures-- An r.m.s. deviation of 1.4 Å is obtained by superimposing the common 1,068 main chain atoms (excluding insertions/deletions) of the Flavobacterium and human GA structures. This is significantly larger than the r.m.s. deviation of 0.26-0.45 Å found between the two human structures (13). Moreover, the r.m.s. deviation between our two bacterial structures is 0.22 Å (see below), similar to that observed between two heterodimers in the asymmetric unit (Table II). A number of peptide fragments within the structure deviate by more than 2 Å (Fig. 1c); most of them are in loops connecting elements of secondary structure. The largest difference of 8.5 Å is near the 7-residue insertion in the bacterial structure. Deviations greater than 2 Å are also observed in the common secondary structural elements (see below). These data are consistent with the observation that molecular replacement using the human structure proved difficult with the data of Flavobacterium GA.3
The human enzyme contains four disulfide bonds (Fig. 2) that are important for protein folding, autoproteolysis, and enzyme activity (15). These four disulfide bonds are conserved among mammalian enzymes. The insect enzyme retains all but one (Cys263-Cys283) of the disulfide bonds (3). However, no conserved disulfide bond is found between the Flavobacterium and eukaryotic GA. Indeed, there are no disulfide bonds among the five cysteines in the bacterial a/b heterodimer. One cysteine pair in the bacterial structure (Cys68-Cys168) has side chains in close proximity that may potentially form a disulfide bond, but this was ruled out based on several observations: (i) Cys68 and Cys168 bind to heavy atoms Hg(OAc)2 and CH3HgCl, respectively; (ii) the initial MIR map indicates that the side chain of Cys168 point away from Cys68; (iii) the simulated annealed omit maps also show these two side chains to be in nonbridged conformations; (iv) a Cys to Ser mutation at either of these two cysteines does not significantly affect either protein stability or enzymatic activity4; and (v) the a- and b-subunits can be separated on a nonreducing SDS protein gel (data not shown). Although the overall protein folds are similar in the bacterial and human structures, the location or length of some secondary structural elements differ. For example, the bacterial enzyme has a unique 310 helix (bH4), whereas the human enzyme carries a C-terminal additional loop on its a-subunit (Fig. 1c). Furthermore, in the bacterial structure, the insertion of Gly14 extends helix aH1 at its N-terminal end by two residues. At the C-terminal end of helix aH1, Ser26 is designated as part of the helix in the human structure, but the equivalent Lys27 in the bacterial structure is not assigned as part of the helix by PROCHECK (22). This is apparently because of a significant deviation of the main chain traces between these two structures (Fig. 3). When these two structures are superimposed by their common secondary structural elements, C
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Active Site and Mechanism--
The loops connecting different
layers of -helices and
-sheets form a deep funnel-shaped active
site centered at the N-terminal Thr152 of the b-subunit
(Fig. 1). The funnel in the bacterial enzyme is wider than that of the
human enzyme, mainly because of deviation of the loop between helix aH2
and strand aS2 as well as lack of the C-terminal loop in the a-subunit
(Fig. 1c). Several conserved residues surround the
nucleophilic center Thr152 of the bacterial active site,
including Thr170, Arg180, Asp183,
Thr203, and Gly204, which are highlighted in
yellow in Fig. 2. These residues had been described to
interact with aspartate, one of the two reaction products (13). As
depicted in Fig. 4a, the human
equivalent to Arg180 forms hydrogen bonds with the
-carboxyl group of aspartate. Both human equivalent residues of
Asp183 and Gly204 make hydrogen bonds with the
-amino group of aspartate. Human residues equivalent to
Thr152, Thr203, and Gly204 also
form hydrogen bonds with the O
1 of aspartate. In addition, human
residue equivalent to Thr170 makes a hydrogen bond with the
O
of Thr152.
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Structure of the T152C Mutant-- Thr152 plays a key role in catalysis (4, 7, 8). Substitution of the N-terminal nucleophile Thr152 by a thiol group (T152C mutant) reduces kcat by 5 orders of magnitude (23). Autoproteolysis in this mutant is also very slow but can be accelerated by hydroxylamine (10). In this study, we have also determined the three-dimensional structure of the T152C mutant in its mature form at 2.1 Å and refined to an Rfree of 28.06% and an Rcryst of 23.32% with all reflections (Table II). The structure of this mutant is essentially identical to that of the wild type enzyme with an r.m.s. deviation of 0.22 Å for all the main chain atoms and 0.25 Å for the active site atoms (Fig. 4b). This indicates that the reduction of reaction rate of this mutant is because of the change of chemical groups at the side chain of residue 152.
The active site of the T152C mutant also has the open conformation as described above. Like the wild type structure, the distance between the CQuaternary Structure of GA--
Bacterial GA forms a dimer of a/b
heterodimers in the crystals (Fig.
5a). A similar quaternary
structure is also observed in the crystals of human GA in different
crystal packings (13). The surface interactions between pairs of
heterodimers are extensive and mainly involve hydrogen bonds and
hydrophobic contacts (Fig. 5, b and c).
Basically, both heterodimers use the same hydrophobic surface for the
(a/b)2 tetramer formation, reminiscent of hand shaking. The
main interface interactions come from the strand aS4, helix bH2, and
the loops between aH3 and aS4, aS4 and aH4, bS2 and bS3, and bH1 and
bH2 in both heterodimers. In addition, the bacterial enzyme has unique
interactions between the 7-residue insertion and the loop connecting
-strands aS2 and aS3. The human structure has substantially more
interactions from the unique C-terminal loop of the a-subunit.
Dimerization of a/b heterodimers sequesters a solvent-accessible
surface area of 1882 Å2 from each a/b heterodimer of the
bacterial GA, compared with 2485 Å2 for the human enzyme.
The smaller interface and thus weaker dimer interaction between two
heterodimers of bacterial GA may explain why no (a/b)2
tetramers of the bacterial GA are observed on sizing columns.5 Nonetheless, the
existence of bacterial (a/b)2 tetramers in the crystals
suggest that the heterotetramers of bacterial GA may also exist in
solution. This heterotetrameric structure of (a/b)2 has
been observed in solution for the human (8), chicken (2), and insect GA
(3).
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DISCUSSION |
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Aspartylglycosaminuria-- The physiological importance of the glycosylasparaginase is revealed by the occurrence of a human genetic disorder, known as AGU, because of a deficiency of this lysosomal hydrolase (1). Many mutations in the GA gene that cause AGU have been reported, and more are likely to be found. However, a major obstacle to studying the consequences of these mutations is the difficulty to obtain recombinant human enzyme in sufficient quantities (28). In this study, four known AGU single mutations have been mapped onto the shared secondary structural elements between the bacterial and human enzymes (Fig. 2). A double mutant (human Arg138 to Gln and Cys140 to Ser) maps outside of the secondary structural elements and appears to result from the loss of a disulfide bond (Cys140-Cys156) that stabilizes a unique loop in the human enzyme. Thus, our work confirms the suitability of the bacterial enzyme as a model to analyze the consequences of mutations in AGU patients at the molecular level.
Structural Comparisons--
Glycosylasparaginase belongs to a
newly classified family of enzymes that have a novel N-terminal
threonine, serine, or cysteine that provides the nucleophile in their
reaction mechanism (11). Previously reported structures of this family
include glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase
from Bacillus subtilis (29), Escherichia coli
penicillin amidohydrolase (30), the 20 S proteasome from the
archaebacterium Thermoplasma acidophilum (31) and yeast
(32), human glycosylasparaginase (13), and the glutaminase domain of
E. coli glucosamine 6-phosphate synthase (27). All of these
enzymes have a similar protein fold comprised of a sandwich of
antiparallel sheets surrounded on either side by layers of
helices. Many of these enzymes are activated by cleavage of the peptide
bond to free the
-amino group to form the N-terminal nucleophile
(10). A different protein fold has recently been described for the
autoprocessing domain of Drosophila Hedgehog protein (33).
It is an all
structure that is distinct from the GA structure but
is related to the intein domain of PI-SceI endonuclease (34,
35).
Enzyme Mechanism-- Crystal structures described in this study, on the other hand, also raise questions about the detail mechanism of GA. Structure of the wild type GA from F. meningosepticum in its mature form has been determined at 2.2 Å resolution. Although the topology of the bacterial enzyme is very similar to that of the human structure, several significant differences have been observed. The active site of Flavobacterium GA is in an open conformation, whereas the human enzyme adopts a closed conformation that grasps the reaction product, aspartate (Fig. 4a). Moreover, the side chain of Thr203 may not be as important in stabilizing the negative oxyanion intermediate as previously suggested (13). This is consistent with a mutagenesis study in which replacement of Thr203 by Ala (T203A mutant) in the bacterial enzyme does not dramatically decrease the reaction rate (23). A three-dimensional structure of the enzyme-substrate complex is necessary to clarify the role of Thr203 side chain in the enzymatic mechanism.
In addition, we also report the structure at 2.1 Å resolution of a T152C mutant wherein the N-terminal nucleophile Thr152 of the b-subunit is replaced with Cys. The T152C mutant has a dramatically reduced rate of autoproteolysis or enzyme catalysis (23) and thus is a good candidate for future crystallographic studies of the precursor structure and enzyme-substrate complex. Similar to the glutaminase domain of glucosamine 6-phosphate synthase, Cys152 in the T152C mutant appears to be in an inactive conformation (27). We propose that binding of substrate would switch the thiol group into an active conformation.Autoprocessing for Enzyme Activation-- Cis-autoproteolysis involves the intramolecular catalytic cleavage of a peptide bond and is required to activate many enzymes (12). In addition to GA, these include penicillin acylase (30), proteasomes (31, 36, 37), as well as the hedgehog family of eukaryotic developmental regulatory proteins (38). Autoproteolytic cleavage also serves as a mechanistic component for protein splicing (35). In contrast to the activation of zymogens, such as chymotrypsinogen and trypsinogen through proteolysis by another trypsin molecule, the autoproteolysis of GA is an intramolecular reaction (10). Human GA is also believed to undergo autoproteolysis to form the active enzyme but with some differences. First, the disulfide bridges in the human enzyme are essential for early folding and for autoproteolytic processing (15). In contrast, there are no disulfide bridges in the bacterial enzyme. Furthermore, part of the 31-residue insertion in the human enzyme is removed from the C terminus of the autoproteolyzed a-subunit in the lysosome by a second cleavage (6). No such trimming occurs for the bacterial enzyme.
This work reveals an (a/b)2 quaternary structure that has been observed in solution or crystals of the eukaryotic GA. Furthermore, an amino acid substitution (equivalent to bacterial Ile186) at this interface in the human enzyme disrupts the dimer formation of the precursor protein and also prevents proteolytic activation of the enzyme (15). Therefore, it appears that in the human enzyme dimerization of precursors is a prerequisite to trigger autoproteolysis. In contrast, only the a/b heterodimer is observed on sizing gels and columns for the bacterial GA.5 Nonetheless, a dimer of a/b heterodimers exists in the crystals of bacterial GA (Fig. 5a) that is similar to the quaternary structure observed in the crystals of human GA (13). This raises the possibility that dimerization of bacterial GA, although it has not been observed yet, might also occur in solution. Further studies are necessary to determine whether dimerization of the single chain precursor proteins occurs and, if so, to determine the significance of this dimerization in autoproteolysis. Unless there is a large conformational change as a result of autoproteolysis, the location of the key cleaved Thr152 in the enzyme active site suggests that the autoproteolytic site is near or overlaps with the active site. In line with intramolecular autoproteolysis, the two active sites in the dimer of GA are facing apart with the autoproteolyzed N-terminal threonines 32 Å away (arrows in Fig. 5a). The size and shape of the active site funnel also appear to be difficult for any proteolytic enzyme to approach Thr152 for peptide bond cleavage. However, it remains unclear how this dimerization triggers autoproteolytic activation of these enzymes. It is still possible that dimerization of precursor proteins results in a conformational change to trigger autoproteolysis. In our group, crystallographic studies on the precursor proteins are underway. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. G. G. Shipley, C. W. Akey, and C. J. McKnight for helpful discussions and comments on the manuscript, T. Cui for sharing unpublished results of Cys to Ser mutations in Flavobacterium GA, and members of the lab for helpful suggestions.
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FOOTNOTES |
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* This work was supported in part by Grant IRG-97 T from the American Cancer Society (to H.-C. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 2GAW and 2GAC) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
§ To whom correspondence should be addressed: Dept. of Biophysics, Boston University School of Medicine, 715 Albany St., Boston, MA 02118-2526. Tel.: 617-638-4023; Fax: 617-638-4041; E-mail: hguo{at}med-biophd.bu.edu.
¶ Present address: Dept. of Biochemistry, University College Cork, Lee Maltings, Cork, Ireland.
The abbreviations used are: GA, glycosylasparaginase; AGU, aspartylglycosaminuria; MIR, multiple isomorphous replacement; r.m.s., root mean square.
2 T. Cui, T. J. O'Loughlin, C. Guan, and H.-C. Guo, manuscript in preparation.
3 H.-C. Guo and Q. Xu, unpublished observation.
4 T. Cui, unpublished results.
5 C. Guan, unpublished results.
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REFERENCES |
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