Identification of Candidate Active Site Residues in Lysosomal beta -Hexosaminidase A*

(Received for publication, April 22, 1996, and in revised form, October 9, 1996)

Maria J. G. Fernandes Dagger §, Sandy Yew §, Daniel Leclerc Dagger , Bernard Henrissat , Constantin E. Vorgias par **, Roy A. Gravel Dagger §Dagger Dagger §§, Peter Hechtman Dagger §Dagger Dagger §§ and Feige Kaplan Dagger Dagger Dagger §§¶¶

From Dagger   McGill University-Montreal Children's Hospital Research Institute, Departments of § Biology, Dagger Dagger  Human Genetics, and §§ Pediatrics, McGill University, Montreal, Canada H3H 1P3,  Centre de Recherches sur les Macromolécules Végétales, CNRS, Grenoble F-38041, Cedex 9, France, and par  European Molecular Biology Laboratory (EMBL), 22603, Hamburg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The beta -hexosaminidases (Hex) catalyze the cleavage of terminal amino sugars on a broad spectrum of glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer of alpha  and beta  subunits (alpha beta ), and Hex B, a homodimer of beta  subunits (beta beta ), have different substrate specificities. The beta  subunit (HEXB gene product), hydrolyzes neutral substrates. The alpha  subunit (HEXA gene product), hydrolyzes both neutral and charged substrates. Only Hex A is able to hydrolyze the most important natural substrate, the acidic glycolipid GM2 ganglioside. Mutations in the HEXA gene cause Tay-Sachs disease (TSD), a GM2 ganglioside storage disorder. We investigated the role of putative active site residues Asp-alpha 258, Glu-alpha 307, Glu-alpha 323, and Glu-alpha 462 in the alpha  subunit of Hex A. A mutation at codon 258 which we described was associated with the TSD B1 phenotype, characterized by the presence of normal amounts of mature but catalytically inactive enzyme. TSD-B1 mutations are believed to involve substitutions of residues at the enzyme active site. Glu-alpha 307, Glu-alpha 323, and Glu-alpha 462 were predicted to be active site residues by homology studies and hydrophobic cluster analysis. We used site-directed mutagenesis and expression in a novel transformed human fetal TSD neuroglial (TSD-NG) cell line (with very low levels of endogenous Hex A activity), to study the effects of mutation at candidate active site residues. Mutant HEXA cDNAs carrying conservative or isofunctional substitutions at these positions were expressed in TSD-NG cells. alpha E323D, alpha E462D, and alpha D258N cDNAs produced normally processed peptide chains with drastically reduced activity toward the alpha  subunit-specific substrate 4MUGS. The alpha E307D cDNA produced a precursor peptide with significant catalytic activity. Kinetic analysis of enzymes carrying mutations at Glu-alpha 323 and Asp-alpha 258 (reported earlier by Bayleran, J., Hechtman, P., Kolodny, E., and Kaback, M. (1987) Am. J. Hum. Genet. 41, 532-548) indicated no significant change in substrate binding properties. Our data, viewed in the context of homology studies and modeling, and studies with suicide substrates, suggest that Glu-alpha 323 and Asp-alpha 258 are active site residues and that Glu-alpha 323 is involved in catalysis.


INTRODUCTION

The beta -hexosaminidases (Hex1, EC 3.2.1.52) are lysosomal hydrolases that catalyze the cleavage of terminal beta -N-acetylglucosamine or beta -N-acetylgalactosamine residues on a broad spectrum of glycoconjugates. The major Hex isozymes in humans are: Hex A, a heterodimer composed of one alpha  and one beta  subunit and Hex B, a homodimer of two beta  subunits. A third isozyme, Hex S, is composed of two alpha  subunits, which are unstable and not normally found in most tissues. The alpha  and beta  subunits are structurally related, sharing 60% amino acid identity in the mature form (1, 2). Both subunits are catalytically active with different but overlapping substrate specificities (3). The beta  subunit, in Hex A and Hex B, hydrolyzes neutral substrates, whereas the alpha  subunit, in Hex A and Hex S, hydrolyzes neutral substrates as well as substrates bearing a negative charge either on the terminal sugar (e.g. GlcNac-SO4) or on a distinct residue (e.g. sialic acid) (4). The latter includes the most important natural substrate, the sialic-acid containing glycosphingiolipid, GM2 ganglioside, found mainly in neuronal tissue. Only Hex A catalyzes cleavage of the terminal beta -N-acetylgalactosamine on GM2 ganglioside in the presence of the substrate-specific protein cofactor, the GM2-activator protein (5).

Mutations in the HEXA, HEXB, and GM2A genes, encoding the alpha  and beta  subunits of Hex A and the GM2 activator, respectively, lead to a group of inherited neurodegenerative diseases, collectively known as the GM2 gangliosidoses, that are characterized by lysosomal accumulation of GM2 ganglioside mainly in neuronal tissue. These disorders range in severity from Tay-Sachs disease (TSD), a progressive and fatal neurodegenerative disorder of infancy, to clinically milder or later onset forms of GM2 gangliosidosis occurring in patients with some residual enzyme activity (reviewed in Gravel et al. (6)).

Although more than 70 mutations at the human HEXA locus, and 14 at the HEXB locus have been described (6), few have revealed information about the location or properties of the alpha  and beta  subunit active sites. A subset of HEXA mutations, known as the B1 mutations, lead to production of normal amounts of mature Hex A, which is deficient in alpha  subunit catalytic activity without affecting beta subunit activity (7). The B1 mutations are thus compatible with normal maturation of Hex subunits and delivery of structurally intact Hex A to the lysosome. The presence of a mature Hex A, unable to hydrolyze charged substrates, suggested that the B1 biochemical phenotype might be associated with mutations at or near the alpha  subunit active site (4, 7, 8). The first B1 mutations described were alpha R178H and alpha R178C (7, 8, 9). In vitro generated mutation at the homologous site to Arg-alpha 178 in the beta  subunit, Arg-beta 211, resulted in production of mature Hex B devoid of catalytic activity (10). More recently, we identified a third B1 mutation, alpha D258H (11). Both Arg-alpha 178 and Asp-alpha 258 have been proposed as candidates for participation at or near the active site of the alpha  subunit.

The mechanism of cleavage of glycosidic bonds by Hex remains unclear. Sinnott (12) has proposed that glycosyl hydrolases employ an acid-catalysis mechanism involving the participation of two acidic residues (a proton donor and a nucleophile) at the active site, as identified in glycoside cleavage by beta -glucosidases (13, 14, 15, 16), alpha -glucosidases (17), beta -glucanases (18, 19, 20), beta -galactosidases (21, 22, 23), and chitinases (24, 25) and the beta -N-acetylglucosaminidase (chitobiase) (26). In contrast, based on inhibition studies using nitrogen-containing substrate inhibitors, Legler and Boothagen (27) and Legler et al. (28) proposed that the mechanism of Hex catalysis involves both an acidic and a basic residue and depends on a transition state in which the glycosidic O of the substrate is joined with the acetyl group of the pyranose ring, a mechanism fundamentally different from that of other glycosidases.

In this study, we investigated candidate active site residues in the alpha  subunit of Hex A through analysis of mutations that affect catalytic activity without disrupting maturation of the enzyme. We introduced conservative mutations at residue Asp-alpha 258, as well as at three glutamic acid residues (Glu-alpha 307, Glu-alpha 323, and Glu-alpha 462) which are evolutionarily invariant in family 20 of glycosyl hydrolases using the classification system of Henrissat (29). We then studied the impact of these substitutions on enzyme activity and maturation after transfection of mutant cDNAs using an SV40-transformed neuroglial (NG) cell line established from a fetus with Tay-Sachs disease which is devoid of endogenously expressed alpha  subunits but produces functional beta  subunits.


EXPERIMENTAL PROCEDURES

Cell Culture

NG141 (NG) and NG125 (TSD-NG), SV40-transformed fetal TSD and normal neuroglial cell lines, respectively, were provided by L. Hoffman and S. Brooks (Kingsbrook Jewish Medical Center, Brooklyn, NY). Cells were cultured in alpha -minimum essential medium with 15% fetal calf serum and antibiotics.

Recombinant Plasmids

The Escherichia coli beta -galactosidase gene, in pSVLbeta gal (Clontech), was used as a reporter gene to control for transfection efficiency. The cloned human HEXA cDNA (1) was inserted into pRCCMV and pREP4 (Invitrogen) to produce pRCCMValpha and pREP4alpha . Preparation of PRCCMValpha involved: 1) subcloning the HEXA cDNA from PSVLalpha into pCRII (Invitrogen) as an XhoI/BamHI fragment, 2) subcloning of an NsiI HEXA cDNA-containing fragment PCRII subclone into the PstI site of pBluescript (Stratagene), 3) subcloning an XhoI/XbaI fragment from pBluescript into a PCRII intermediate, and 4) cloning a NotI/XbaI HEXA cDNA-containing fragment from the PCRII subclone into PRCCMV. Preparation of pREP4alpha involved cloning the XhoI/BamHI HEXA cDNA-containing fragment from PSVLalpha into pREP4. Construction of pCMValpha required creating a HEXA cDNA flanked by NotI sites. The HEXA cDNA insert in pRCCMValpha has a NotI site at the 5' end of the gene. This insert was subcloned into the BamHI site of pBluescript (KS-, Stratagene) introducing a second NotI site downstream from the 3' end of the gene. This step permitted the cloning of the insert into the unique NotI site of pCMValpha . All plasmids were purified on Qiagen columns prior to transfection.

Site-directed Mutagenesis

Mutations were introduced into the HEXA cDNA using a modified protocol of the Clontech Transformer Mutagenesis Kit. The second screening step for mutant clones was omitted. Mutant clones were identified after the first screen by PCR amplification of samples of isolated disrupted bacterial colonies followed by restriction enzyme digestion or allele-specific hybridization of amplified product to identify mutant genotypes. Mutations were introduced into pSVLalpha , a cassette containing the altered sequence was subcloned into pBS(KS)HEXA, and the full-length mutant cDNA was subsequently subcloned into pCMV. Mutant pCMValpha inserts were sequenced (Pharmacia T7 sequencing kit) and the plasmids purified on Qiagen columns prior to transfection.

Transfection

For transient expression in cell line NG 125, subconfluent T175s (175 cm2, Sarstedt) were harvested by trypsinization and washed twice with 1 × phosphate-buffered saline. The cell pellet was resuspended in Optimem medium (Life Technologies, Inc., containing 5% fetal bovine serum) to obtain a final concentration of 6 × 106 cells/ml. Cell suspension (800 µl), 20 µg of pCMValpha , and 2 µg of pSVLbeta gal were placed in a 0.4-cm cuvette, mixed, placed on ice for 5 min, and pulsed (500 microfarads, 400 V) using a Bio-Rad electroporation apparatus. The time constant was between 16 and 18 ms. After 15 min on ice, 800 µl of alpha -minimum essential medium (without antibiotics) was added to the suspension. For the beta -galactosidase qualitative assay, 250 µl of transfected cell suspension were plated on 12 multiwell plates. The remainder of the transfected cell suspension was grown for 48 h in a T75 (75 cm2).

Enzyme and Protein Assays

A qualitative beta -galactosidase assay was performed to determine the percentage of surviving cells which expressed bacterial enzyme. Multiwell-plated cells were incubated as described by Lake (30). After 24-h incubations the number of blue cells was estimated microscopically. Harvested cells were lysed by freeze-thawing in 0.25 M Tris-HCl (pH 7.4) and protein determined by the Bradford method (Bio-Rad). A fluorescent assay (31) was adapted for quantitation of beta -galactosidase activity in transfected cell lysates. The reaction mixture contained 3 µl of 100 × magnesium solution (4.5 M 2-mercaptoethanol, 0.1 M MgCl2), 100 µl of 0.5 mM 4 methyl-umbelliferyl beta -D-galactoside, and approximately 2-5 µg of lysate protein in 0.1 M sodium phosphate buffer (pH 7.5) in a volume of 334 ml. After incubation at 37 °C for 15 min, fluorescence, due to release of 4MU, was determined using a Perkin-Elmer spectrofluorimeter (excitation wavelength, 360 nm; emission, 447 nm). Hexosaminidase activity was also determined fluorometrically using either 4MUGS (alpha  subunit substrate) (32) or the 4MUG (beta  subunit) substrate.

Western Blot Analysis

The enhanced chemiluminescence (ECL) Western blotting kit from Amersham Corp. was used to detect the presence of the Hex A alpha  subunit with a polyclonal rabbit anti-human Hex A antibody. Both the primary and secondary (rabbit Ig, horseradish peroxidase-labeled antibody) antibodies were used at a 1:5000 dilution.

Chromatofocusing of Transfected Cell Extracts

The hexosaminidase isoenzyme profile in cell lysates was determined by chromatofocusing using the Pharmacia Polybuffer Exchanger (PEB) system according to a modified protocol of O'Dowd et al. (33). Transfected cell extracts were freeze-thawed (three times) in 0.025 M imidazole buffer (pH 7.4), and the protein concentration was determined by the Bradford method. All steps were carried out at 7 °C. Approximately 1 ml of PEB74 slurry was used to prepare a column in a 1-ml syringe. The column was washed with the equivalent of 1.5-2 × bed volume with 0.025 M imidazole. The protein extract was added after passing 2 × 100 µl of Polybuffer (pH 4.0) through the column. Four hundred-microliter fractions were collected. Sodium citrate buffer (0.13 M, pH 3.46) was used to elute Hex S after a pH of 4.0 was reached using a pH gradient.

Kinetic Analysis

The Km and Ki for wild type and mutant Hex A proteins were determined using 4MUGS substrate at concentrations from 1.0 to 7.5 mM. For inhibition studies, the competitive inhibitor N-aetylglucosamine-6-phosphate (Sigma) was added to a final concentration of 10 mM.


RESULTS

Expression of the HEXA Gene in TSD-NG Cells

The endogenous activity of NG cells toward the alpha -specific substrate 4MUGS was 715 ± 13 nmol/mg/h. In untransfected TSD-NG cells, the rate of 4MUGS hydrolysis is <1% of that in NG cells. This trace activity is probably due to the residual action of Hex B on 4MUGS (32).

In order to maximize alpha  subunit expression through transfection, several vectors carrying the HEXA gene cDNA were assessed for their ability to drive expression of enzymatic activity when transfected into TSD-NG cells. Transfection efficiency (percent surviving cells catalyzing 5-bromo-4-chloro-3-indoyl beta -D-galactoside hydrolysis) was determined to be 10-20%. Hexosaminidase activity in cells transfected with 20 µg of plasmids pSVLalpha , pRCCMValpha , pREP4alpha , or pCMValpha was at least 10-fold higher than activity in mock-transfected cells (Table I). The highest level of expression (approximately 1000 × mock-transfected cells) was achieved when cells were transfected with pCMValpha . Further analysis of Hex activity in TSD-NG cells at 24, 48, and 72 h post-transfection with pCMValpha (Table I) showed that activity continued to increase throughout the 72-h period. The pCMValpha plasmid expressed over a 48-h incubation period post-transfection was selected for all subsequent experiments.

Table I.

HEXA gene expression in TSD-NG cells


Vector Time harvesteda Hex activityb

h nmol/h/mg
Untransfected NG cells 715 (±13)
Untransfected TSD-NG cells 3 (±2)
pSVLalpha c 48 43 (±15)
pRCCMValpha 48 358 (±52)
pREP4alpha 48 611 (±12)
pCMValpha 24 1825 (±246)
pCMValpha 48 3986 (±529)
pCMValpha 72 7145 (±365)

a  Hours post-transfection.
b  Nanomoles of 4MUGS hydrolyzed/h/mg of protein.
c  All transfections into TSD-NG cells.

Western blot analysis of transfected (pCMValpha and pSVLbeta gal) and mock-transfected (pSVLbeta gal) cell extracts confirmed that the increase in 4MUGS activity in TSD-NG cells is associated with alpha  subunit expression (Fig. 1, lane N). Both precursor and mature alpha  subunit are absent in mock-transfected TSD-NG cell extracts (Fig. 1, lane M) and present in cells transfected with pCMValpha .


Fig. 1. Western blot analysis of TSD-NG cells transfected with A, pCMValpha G269S (HEXA mutation associated with adult onset TSD); J, pCMValpha G250D (HEXA mutation associated with juvenile onset TSD); I, pCMValpha R170W (HEXA mutation associated with infantile classical TSD); and N, pCMValpha (wild type alpha  subunit). alpha p and alpha m indicate precursor and mature forms of Hex A alpha  subunit. Lane M represents mock-transfected cells.
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In order to determine whether the alpha  subunits encoded by the HEXA cDNA were expressed as the heterodimeric enzyme Hex A (alpha beta ) or the homodimeric species Hex S (alpha alpha ), the Hex isozyme forms in transfected TSD-NG cell lysates were resolved by chromatofocusing. Fig. 2a illustrates the chromatofocusing profile of TSD-NG cells transfected with pCMValpha and assayed with 4MUG (all Hex) and 4MUGS (Hex A and Hex S) to detect all Hex isozyme species. All three hexosaminidase isoenzymes were present, and eluted at their expected pIs. Hex S was the isoenzyme present in greatest abundance followed by Hex A and Hex B respectively. This high expression of the nonphysiological Hex S in TSD-NG cells is the most likely due to the massive overexpression of alpha  subunits in the face of limiting, endogenously produced beta subunits. Total Hex activity in pCMValpha transfected TSD-NG cells (harvested at 48 h post-transfection) is 5-6-fold greater than endogenous Hex activity in untransfected normal NG cells (Table I). Given an efficiency of transfection of 10-20%, the transfected cells express up to 60-fold greater Hex activity than normal untransfected NG cells. Despite overwhelming alpha  subunit synthesis, a significant proportion (22%) of the transfected gene product is expressed as the Hex A heterodimer as shown by the chromatofocusing profile (Fig. 2).


Fig. 2. Chromatofocusing of NG cell extracts transfected with pCMValpha , pCMValpha E323D, and pCMValpha E462D and assayed with the synthetic substrates 4MUG (black-square[sbond--black-square) and 4MUGS (open circle ----open circle ). Activity peaks for Hex B, Hex A, and Hex S are indicated. (----) pH gradient.
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Expression of HEXA Mutations

In order to evaluate the expression of mutant alpha  subunits in TSD-NG cells, we initially examined mutations known to cause the infantile acute (alpha R170W) (11) subacute (alpha G250D) (35) and chronic (alpha G269S) (37) forms of GM2 gangliosidosis. The mutant cDNAs were expressed in TSD-NG cells (Table II) and cell lysates assayed for Hex A specific activity using 4MUGS. Hex A activity in lysates of alpha G269S- and alpha G250D-transfected TSD-NG cells was <4% and 1.5%, respectively, of the activity measured after transfection with wild type pCMValpha . These Hex A activities were 9- and 4-fold greater than mock-transfected activity measured with the 4MUGS substrate. In contrast, expression of alpha R170W exhibited no significant 4MUGS activity above that of mock transfected cells. Western blot analysis showed that the alpha  subunit synthesized by all three mutant cDNAs appeared in the precursor form (Fig. 1, lanes A, J, and I). Only the alpha G269S mutation appeared to be associated with expression of a small amount of mature alpha  subunit (Fig. 1, lane A). These results are compatible with previous findings on alpha G269S (34) and alpha G250D (35) in COS cell expression studies and demonstrate complete inactivation of Hex A by the alpha R170W mutation consistent with the infantile TSD phenotype. In the latter case, the R170W substitution is associated with expression of an alpha  subunit precursor (Fig. 1, lane I) but not with its maturation and targeting to the lysosome.

Table II.

Expression of GM2 gangliosidosis mutations


Vector Age of onset of TSDa Hex/beta -galactosidase activityb

pCMValpha Normal 49.0 (±15)
pCMValpha G269S Adult 1.5 (±0.8)
pCMValpha G250D Juvenile 0.67 (±0.3)
pCMValpha R170W Infantile 0.20 (±0.08)
Mock 0.16 (±0.02)

a  Age of onset of TSD in patients carrying indicated mutations.
b  The numbers express a ratio. Hex and beta -galactosidase activity were expressed as nanomoles/h/mg of protein.

Analysis of Putative Active Site Residues

We next examined the expression in TSD-NG cells of mutations at amino acid residues which are candidates for participation in the Hex A alpha  subunit catalytic site. To assess the role of mutation at residue Asp-alpha 258 (alpha D258H), first described in association with the B1 biochemical phenotype of TSD, HEXA cDNA constructs carrying the conservative or isosteric substitutions alpha D258H, alpha D258E, or alpha D258N were expressed in TSD-NG cells and Hex A activity measured in transfected cell lysates. All three mutations resulted in negligible or trace amounts of Hex A activity, with alpha D258N exhibiting 2-fold background and the others the same activity as measured in mock-transfected cells (Table III, Experiment 1). Western blot analysis of transfected cell extracts (Fig. 3), revealed that maturation of pro-alpha chains carrying the alpha D258H mutation was reduced. In contrast, expression alpha D258N and alpha D258E mutant cDNAs resulted in production of mature alpha  subunit. Chromatofocusing of alpha D258N cell extracts revealed that this mutation significantly reduced 4MUGS hydrolysis by Hex A and Hex S (data not shown). The 4MUG hydrolysis of Hex A was reduced to a lesser extent. This result was expected since 4MUG hydrolysis is catalyzed by both the alpha  and beta  subunits of Hex A. 

Table III.

Effect of substitutions at putative active site residues on Hex A activity


Genotype Hex/beta -galactosidase ratio

Experiment 1 
  Wild type 88.0 (±32)
  alpha D258H 0.15 (±0.04)
  alpha D258E 0.15 (±0.05)
  alpha D258N 0.37 (±0.06)
  Mock 0.13 (±0.07)
Experiment 2 
  Wild type 36 (±14)
  alpha E307D 3.6 (±0.5)
  alpha E323D 0.2 (±0.09)
  alpha E462D 0.85 (±0.13)
  Mock 0.06


Fig. 3. Western blot analysis of TSD-NG cells transfected with pCMValpha D258H (D/H), pCMValpha D258E (D/E), pCMValpha D258N (D/N,) and pCMValpha (N). Lane M represents mock-transfected cells.
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We then studied the effects of mutation at conserved residues Glu-alpha 307, Glu-alpha 323, and Glu-alpha 462, predicted as candidate active site residues by homology studies and hydrophobic cluster analysis of family 20 glycosyl hydrolases. Fig. 4, a and b, illustrate multiple alignment of the two regions of family 20 enzymes which produce significant alignment. Asterisks indicate the three evolutionarily invariant glutamate residues identified as candidate active site residues using the classification system of Henrissat. Other invariant amino acids are also indicated.


Fig. 4. Multiple alignment of two regions of family 20 glycosyl hydrolases (a and b). Asterisks (*) indicate invariant glutamate residues Glu-alpha 307, Glu-alpha 323, and Glu-alpha 424. Residues at which B1 mutations occur (Arg-alpha 178 and Asp-alpha 258) are marked by (black-square). Other invariant residues are also indicated (·).
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HEXA cDNA constructs carrying the conservative substitutions alpha E307D, alpha E323D, or alpha E462D were expressed in TSD-NG cells and Hex activity measured in transfected cell lysates. All three substitutions had dramatic effects on catalytic activity (Table III, Experiment 2). alpha E323D did not exhibit any 4MUGS activity above background whereas alpha E462D and alpha E307D exhibited 14- and 60-fold 4MUGS activity above the background activity obtained in mock transfected cells respectively. Western blot analysis of transfected cell extracts showed that only the alpha E323D and alpha E462D mutations were compatible with synthesis of significant amounts of precursor and mature alpha  subunit (Fig. 5). Chromatofocusing of alpha E323D and alpha E462D cell extracts confirmed that the Hex A isoenzyme is processed normally but has a catalytically defective alpha  subunit that is unable to hydrolyze 4MUG or 4MUGS (Fig. 2, b and c). The alpha E307D mutation, however, dramatically reduces alpha  subunit maturation.


Fig. 5. Western blot analysis of TSD-NG cells transfected with pCMValpha E307D (307), pCMValpha E323D (323), pCMValpha E462D (462), and pCMValpha (N). Lane M represents mock-transfected cells.
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Kinetic experiments were performed to evaluate the impact of the alpha E323D mutation on the binding affinity of Hex A for the substrates 4MUGS and for the competitive inhibitor N-acetylglucosamine-6-PO4 (Fig. 6). The Lineweaver-Burke plots for the wild type enzyme are monophasic, representing the contribution of a single active site to the hydrolysis of 4MUGS. In contrast, the kinetics of hydrolysis of this substrate by the Hex A alpha E323D enzyme are biphasic revealing a significant contribution of a second active site (the beta  subunit) at high substrate concentrations. When the kinetic parameters of the high affinity site are evaluated the Km for 4MUGS is 2.5 mM for Hex A alpha E323D compared to 1.7 mM for the wild type enzyme. Similiarly Ki values for the competitive inhibitor are 15.6 mM for the alpha E323D-substituted Hex A and 11.0 mM for wild type Hex A. We showed, previously, that the Km for Hex A in fibroblasts from a patient carrying the D258H allele (and a null allele) was identical to that of wild type enzyme (7).


Fig. 6. Lineweaver-Burk plots of wild type and alpha E323D mutant Hex A for hydrolysis of 4MUGS in the absence (black-diamond ), or presence (black-triangle) of 10 mM N-aetylglucosamine-6-phosphate. Km for wild type Hex A = 1.7 mM. Km for alpha E323D Hex A = 2.5 mM. Ki for wild type Hex A = 11.0. Ki for alpha E323D Hex A = 15.6 mM.
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DISCUSSION

beta -Hexosaminidase catalyzes the hydrolysis of its glycoside substrates by a "retaining" mechanism in which the anomeric configuration of the substrate remains unchanged (12, 36). Most retaining glycosidases employ an acid catalysis mechanism in which two amino acids function respectively as proton donor and nucleophile (12). Alternatively, glycosaminidases may employ a second mechanism, termed substrate-assisted catalysis, in which an acidic residue and a basic residue participate in substrate cleavage. In this study, we attempted to identify acidic residues in the Hex A alpha subunit which may participate in the active site of Hex A.

Since our studies involved the use of a novel TSD-NG expression system, we first evaluated expression in this system of wild type and mutant HEXA cDNAs. Transient expression of mutant HEXA cDNAs in COS1 cells (reviewed in Brown and Mahuran (34)) has several disadvantages which are overcome in the TSD-NG expression system. COS1 cells exhibit a high level of endogenous Hex A and Hex B activity which compromises the analysis of low levels of Hex activity expressed by mutant HEXA cDNAs. Furthermore, because the cells are of heterologous origin, human HEXA gene mutations are expressed in COS1 cells as the non-physiological isozyme Hex S (7, 37, 38), an alpha alpha homodimer which differs from Hex A both in stability and substrate specificity. As a result, the activity of mutant alpha  subunits produced following transfection of mutant HEXA cDNAs, does not correlate well with clinical severity of disease or biochemical phenotypes observed in patients (34, 35).

In contrast, TSD-NG cells, produce no endogenous alpha  subunits. Endogenous expression of human beta  subunits in these cells, however, permits dimerization with transfected human alpha  subunits, to form active Hex A. When wild type alpha  subunits are overexpressed in TSD-NG cells, beta  subunit concentration limits the formation of Hex A. As the beta  subunits become depleted, excess alpha  subunits dimerize to form Hex S, distinguishable from Hex A by chromatofocusing. The presence of both the precursor (67 kDa) and mature (54 kDa) forms of alpha  subunit following transfection confirms that newly synthesized alpha  subunit can be normally processed and targeted to the lysosome, but also indicates that excess alpha  subunits are poorly processed in the absence of beta  subunits.

We evaluated the expression in TSD-NG cells of alpha  subunits carrying mutations associated with acute (alpha R170W), subacute (alpha G250D), and chronic (alpha G269S) forms of GM2 gangliosidosis, respectively, and demonstrated that residual enzyme activity was inversely correlated with disease severity. Results for the alpha R170W mutation confirmed our previous report (11), that this mutation causes the classical infantile form of TSD. We proceeded to use this expression system to evaluate the role of candidate active site residues.

The four acidic amino acids we examined were chosen on the basis of (a) occurrence in the B1 variant of Tay-Sachs disease (Asp-alpha 258) or (b) conservation in family 20, a sequence related family of glycosyl hydrolases (alpha 307, alpha 323, and alpha 462). The TSD-B1 phenotype is believed to be caused by mutations which lead to substitution of residues with catalytic function (4, 7, 10). These include the basic residue Arg-alpha 178 (4, 8, 9, 10) (which is invariant in family 20 enzymes) and the acidic Asp-alpha 258 (7, 10). Conservative substitutions at these positions have little affect on Km but dramatically reduce the Vmax of Hex A (7). The residues Glu-alpha 307, Glu-alpha 323, and Glu-alpha 462 were selected on the basis of their evolutionary invariance in family 20 glycosyl hydrolases (39), a classification system (29) based on amino acid sequence similarity. Members of homology families generally share folding characteristics and catalytic residues within families are strictly conserved (40). The three glutamate residues are invariant at homologous positions in all members of family 20. The position corresponding to Asp-alpha 258 shows variation in a single enzyme which carries the functionally related acidic residue glutamate at this position.

Analysis of residual enzymatic activity and alpha  subunit maturation in cells transfected with HEXA cDNAs carrying conservative or isosteric substitutions at residues Glu-alpha 307 and Glu-alpha 462 led us to conclude that these residues are unlikely to have catalytic functions. Residual Hex A activity associated with both mutations was too high to be compatible with the loss of either an acid catalyst or a proton donor. Furthermore, the alpha E307D substitution prevented alpha  subunit maturation (Fig. 5). In contrast, both the isosteric substitution alpha D258N and the conservative substitution alpha E323D were associated with production of mature Hex A with drastically reduced enzyme activity. Since maturation of the alpha  subunit is dependent on dimerization with the beta  subunit, we were also able to conclude that the alpha D258H and alpha E323D mutations are compatible with production of normal amounts of alpha beta heterodimer and that loss of activity is not the result of compromised dimerization. Furthermore, kinetic analysis of residual Hex A activity in alpha E323D transfected cells (Fig. 6) or alpha D258H in patient fibroblasts (7) demonstrates that loss of enzymatic activity is the consequence of decrease in kcat rather than a change in Km.

Our proposal that Asp-alpha 258 and Glu-alpha 323 are catalytic residues is in accord with results of other structure/function studies. The x-ray crystallographic structure for Serratia marcescens chitobiase, the first member of family 20 glycosyl hydrolases to be crystallized, was recently reported (26, 40, 42). Residues Glu-519 and Glu-740 of chitobiase are not located at or near the active site of the bacterial enzyme suggesting, by analogy, that the homologous residues, Glu-alpha 307 and Glu-alpha 462, in the alpha  subunit of Hex A would also not be present at the the alpha  subunit active site. Most significantly, the x-ray crystallographic data (26) identify Glu-540, the residue corresponding to Glu-alpha 323, as the acid catalyst in the S. marcescens enzyme.

Members of family 20 glycosyl hydrolases share a conserved central region which aligns in all members of the family (Fig. 4). According to the crystal structure of chitobiase, this region comprises the catalytic domain which has an alpha /beta barrel fold (26). The modeling of the catalytic domain of Hex A (Fig. 7) (26), brings Arg-alpha 178, Asp-alpha 258, and Glu-alpha 323 in proximity within the substrate binding cleft (facing toward the center of the alpha /beta barrel) and spatially arranged to facilitate catalysis. In contrast, Glu-alpha 307, Glu-alpha 462, and Asp-alpha 163 which corresponds to Asp-beta 196, a residue recently proposed as an active site residue in the beta  subunit (44), appear to be remote from the catalytic domain. While it is likely that the model, illustrated in Fig. 7, will contain some errors in its proposed structure of Hex A, the high degree of conservation of residues in and around the active site suggest that the model is much less prone to errors in the active site pocket of the enzyme.


Fig. 7. Predicted structure of the (beta alpha )8 of hexosaminidase showing the apposition of residues Arg-alpha 178, Asp-alpha 258, and Glu-alpha 323 in the active site pocket.
[View Larger Version of this Image (60K GIF file)]


A role for Glu-alpha 323 at the Hex A active site is also supported by the studies of Liessem et al. (43). Using a mechanism-based pyrrolidine substrate analog, this group identified residue Glu-beta 355, which corresponds to Glu-alpha 323, as the only reactive residue at or near the beta  subunit active site. The role of Arg-alpha 178 is less clear. Given the high pKa of arginine (12.0) it is unlikely that this residue functions as a nucleophile at the pH optimum of Hex (3.9-4.2). The role of active-site arginine may be to maintain the acid catalyst in its protonated state at a pH which is significantly higher than the pKa for dicarboxylic amino acids.

The extensive sequence similarity of the alpha  and beta  subunits underscores the structural and catalytic similarity of two subunits of Hex A. Chimeric Hex enzymes, generated by the fusion of different segments of the alpha  and beta  subunits have recently been used to identify domains required for substrate specificity (41, 45). The structural modelling of Hex should now permit the more precise localization of other residues involved in the active site as well as in other functions of the enzyme, such as dimerization, substrate binding and activator recognition.


FOOTNOTES

*   This work was supported by grants from the Medical Research Council of Canada (to P. H. and F. K.) and the Canadian Genetic Diseases Network (to R. A. 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.
**   On leave at Athens University.
¶¶   To whom correspondence should be addressed: McGill University-Montreal, Children's Hospital Research Institute, 2300 Tupper St., Montreal, PQ Canada H3H 1P3. Tel.: 514-934-4417; Fax: 514-934-4329.
1    The abbreviations used are: Hex, hexosaminidase; TSD, Tay-Sachs disease; HEXA, HEXB, and GM2A, the genes for the alpha  and beta subunits of Hex A and the GM2 ganglioside activator protein, respectively; 4MU, 4-methylumbelliferone; 4MUG, 4-methylumbelliferone beta -N-acetylglucosamine; 4MUGS, 4-methylumbelliferone N-acetylglucosamine-6-sulfate; NG, neuroglial; PCR, polymerase chain reaction.

Acknowledgments

We thank Franelli Yadeo and Daniel Phaneuf for advice and technical assistance.


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