Evidence for the Involvement of Glu-355 in the Catalytic Action of Human beta -Hexosaminidase B*

(Received for publication, December 5, 1996)

Mark Pennybacker §, Christina G. Schuette §par , Bernd Liessem , Stefan T. Hepbildikler , Jennifer A. Kopetka , Matthew R. Ellis , Rachel Myerowitz **, Konrad Sandhoff and Richard L. Proia ‡‡

From the  Section on Biochemical Genetics, Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the  Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn Germany, and the ** Biology Department, St. Mary's College of Maryland, St. Mary's City, Maryland 20686

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
Note Added in Proof
REFERENCES


ABSTRACT

In a previous study the photoactivable affinity probe, 3-azi-1-[([6-3H]2-acetamido-2-deoxy-1-beta -D-galactopyranosyl)thio]-butane, was used to identify the active site of beta -hexosaminidase B, a beta -subunit dimer (Liessem, B., Glombitza, G. J., Knoll, F., Lehmann, J., Kellermann, J., Lottspeich, F., and Sandhoff, K. (1995) J. Biol. Chem. 270, 23693-23699). The probe predominately labeled Glu-355, a highly conserved residue among hexosaminidases. To determine if Glu-355 has a role in catalysis, beta -subunit mutants were prepared with the Glu-355 codon altered to either Ala, Gln, Asp, or Trp. After expression of mutant proteins using recombinant baculovirus, the enzyme activity associated with the beta -subunits was found to be reduced to background levels. Although catalytic activity was lost, the mutations did not otherwise affect the folding or assembly of the subunits. The mutant beta -subunits could be isolated using substrate affinity chromatography, indicating they contained intact substrate binding sites. As shown by cross-linking with disuccinimidyl suberate, the mutant beta -subunits were properly assembled. They could also participate in the formation of functional beta -hexosaminidase A activity as indicated by activator-dependent GM2 ganglioside degradation activity produced by co-expression of the mutant beta -subunits with the alpha -subunit. Finally, the mutant subunits showed normal lysosomal processing in COS-1 cells, demonstrating that a transport-competent protein conformation had been attained. Collectively the results provide strong support for the intimate involvement of Glu-355 in beta -hexosaminidase B-mediated catalysis.


INTRODUCTION

The human lysosomal beta -hexosaminidases (EC 3.2.1.52) are composed of two structurally related subunits, alpha  and beta  (for reviews see Refs. 1 and 2). After synthesis, the precursor subunits dimerize and form three isozymes: beta -hexosaminidase A (alpha beta ), beta -hexosaminidase B (beta beta ), and beta -hexosaminidase S (alpha alpha ). Proper folding and assembly of the subunits is required for expression of catalytic activity and for transport of the enzymes out of the endoplasmic reticulum (3, 4). After reaching their final destination within lysosomes, the precursor polypeptides undergo proteolytic processing to a mature lysosomal form.

The beta -hexosaminidases degrade glycoproteins, glycolipids, and proteoglycans through the release of terminal beta -glycosidically linked N-acetylglucosamine or N-acetylgalactosamine residues. Each subunit possesses an active site within the context of the dimeric structure of the holoenzyme. The alpha  and beta  active sites, although functionally very similar, exhibit differences in their ability to hydrolyze sulfated substrates (5). Significant substrate specificity differences also exist between the isozymes. Most notably, only the heterodimer beta -hexosaminidase A together with the GM21 activator protein is able to degrade GM2 ganglioside at significant rates (6). The GM2 activator protein functions through binding the ganglioside substrate and interacting with beta -hexosaminidase A. The release of the terminal N-acetylgalactosamine residue from the ganglioside is accomplished via the alpha -chain active site (5, 7, 8). However, the beta -subunit contributes essential functions in this reaction, possibly in promoting the interaction with the activator protein (9).

Mutations in the HEXA (alpha -subunit) and HEXB (beta -subunit) genes cause Tay-Sachs and Sandhoff diseases, respectively. Defects in the GM2A gene result in GM2 activator deficiency. In each of these genetic disorders there is a massive accumulation of GM2 ganglioside and related glycolipids in neuronal lysosomes, leading to severe neurodegeneration. In addition to GM2 ganglioside and related glycolipids, glycosaminoglycans are also critical substrates as demonstrated by the severe mucopolysaccharidoses phenotype exhibited in mice lacking both subunits of hexosaminidase (10).

Although much progress has been made in understanding the genetics and biochemistry of the hexosaminidases, details concerning the structures of the active sites and mechanisms of catalysis have only recently emerged. The first biochemical evidence localizing an active site was presented in a study by Liessem et al. (11) using the photoaffinity reagent, [3H]ATB-GalNAc. This probe was shown to label Glu-355 of the human beta -subunit, implicating this amino acid in the architecture of the active site. To determine if Glu-355 has a role in beta -hexosaminidase B-mediated catalysis, we altered this residue by site-directed mutagenesis. The mutant beta -subunits lost catalytic activity but retained their ability to bind a substrate analogue, form homo- and heterodimers, function in the context of beta -hexosaminidase A in activator-dependent GM2 degradation, and undergo intracellular transport to lysosomes. These results, together with affinity labeling of Glu-355 and the recently described structure of an evolutionarily related chitobiase (12), provide compelling evidence that Glu-355 has an essential role in the catalytic mechanism of human beta -hexosaminidase B.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

The mutations were made with the Sculptor in vitro mutagenesis system from Amersham Corp. (RPN 1526) using the human beta -subunit cDNA that had been cloned into M13mp18 (13). The mutant oligonucleotides (antisense) were as follows: E355A, CCCAACATTTAAATTCCAC<UNL>TGC</UNL>ATCTCCTCCCAAATG; E355Q, CCCAACATTTAAATTCCAC<UNL>TTG</UNL>ATCTCCTCCCAAATG; E355D, CCCAACATTTAAATTCCAC<UNL>ATC</UNL>ATCTCCTCCCAAATG; E355W, CCCAACATTTAAATTCCAC<UNL>TGC</UNL>ATCTCCTCCCAAATG.

The resultant mutant cDNAs were cloned into the XbaI and SacI sites of the pSVL vector (Pharmacia Biotech Inc.) for expression in COS-1 cells. For the production of baculovirus, the mutant cDNAs were excised from the pSVL vector and subcloned into the XbaI/SmaI sites of the baculovirus shuttle vector pVL1392 (Pharmingen). All mutant constructs were verified by DNA sequencing.

Expression of Mutant Constructs

Clonal recombinant baculovirus containing each of the mutant cDNAs was produced as described except that the virus producing the mutant cDNAs was identified by production of immunoreactive beta -subunits rather than by enzymatic activity (9). The recombinant baculovirus containing wild-type beta -subunit was described previously (14). Production of expression media by infection of insect cells (High Five and SF21) and co-expression of the mutant beta -subunit cDNAs with the alpha -subunit was accomplished as described by Pennybacker et al. (9). COS-1 cells were transfected with mutant cDNAs in the pSVL vector using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. [35S]Methionine labeling and immunoprecipitation have been described (13).

Assay of Mutant Proteins

Enzyme activity was determined with MU-GlcNAc and MU-GlcNAc-6-SO4 as described previously (9). Protein concentrations were measured according to the method of Bradford (15) using bovine serum albumin as a standard. [3H]GM2 ganglioside, labeled in the GalNAc moiety, was used to determine GM2 degrading activity in the presence of recombinant GM2 activator protein (9).

Affinity Chromatography

The affinity gel was prepared as described by Izumi and Suzuki (16) using 2-acetamido-N-(epsilon -aminocaproyl)-2-deoxy-beta -D-glucopranosylamine as an affinity ligand. Small columns were prepared with a 1-ml bed volume and were equilibrated in 10 mM phosphate buffer (pH 6.0) containing 200 mM NaCl. Samples (0.5 ml) of dialyzed and concentrated expression medium were loaded on the affinity columns. Nonspecifically bound protein was removed with 2.5 ml of 10 mM phosphate buffer (pH 6.0) containing 100 mM NaCl. Specifically bound proteins were eluted with 2 ml of 150 µM delta -lactone in equilibration buffer. Proteins were analyzed by 10% Tricine polyacrylamide gels as described by Schägger and von Jagow (17). In the case of E355D, wash-through and eluate were concentrated approximately 5-fold before electrophoresis. Protein bands were visualized by silver staining as described by Blum et al. (18). For Western blotting, equal volumes of eluate were resolved by SDS-polyacrylamide gel electrophoresis as described above. Proteins were transferred to polyvinylidene difluoride membrane. The membranes were blocked overnight at 4 °C and then incubated with goat anti-hexosaminidase B for 1.5 h at room temperature. The membranes were washed with Tris buffer (40 mM Tris-HCl, 340 mM NaCl, 0.1% Nonidet P-40, 0.01% NaN3, pH 7.4). After 1 h of incubation with rabbit anti-goat IgG coupled to alkaline phosphatase followed by washing in Tris buffer, the membrane was developed using 0.7 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.2 mM nitro blue tetrazolium in carbonate buffer (100 mM carbonate, 1 mM MgCl2, 0.01% NaN3, pH 9.8).

Cross-linking

Disuccinimidyl suberate (DSS, 10 mM) was freshly prepared in dimethyl sulfoxide. beta -Subunit preparations (30 ng in 75 mM phosphate buffer, pH 7.5) were incubated in the presence of 0.78 mM DSS at 37 °C for 30 min. Control samples contained an equivalent amount of dimethyl sulfoxide without the cross-linker. The reaction was stopped by the addition of Tris-HCl (pH 7.5) to 27 mM. The samples were electrophoresed on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After probing the membrane with anti-hexosaminidase B, detection was accomplished with the Vistra ECF Western blotting kit (Amersham). Probed membranes were scanned with the Storm 860 Fluorimager from Molecular Dynamics.


RESULTS

The codon specifying Glu-355 in the beta -hexosaminidase beta -subunit cDNA was altered to produce cDNAs with the following changes: E355A, E355W, E355Q, and E355D. The cDNAs were recombined into baculovirus for expression in insect cells. We previously showed that precursor enzyme can be recovered from the expression medium for analysis in a number of biochemical assays (9). The expression medium from cells infected with each recombinant virus was assayed for enzymatic activity with the synthetic substrate, MU-GlcNAc, and for beta -subunit protein by Western blotting (Fig. 1). Abundant beta -hexosaminidase activity was detected in medium from insect cells infected with virus expressing the unaltered beta -subunit (Glu-355). In contrast, expression media containing the Glu-355 mutant beta -subunits resulting from infection with baculovirus carrying any of the mutant cDNAs (E355A, E355Q, E355W, E355D) displayed very low levels of activity (<= 3%) relative to wild-type beta -hexosaminidase B after normalization to the amount of beta -subunit protein present.


Fig. 1. Enzyme activity of Glu-355 (E355) beta -subunit mutants. Cultures of insect cells were infected with recombinant baculovirus containing the unaltered beta -subunit (Glu-355 (E355)) or beta -subunit mutants (E355Q, E355A, E355W, E355D). A portion of each expression medium was used to determine hexosaminidase activity with the synthetic substrate, MU-GlcNAc. Another portion was subjected to Western analysis with antibody to beta -hexosaminidase B (inset). The probed membrane was scanned with a Molecular Dynamics Storm 860 Fluorimager, and the relative amount of beta -subunit in each sample was determined with Image Quant 1.1 software. The activity of each sample was normalized to the amount of beta -subunit protein. The wild-type beta -subunit (Glu-355) was considered to express 100% activity. Determination was carried out in duplicate, and the deviation did not exceed 5%.
[View Larger Version of this Image (29K GIF file)]


The expression media were passed through miniaffinity columns containing the ligand, 2-acetamido-N-(epsilon -aminocaproyl)-2-deoxy-beta -D-glucopranosylamine (Fig. 2). This is the classical affinity ligand used for purification of beta -hexosaminidase via its substrate binding site (16, 19, 20). After washing the columns, elution was carried out with the high affinity competitive inhibitor, delta -lactone, a transition state analogue with a submicromolar Ki. If the mutant subunits retained their substrate binding sites, they should bind to the columns and then be eluted specifically with delta -lactone. The wash-through fraction obtained from each expression medium showed a complex mixture of protein species after electrophoresis on SDS-gels and visualization by silver staining. As expected, a Mr 63,000 polypeptide, the precursor size of the beta -subunit (20), was specifically eluted from the affinity column that had been loaded with expression medium containing beta -hexosaminidase B (Glu-355 (E355), Fig. 2A). Similar results were obtained with the expression media obtained by infection with virus carrying the mutant beta -subunit cDNAs even though only minimal enzyme activity had been found. In each case the Mr 63,000 polypeptide was proven to be the hexosaminidase beta -subunit by virtue of its reactivity with antiserum prepared against beta -hexosaminidase B (Fig. 2B). No comparable peptide was found in the medium from cells infected with wild-type virus (control).


Fig. 2. Purification of beta -subunit Glu-355 (E355) mutants by substrate affinity chromatography. Expression media prepared by infection of insect cells with baculovirus containing the unaltered beta -subunit (Glu-355 (E355)), beta -subunit mutants (E355Q, E355A, E355W, E355D), and the wild-type baculovirus (Control) were passed through mini-columns of 2-acetamido-N-(epsilon -aminocaproyl)-2-deoxy-beta -D-glucopyranosylamine coupled to Sepharose. The wash-through was collected. After rinsing the column, elution was carried out with 150 µM delta -lactone. Samples of the wash-through and elution were electrophoresed on an SDS-polyacrylamide gel. The gels were then silver-stained to reveal proteins (panel A). Portions of the elution fractions were subjected to Western analysis using antibody directed at beta -hexosaminidase B (panel B).
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The affinity-purified preparations were used for determination of kinetic parameters with the synthetic substrate, MU-GlcNAc (Table I). The recombinant hexosaminidase B displayed a Vmax and Km similar to the purified placental enzymes. A low level of activity was detected in the preparation derived from the wild-type virus infection (control). This background activity likely represents a previously described insect cell activity released during viral infection (21) that apparently binds to the affinity column. The Vmax values of each of the affinity-purified mutant preparations were reduced to near the background level of the control virus preparation. The Km values were also close to that of the background insect cell enzyme and may be the result of interference by this endogenous activity.

Table I.

Km and Vmax values of recombinant beta -hexosaminidase beta -subunit preparations purified by substrate affinity chromatography

Measurements were made with MU-GlcNAc. Glu-355 (E355) is the unaltered beta -subunit. Control refers to the preparation obtained by infection of insect cells with the wild-type baculovirus and represents the endogenous insect cell activity.
Enzyme Kma Vmaxa,b

mM µmol/min/mg
 beta -Hexosaminidase A (placental) 0.70 138
 beta -Hexosaminidase B (placental) 0.57 250
E355 0.63 174
E355Q 0.40 5.5
E355A 0.63 9.7
E355W 0.63 8.6
E355D 0.59 6.5
Control 0.56 8.9

a Determinations were carried out in duplicate; deviation did not exceed 5%. Km and Vmax values were calculated from double-reciprocal plots of reaction velocity versus substrate concentration.
b Vmax values were calculated from protein concentrations determined by the Bradford method. Due to the low amount of protein recovered from the affinity column, the mutant preparation Vmax values have an experimental error of <= 50%.

The mutant subunits were tested to ensure their structural and functional integrity in light of their catalytic deficiency. First, mutant beta -subunit preparations were cross-linked with DSS to determine if they had assembled, a state required for enzymatic activity to be expressed (4). As seen in Fig. 3, the catalytically impaired mutant proteins were indistinguishable from catalytically active beta -hexosaminidase B (Glu-355) in their capacity to be cross-linked by DSS.


Fig. 3. Cross-linking of beta -subunit Glu-355 (E355) mutants. Samples of baculovirus expressed beta -hexosaminidase B (Glu-355 (E355)) and beta -subunit mutants (E355Q, E355A, E355W, E355D) were incubated with or without DSS. The samples were electrophoresed on an SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and subjected to Western analysis using antibody directed at beta -hexosaminidase B. The protein band marked by the asterisk appears after the incubation at 37 °C and likely represents a proteolytic breakdown product.
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Next, we tested whether the catalytically deficient mutant beta -subunits could function in the context of the heterodimer beta -hexosaminidase A in activator-dependent GM2 ganglioside degradation. The alpha -subunit was co-expressed with each beta -subunit to produce heterodimers. The resulting expression media were assayed for the capacity to hydrolyze labeled GM2 ganglioside in the presence of activator protein. All mutant beta -chains, in concert with the alpha -subunit, were competent in their capacity to degrade the ganglioside (Fig. 4). The GM2 ganglioside-degrading activities of the expression media containing the mutant heterodimers ranged from 77 to 120% of the activity of co-expressed wild-type alpha - and beta -subunits (Fig. 4).


Fig. 4. Activator-dependent hydrolysis of GM2 ganglioside by heterodimers of the alpha -subunit and beta -subunit Glu-355 (E355) mutants. Insect cells were co-infected with recombinant baculovirus containing alpha -subunit together with the unaltered beta -subunit or mutant beta -subunits (E355Q, E355A, E355W, E355D). The Control sample was from an infection with wild-type baculovirus. The expressed enzyme in the culture media was assayed for degradation of [3H]GM2 ganglioside in the presence of recombinant GM2 activator protein. The activity is expressed as µmol of GM2 ganglioside cleaved/h/unit of MU-GlcNAc-6-SO4 activity. Experiments were carried out in duplicate, and deviations did not exceed 10%.
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Finally, the mutant subunits were expressed in COS-1 cells to determine their intracellular fate (Fig. 5). After transfection, the mutant constructs did not produce enzyme activity above background levels (not shown). However, the mutant constructs did direct the production of Mr 63,000 beta -subunit precursors (Fig. 5, Pulse) that were converted to the mature, lysosomal forms (beta a-chain, Mr 29,000 and beta b-chains, Mr 24,000) upon chase in a manner indistinguishable from the unaltered beta -subunit (Glu-355) (Fig. 5, Chase) (13).


Fig. 5. Lysosomal processing of beta -subunit Glu-355 (E355) mutants in COS-1 cells. The unaltered beta -subunit (Glu-355 (E355), mutant beta -subunits (E355Q, E355A, E355W, E355D), and the empty pSVL vector were transfected into duplicate sets of COS-1 cells as indicated. After 48 h the cells were incubated with [35S]methionine for 2 h. One set of cultures was harvested (Pulse). The other was incubated further with unlabeled growth medium for 18 h before harvesting (Chase). The extracts of the radiolabeled cells were subjected to immunoprecipitation with antibody directed at beta -hexosaminidase B followed by SDS-polyacrylamide gel electrophoresis.
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DISCUSSION

Using [3H]ATB-GalNAc as a photoaffinity label for the substrate binding site of beta -hexosaminidase B, Liessem et al. (11) showed a specific incorporation into Glu-355 of the beta -subunit. This result, the strongest biochemical evidence to date in the identification of an active site, focused our attention on this evolutionarily conserved amino acid. We have now extended these studies by analyzing mutant beta -subunits with Glu-355 converted into four other amino acids, Ala, Asp, Trp, and Gln. Each of the mutant proteins showed an abrogation of enzymatic activity to near background levels. Despite the catalytic deficit, other functions and properties associated with the beta -subunit were normal, indicating that the mutations did not adversely affect the folding and quaternary structure of the protein.

The mutant beta -chains underwent normal proteolytic maturation in COS-1 cells indicative of lysosomal delivery. The capacity to pass through the endoplasmic reticulum "quality control" system is evidence for the correct folding and assembly of subunits (22). Mutations in hexosaminidase subunits impairing their folding and assembly leads to premature degradation before lysosome delivery (2). We also directly demonstrated that the subunits were in an assembled state. The mutant beta -subunits were indistinguishable from wild-type beta -subunits in their capacity to be chemically cross-linked. Activator-dependent GM2 ganglioside degradation, a functional assay for heterodimer formation with the alpha -subunit, was also found to be unimpaired. Since ganglioside degradation is mediated through the alpha -subunit active site, it would be expected that a defective beta  active site should not affect hydrolysis. However, the presence of the beta -subunit is essential for ganglioside hydrolysis, perhaps by contributing to the interaction with the activator-ganglioside complex. It is clear that the presence of the Glu-355 mutations did not impair these essential functions.

We found that the mutant proteins could be isolated using a substrate affinity column. This result indicates that the substrate binding site on the mutant proteins is sufficiently intact for interaction with the low affinity ligand, 2-acetamido-N-(epsilon -aminocaproyl)-2-deoxy-beta -D-glucopranosylamine (Ki ~0.5 mM). Mutant proteins that were affinity-isolated through their substrate binding site were catalytically impaired. The presence of substrate binding in association with a large decrease in enzymatic activity is, in itself, strong evidence for the involvement of Glu-355 in the catalytic mechanism of beta -hexosaminidase B. Due to the high degree of sequence similarity between the alpha - and beta -subunits and the functional similarities of their active sites, it is expected that alpha -subunit Glu-323, the counterpart of beta -subunit Glu-355, would also be involved in catalysis mediated by the alpha -subunit. Mutagenesis of alpha -subunit Glu-323 also impairs catalytic activity (not shown).

Recently, the crystal structure of Serratia marcescens chitobiase, a member of the glycosyl hydrolase family that also includes the human beta -hexosaminidases, was determined (12). Based on the structure of the enzyme-substrate complex, an acid-base substrate-assisted catalytic mechanism was proposed in which Glu-540 of the chitobiase functions as the catalytic acid while the polar acetamido group of the sugar substrate serves as a nucleophile in the reaction. Homology modeling of the catalytic domains of the human enzymes suggests that they adhere to the same catalytic mechanism as proposed for chitobiase (12). Significantly, sequence alignments show that Glu-540, the proposed proton donor in chitobiase, coincides with Glu-323 of human alpha -subunit, the equivalent of Glu-355 in the human beta -subunit. Our results, in combination with the structure of the related chitobiase, would indicate that Glu-355 serves as the proton donor during the catalytic action of beta -hexosaminidase B.

Previously, two other amino acids, Arg-211 and Asp-196, were suggested to be involved in the beta -hexosaminidase B catalytic mechanism (23, 24). However, current evidence does not support a direct role for either of these amino acids in this process. The structure of chitobiase indicates that the counterpart of Arg-211 is involved in substrate binding and not in the catalytic mechanism (12). Asp-196 was suggested to serve as the acid catalyst in beta -hexosaminidase B (24). This seems very unlikely given the very strong evidence summarized here supporting Glu-355 for this role and because all proton donors in glycosidases analyzed thus far have been found to be glutamic acid (25).

In conclusion, three independent lines of evidence provide compelling support for the involvement of Glu-355 in the catalytic mechanism mediated by beta -hexosaminidase B. First, Glu-355 was uniquely labeled by an active-site affinity probe. Second, the structure of an evolutionarily related enzyme suggests that the counterpart of Glu-355 functions as the catalytic acid in an acid-base enzyme mechanism. Third, the data presented in this report show that mutant beta -subunits with Glu-355 altered to any of four other amino acids display minimal beta -hexosaminidase B activity even though other properties of the beta -subunit, including formation of the substrate binding site, subunit assembly, activator-dependent GM2 degradation by beta -hexosaminidase A, and intracellular transport, remain intact.


FOOTNOTES

*   This work was supported in part by Grant SFB 284 from the Deutsche Forschungsgemeinschaft.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.
§   Both authors contributed equally to this work.
par    Recipient of a graduate student fellowship by the Boehringer Ingelheim Foundation.
‡‡   To whom correspondence should be addressed: Bldg. 10, Rm. 9D-20, 10 Center Dr., MSC 1810, NIH, Bethesda, MD 20892-1810. Tel.: 301-496-6774; Fax: 301-496-9878. 
1   The abbreviations used are: GM2 ganglioside, GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1Cer; HEXA, the gene encoding the alpha -subunit of beta -hexosaminidase; HEXB, the gene encoding the beta -subunit of beta -hexosaminidase; GM2A, the gene encoding the GM2 activator protein; MU-GlcNAc, 4-methylumbelliferyl-2-acetamido-2-deoxy-beta -D-glucopyranoside; MU-GlcNAc-6-SO4, 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta -D-glucopyranoside; [3H]ATB-GalNAc, 3-azi-1-[([6-3H] 2-acetamido-2-deoxy-1-beta -D-galactopyranosyl)thio]-butane; DSS, disuccinimidyl suberate; delta -lactone, 2-acetamido-2-deoxy-beta -D-glucono-1,5-lactone; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

Acknowledgments

We thank Janet Yancey-Wrona for her comments on the manuscript. We also thank Dr. Thomas Kolter for the synthesis of radiolabeled GM2 ganglioside and Thorsten Lemm for expression of recombinant GM2 activator protein.


Note Added in Proof

After this manuscript was accepted for publication, Fernandes et al. (26) reported that mutagenesis of Glu-323 of the alpha -subunit, the equivalent of Glu-355 of the beta -subunit, impairs catalytic activity, supporting our conclusions.


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