Benign HEXA Mutations, C739T(R247W) and C745T(R249W), Cause beta -Hexosaminidase A Pseudodeficiency by Reducing the alpha -Subunit Protein Levels*

(Received for publication, February 5, 1997, and in revised form, April 3, 1997)

Zhimin Cao Dagger , Emmanuel Petroulakis , Timothy Salo and Barbara Triggs-Raine §

From the Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha -subunit of beta -hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alpha alpha ) and Hex A (alpha beta ) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta -N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha -subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta -N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta -N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha -subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha -subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.


INTRODUCTION

The lysosomal hydrolase, beta -hexosaminidase (beta -N-acetylhexosaminidase, EC 3.2.1.52), has two major isoenzyme forms, A (Hex A)1 and B (Hex B), and a minor form, S (Hex S) (reviewed in Ref. 1). These isozymes are dimers formed from the alpha -subunit encoded by HEXA and/or the beta -subunit encoded by HEXB. The primary natural substrate of this enzyme, GM2 ganglioside, is only hydrolyzed by the alpha beta dimer, Hex A, with the help of the GM2 activator protein (GM2A gene), which binds and solubilizes the sphingolipid for hydrolysis (2, 3). The presence of mutation on both alleles of any one of the genes, HEXA, HEXB, or GM2A, can result in a loss of Hex A (alpha beta ) activity in vivo and GM2 gangliosidosis (reviewed in Ref. 4).

HEXA mutations that cause a complete Hex A deficiency and Tay-Sachs disease, have a higher frequency among Ashkenazi Jews (5) and French Canadians of Eastern Quebec (6). Screening programs for the prevention of this disease have been established (7). They utilize a synthetic substrate, 4-methylumbelliferyl-beta -N-acetylglucosaminide (4-MUG), in combination with a heat denaturation step, to differentiate the activities of the heat-stable Hex B (beta beta ) from Hex A (alpha beta ) (8, 9). Through these programs, healthy individuals with a low in vitro Hex A activity (i.e. pseudodeficient) have been identified (10-15). Pseudodeficiency complicates prenatal diagnosis because a pseudodeficient fetus may be wrongly diagnosed as affected (14, 16).

Two benign mutations, C739T(R247W) and C745T(R249W), have been identified in subjects with Hex A pseudodeficiency (17, 18). Pseudodeficient subjects are compound heterozygotes, typically having the C739T mutation on one chromosome and a common Tay-Sachs disease mutation on the other. Studies of the percentage of Hex A in their samples using the synthetic substrate 4-MUG, revealed very low levels in serum (0-15%), and higher levels in leukocyte (13-24%) and fibroblast (8-26%) samples (10-15). Using GM2 ganglioside, fibroblast-loading studies and enzyme assays gave results in the low normal range (10, 11, 13, 14, 17). Metabolic labeling studies in fibroblasts from two subjects harboring the benign mutation C739T showed that the alpha -subunit protein was processed to its mature lysosomal size (13, 14). Although this analysis was not quantitative, the protein level was consistent with that which might be expected in a Tay-Sachs disease heterozygote. Some investigators proposed that the enzyme's capacity to hydrolyze the synthetic, but not the natural, substrate was reduced (10, 11, 17). Others suggested that there was a reduction in the activity of the enzyme toward both synthetic and natural substrates and that a differential tissue distribution accounted for the very low serum levels (13, 14).

Our aims were to determine 1) if the benign mutations are the primary cause of Hex A pseudodeficiency; 2) if the benign mutations can be differentiated from disease-causing mutations using a transient expression system; and 3) how the benign mutations lead to reduced Hex A activity.


EXPERIMENTAL PROCEDURES

Plasmids, Reagents, and Cell Lines

The vectors pBluescript (pBS) and pSVL were purchased from Stratagene and Pharmacia Biotech, Inc., respectively. The beta -subunit expression vector pCD43 (19) and pTK18 plasmid (20) were gifts from Dr. Roy Gravel (McGill University, Montreal). The beta -galactosidase expression vector, pRc/CMV-beta -gal, was provided by Dr. D. Litchfield (University of Western Ontario, London, Canada). Double-stranded DNA sequencing kits were obtained from Pharmacia; Geneclean II kits from BIO/CAN Scientific (Mississauga, Canada); restriction and DNA modifying enzymes from New England Biolabs or Life Technologies, Inc.; Nucleobond AX kits from the Nest Group Inc. (Southport, MA); alpha -minimum essential medium (alpha -MEM), phosphate-free medium, fetal calf serum, antibiotics, and rabbit polyclonal antiserum against human fibronectin from Life Technologies, Inc.; PansorbinTM from Calbiochem; methionine/cysteine-free minimum essential medium (Met/Cys-free medium) from ICN Pharmaceuticals; 4-MUG and 4-methylumbelliferyl-6-sulfo-beta -N-acetylglucosaminide (4-MUGS) from Toronto Research Chemicals Inc. (Toronto, Canada); dialyzed fetal bovine serum, 4-methylumbelliferyl-beta -galactoside, N-ethylmaleimide, E-64, and leupeptin from Sigma; 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc) from Boehringer Mannheim; and DEAE-Cellulose resin from Pharmacia. A polyclonal antibody against human placental Hex A, provided by Dr. Don Mahuran (University of Toronto) and prepared by Dr. Greg Lee (University of British Columbia, Vancouver, Canada), was a gift from Dr. Roy Gravel (McGill University, Montreal, Canada). The anti-alpha -subunit and anti-Hex B specific polyclonal antibodies prepared in goat (21) were gifts from Dr. Rick Proia (National Institutes of Health). ECL Western blotting detection reagents and [35S]dATP were from Amersham Corp. Tran35S-label (L-[35S]methionine/L-[35S]cysteine) was from ICN Pharmaceuticals. 32P and Entensify solutions were from DuPont NEN. COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD). Fibroblast cell lines from individuals with Hex A pseudodeficiency, TC72 (13) and GM04863 (14), were from Dr. George Thomas (the Kennedy Institute, Baltimore, MD) and the NIGMS (National Institutes of Health) Human Genetic Mutant Cell Repository (Camden, NJ), respectively. These samples were identified as subjects B (GM04863) and F (TC72) in a previous study (17), where they were shown to be compound heterozygotes for the C739T(R247W) mutation and a second mutation associated with Tay-Sachs disease. The second allele in GM04863 was 1278ins4 (22), and in TC72 it was 1073+1Gright-arrowA (23, 24). The control fibroblast cell lines included normal WP09 (Winnipeg), normal MCH065, Tay-Sachs disease WG1881 (both from the Repository for Mutant Human Cell Strains, Montreal, PQ) and Sandhoff disease GM00294 (NIGMS Human Genetic Mutant Cell Repository, Camden, NJ).

Vector Construction and Site-directed Mutagenesis

The alpha -subunit cDNA fragment from pTK18 (NarI/PstI) was subcloned into pBS+ (AccI/PstI) to create pHHEXA2 and into pSVL to create alpha pSVL as described (25). The C739T mutation was introduced into the alpha pSVL cDNA by replacing a NdeI/SnaBI fragment with the same fragment from a polymerase chain reaction product generated from fibroblast cDNA containing C739T. The C745T mutation was introduced with the oligonucleotide 5'-TACGCACGGCTCTGGGGTATCCG-3' using the Unique Site Elimination (USE) mutagenesis method (26). The other mutations were introduced into pHHEXA2 single-stranded DNA following instructions from Bio-Rad, based on the procedure of Kunkel et al. (27) and using T7 polymerase (28). The 5'-end phosphorylated oligonucleotides 5'-TCCTGGGGACCAAGTATCCCTGGA-3', 5'-ACGGCTCCGGGATATCCGTGTGC-3', and 5'-GCTTTCCTCACTGGGGCTTGCTG-3' were used to create the substitutions G805A(G269S) (29, 30), G749A(G250D) (31), and C508T(R170W) (32), respectively. The reaction mix was separated by electrophoresis on a 0.8% agarose gel, the newly synthesized DNA was excised and purified using the Geneclean II kit, and 20-40% of the product was transformed into Escherichia coli/DH5alpha . To identify which colonies contained the mutant vector, the relevant region was polymerase chain reaction-amplified and restriction enzyme-digested with an enzyme whose site was created or destroyed by the mutation (the base change G749A creates an EcoRV site; C508T destroys a HpaII site; G805A creates a ScaI site when an antisense oligonucleotide, 5'-CAAGGAGTCAGTAATCCAGAGTAC-3' containing a single base substitution and base deletion, is used in the polymerase chain reaction). The mutagenized alpha -subunit cDNA fragments were subcloned into pSVL (BamHI/XhoI) to create the various mutant alpha pSVL(s). In all cases, the entire cDNA insert was sequenced to confirm the presence of the desired, but no additional, base changes.

Cell Culture

COS-7 and human fibroblast cell lines were cultured in alpha -MEM containing 10% fetal bovine serum and penicillin (100 units/ml)/streptomycin (100 µg/ml) at 37 °C in 5% CO2. For some experiments, 200 µM leupeptin was added to the alpha -MEM when the cells were about 60% confluent.

DNA Transfection

Plasmid DNA for transfection was isolated using Nucleobond AX columns and quantitated by gel electrophoresis and absorbance at 260 nm. COS-7 cells, grown to 60-80% of confluence, were collected by trypsinization, washed, and resuspended in PBS containing 2.7 mM KCl, 1.1 mM KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4, pH 7.6. Approximately 3 × 106 cells were mixed with the DNA in total volume of 400 µl of PBS for transfection (6.5 µg of alpha pSVL, or its derivatives, alone for Hex S expression, and together with 2.3 µg of pCD43 for Hex A plus S expression). Two µg of pRc/CMV-beta -gal vector, a concentration that did not influence the expressed Hex S and Hex A plus S activities, was co-transfected to express beta -galactosidase as a measure of transfection efficiency. Expression vectors were introduced into the cells by electroporation using an Electro Cell Manipulator 600 (BTX Inc., San Diego, CA) with the settings of 150 charging volts, 48 ohms, and 1200 microfarads (33). The medium was changed at 24 h, and the cells were harvested at 72 h post-transfection.

Preparation of Cell and Medium Extracts

Cell extracts for enzyme assay were prepared differently from that for immunoprecipitation. For enzyme assays, the cell monolayer was washed with ice-cold PBS, scraped off into TEN buffer (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl) and pelleted by centrifugation. Cells were resuspended in a 20 mM Tris-HCl, pH 7.0, and lysed by three rounds of freezing (-70 °C) and thawing (37 °C), and cell debris was removed by centrifugation at 14,000 × g for 20 min. For immunoprecipitation, cell and medium extracts were prepared according to a protocol by Proia et al. (34) except that lysis buffer and the solutions used in the preparation procedures contained 1 mM Pefabloc.

Protein Determination and Enzyme Assay

Protein concentrations were determined by the Bradford method (35) using gamma -globulin as the standard. Hex S and Hex A plus S activities were measured using 4-MUGS or 4-MUG as a substrate (9). beta -Galactosidase activity was determined using 4-methylumbelliferyl-beta -galactoside as a substrate (36).

Separation of Hex A, B, and S Isozymes

Previously described protocols were modified to separate Hex A from Hex B and Hex S (1, 37). A DEAE-cellulose column (1.4-ml bed volume) was equilibrated with 10 mM phosphate buffer, pH 6.0, and 400-900 µl (1.2-3.0 mg of protein) of cell extract was loaded. After a 20-min incubation at 4 °C, the column was sequentially eluted with 6 ml of the phosphate buffer, 20 ml of a linear NaCl gradient (0-0.215 M), 5 ml of 0.215 M NaCl, 5 ml of 0.3 M NaCl, and 5 ml of 0.5 M NaCl, all in the phosphate buffer. One-ml fractions were collected, and the activity in each fraction was measured using 4-MUG and 4-MUGS. The fractions representing each isoenzyme form were pooled, dialyzed, and concentrated; their identity was determined by Western blot analysis. The fractions corresponding to Hex A were used for Km and Vmax determination.

Western Blot Analysis

Cell extract proteins (equal amount of normalized COS-7 protein, typically 25 µg) were separated by SDS-PAGE as described (38). Fibroblast extracts were loaded (20-50 µg) on gels as controls. The proteins were transferred to a nitrocellulose membrane (MSI) according to Sambrook et al. (39). The anti-Hex A antibody (1:5,000) was used to probe the blot, and the bound antibody was detected using a 1:10,000 dilution of anti-rabbit horseradish peroxidase-conjugated antibody and the ECL solutions.

Cell Metabolic Labeling and Immunoprecipitation

Procedures for cell labeling were based on the protocol of Hasilik and Neufeld (40) except that PBS was used to wash the cell monolayer, Met/Cys-free medium supplemented with antibiotics and L-glutamine was used to deplete the intracellular pool of methionine and cysteine, and 20 µl (240 µCi) of Tran35S-label was employed for labeling the cells in 2.9 ml of Met/Cys-free medium supplemented with 150 µl of dialyzed fetal bovine serum. For pulse-chase experiments, 3.6 ml of alpha -MEM supplemented with 10% fetal bovine serum, antibiotics, 0.075 mg/ml L-methionine, and 0.5 mg/ml L-cysteine was used to replace the labeling medium. In the experiments with protease inhibitors, the cells were cultured in the presence of appropriate inhibitors throughout pulse and chase. In the experiments with NH4Cl, the cells were cultured in 3.95 ml of Met/Cys-free medium with 30 µl (360 µCi) of Tran35S-label and 10 mM NH4Cl.

For cell labeling with 32Pi, phosphate-free medium supplemented with sodium pyruvate and L-glutamine was used to rinse the cell monolayer and to deplete the intracellular phosphate. Cells were labeled with a mix of 3.8 ml of this medium, 150 µl of dialyzed fetal bovine serum, and 1 µl of 32P (500 mCi/ml).

For immunoprecipitation, two methods were used. The first method was according to Proia et al. (34) except that three different polyclonal antibodies, against human alpha -subunit, Hex B, and Hex A, were used for immunoprecipitation. In the second method, total alpha -subunit was first immunoprecipitated using anti-Hex A antibody and following the method of Proia et al. (34). The immunoprecipitated complexes were then dissociated by suspending the antibody-antigen-Pansorbin cell pellet in 50 µl of a denaturing solution (20 mM Tris-HCl, pH 7.4, 1% SDS, 20 mM dithiothreitol) and incubating at 100 °C for 10 min followed by a centrifugation. This process was repeated once, and supernatants were combined and mixed with 0.9 ml of lysis buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P40, 0.02% NaN3, 1 mM Pefabloc) containing 1% bovine serum albumin and 4 mM N-ethylmaleimide. N-Ethylmaleimide was used as a chelator of dithiothreitol in the denaturing solution. The total alpha -subunit in the mix was then immunoprecipitated using 4 µl of anti-alpha -subunit antibody and following the method of Proia et al. (34). The process was repeated once to completely recover the alpha -subunit.

High Porous Gradient SDS-PAGE

High porous 5-12% gradient polyacrylamide separating gels (0.075 × 12 × 16 cm) and 4% polyacrylamide stacking gels were prepared and run as described previously (41, 42).

Fluorography and Autoradiography

Signals from 35S-labeled proteins were detected by fluorography according to the instruction provided by the manufacturer of DuPont Entensify solution.

Autoradiography was used to detect the signals of the 32P-labeled proteins. The gel was incubated twice for 30 min in 50 ml of fixing solution (acetic acid:methanol:H2O = 10:20:70), dried, and exposed to Kodak X-Omat film.


RESULTS

To determine the effects of the benign mutations, C739T(R247W) and C745T(R249W), on Hex A activity and the alpha -subunit protein, both a transient expression system and fibroblast cell lines from normal and Hex A pseudodeficient subjects were employed. Hex S was expressed and analyzed in this study because it is an alpha -subunit dimer, and if the benign mutation was present on both of the subunits in Hex S, their effects might be more obvious than on Hex A (alpha beta ). Hex A was also expressed to mimic the physiological form of the enzyme.

Analysis of Expressed Hex S Activity and alpha -Subunit Protein

To determine the effects of the benign mutations on the activity of Hex S and the alpha -subunit protein level, normal and mutant Hex S was expressed in COS-7 cells. Four mutant vectors, containing C739T(R247W), C745T(R249W), G805A(G269S), or C508T(R170W) mutations associated with Hex A pseudodeficiency (two vectors), adult onset and infantile forms of GM2 gangliosidosis, respectively, were expressed. The results showed that normal Hex S activity (1026 ± 200 nmol/h/mg of protein) was expressed to a level more than 10 times that of the beta -hexosaminidase activity in COS-7 cells (82 ± 10 nmol/h/mg of protein). For comparison, the wild-type Hex S activity, after subtracting the COS-7 cell background and normalization, was expressed as 100%. The activities of the various mutant Hex S isozymes were converted to a percentage of the wild-type (Fig. 1). Data revealed that the activity of Hex S harboring the benign mutations, R247W and R249W, was about 20-35% of the wild-type Hex S activity and was substantially higher than that of Hex S containing the G269S substitution that is associated with adult onset GM2 gangliosidosis.


Fig. 1. Expression of Hex S (alpha alpha ) (A) and Hex A plus S (B) activities in COS-7 cells. Both Hex S and Hex A plus S were expressed in COS-7 cells. Normal and mutant alpha pSVL (6.5 µg) were transfected alone for Hex S expression or together with pCD43 (2.3 µg) for Hex A plus S expression. The expressed Hex S and Hex A plus S activities were determined using the synthetic substrate 4-MUGS. The specific activities were normalized after the subtraction of the mock-transfected background levels. Normalization was based on the co-expressed beta -galactosidase activity. The average wild type specific activity was defined as 100%, and the mutant levels are shown as a percentage of the wild type level. The number of experiments (n) represented by each bar is shown. Each experiment contained duplicate transfections. The error bars represent the sample S.D.
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The level of the mature alpha -subunit protein containing the benign mutations was also decreased to about 40% of the normal level (Fig. 2), consistent with its reduced enzyme activity. No detectable mature form, but a normal level of the precursor form, of the alpha -subunit corresponding to the G269S- and R170W-containing Hex S was observed.


Fig. 2. Levels of expressed alpha -subunit protein containing the benign and disease-causing mutations. The alpha -subunit cDNA and its various mutant derivatives were transfected alone to express Hex S (A) or together with the beta -subunit to express Hex A plus S (B and C). The amount of protein loaded for each sample within a panel (A and B, 20-30 µg; C, 5-10 µg) was normalized to the co-expressed beta -galactosidase activity, with the exception of the mock (25-30 µg) and fibroblast (30-50 µg) samples. The alpha - and beta -subunits of beta -hexosaminidase were detected with a polyclonal anti-Hex A antibody. A, lane 1, alpha pSVL (normal); lane 2, C739Talpha pSVL (benign); lane 3, C745Talpha pSVL (benign); lane 4, G805Aalpha pSVL (adult onset); lane 5, C508Talpha pSVL (infantile); lane 6, MCH065 fibroblast (normal); lane 7, WG1881 fibroblast (Tay-Sachs disease); lane 8, GM00294 fibroblast (Sandhoff disease); lane 9, mock-transfected COS-7 cells (background). B, lane 1, alpha pSVL (normal); lane 2, C739Talpha pSVL (benign); lane 3, C745Talpha pSVL (benign); lane 4, G805Aalpha pSVL (adult onset); lane 5, G749Aalpha pSVL (juvenile); lane 6, C508Talpha pSVL (infantile); lane 7, mock-transfected COS-7 (background); lane 8, WP09 fibroblast (normal), lane 9, WG1881 fibroblast (Tay-Sachs disease). C, lanes 1-7, the same as in B; lane 8, WG1881 fibroblast (Tay-Sachs disease); lane 9, GM00294 fibroblast (Sandhoff disease). ×, cross-reacting protein of unknown identity.
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Analysis of Expressed Hex A plus S Activity and alpha -Subunit Protein

To examine the effects of the benign mutations, C739T(R247W) and C745T(R249W), on Hex A plus S activity and the alpha -subunit protein, normal and mutant Hex A plus S were transiently expressed by introducing pCD43, together with alpha pSVL or its mutant variants (including the G749Aalpha pSVL construct, which contains a mutation associated with juvenile onset GM2 gangliosidosis) into COS-7 cells. Once again, the specific activity of the expressed normal Hex A plus S was about 10-fold above the COS-7 cell background (Fig. 1). The specific activity of Hex A plus S harboring the benign mutation, R247W and R249W, was about 38 and 22% of that of the normal Hex A plus S, respectively (Fig. 1). Hex A plus S with the adult onset disease G269S mutation in the alpha -subunit had about 11% of the normal level of activity, significantly less than either benign mutation. The activity of Hex A plus S containing the G749A(G250D) mutation was less than 4% of the normal, consistent with a juvenile onset clinical phenotype (43). The Tay-Sachs disease mutation R170W expressed a specific activity similar to the COS-7 cell background (Fig. 1).

The levels of the mature alpha -subunit protein expressed as Hex A plus S were also correspondingly decreased by the benign mutations (Fig. 2, B and C), but the level of the precursor alpha -subunit containing the benign mutations was similar to that of the normal. The mature alpha -subunit was not detected in the extracts prepared from expression of disease-causing mutations, although a significant amount of the precursor was detected. The differences between the benign mutations and disease-causing mutations appeared more obvious at the level of the mature alpha -subunit protein than at the activity level.

The co-transfection of pCD43 and alpha pSVL in COS-7 cells under the conditions used in these studies (see "Experimental Procedures") resulted in the formation of both Hex A and Hex S; Hex A was predominant, although there was a significant level of Hex S produced (Fig. 3). Anion exchange chromatography showed that Hex A was formed when the beta -subunit was co-expressed with the alpha -subunit carrying the benign mutations (data not shown).


Fig. 3. Separation of the beta -hexosaminidase isoenzyme forms Hex A, Hex B, and Hex S by anion exchange chromatography. Cell extract (1.7 mg of protein) prepared from alpha -/beta -subunit cDNA co-transfected COS-7 cells was loaded onto a DEAE-cellulose column (1.4-ml bed volume), that had been equilibrated with 10 ml of 10 mM phosphate buffer, pH 6.0, and the column was eluted with a linear and then step gradient of NaCl extending to 0.5 M NaCl. The linear NaCl gradient is shown as triangle -·-triangle . Start points for the steps of the gradient are denoted by a down arrow, the number at the top of the arrow indicates the molar concentration of NaCl. Column fractions representing each peak of activity (numbered 1-4) were pooled and concentrated; ~20 µg of fractions 1 and 2 and ~40 µg of fractions 3 and 4 were separated by SDS-PAGE followed by Western blot analysis to determine the identity of the peaks using the anti-Hex A antibody. Inset, lane 1, no NaCl (Hex B); lane 2, 0-0.215 M NaCl (Hex A); lane 3, 0.3 M NaCl (Hex S); lane 4, 0.5 M NaCl (Hex A-related; function unknown); lane 5, extract of COS-7 cells transfected with pCD43/alpha pSVL (normal); lane 6, WG1881 fibroblast (Tay-Sachs disease); lane 7, GM00294 fibroblast (Sandhoff disease).
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The level of normalized Hex S and Hex A plus S activities was found to be influenced by the transfection efficiency. When transfection efficiency was low, higher levels of both beta -hexosaminidase activity and alpha -subunit protein were expressed from mutant vectors in comparison with the normal vector. In Fig. 2B, expressed levels of alpha -subunit protein were higher than those in Fig. 2C, where the transfection efficiency was higher.

Analysis of Hex A in Cultured Fibroblasts

The Hex A activity and the alpha -subunit protein in fibroblasts from Hex A pseudodeficient subjects were compared with that from the normal. Hex A-pseudodeficient fibroblasts had 36-41% of normal Hex A activity (Table I) and a comparable level of mature alpha -subunit protein (data not shown). This is consistent with previous studies where pseudodeficient fibroblasts had Hex A activity that was 23-26% of the total beta -hexosaminidase activity compared with 49-65% of Hex A for the normal (13). Leupeptin, a lysosomal protease inhibitor at 200 µM (44) did not increase the percentage of the specific activity of Hex A (Table I) or the level of mature alpha -subunit protein (data not shown) in either the normal or mutant fibroblasts.

Table I. beta -Hexosaminidase A activity in cultured fibroblast cells

The activities were determined by incubating a 30-µl reaction mix containing 4-8 µg of cell extract protein and 20 µl of 4 mM 4-MUGS in citrate-phosphate buffer, 0.3% bovine serum albumin, pH 4.4, for 120 min at 37 °C. The activities are defined as nmol of 4-MUGS hydrolyzed/h/mg of protein. The data represent the average of four experiments using three plates of each fibroblast cell line. The enzyme assay on each plate was carried out in triplicate.

MCH065 TC72 GM04863 WG1881 GM00294

 -Leupeptina 332  ± 64 120  ± 16 137  ± 19 13  ± 3 43  ± 3
Percentageb 100 36 41 4 13
+Leupeptinb 419  ± 52 141  ± 8 154  ± 5 15  ± 2 78  ± 5
Percentageb 100 34 37 4 19

a ± leupeptin indicates the presence (+) or absence (-) of leupeptin in the cell culture medium.
b Hex A activities of the Hex A pseudodeficient cell lines, TC72 and GM04863, a Tay-Sachs disease cell line WG1881, and a Sandhoff disease cell line, GM00294, are presented as a percentage of the activity of a normal fibroblast cell line MCH065 (100%).

Isolation and Property Studies of Mutant Hex A

Hex A was separated from Hex B and Hex S as shown in Fig. 3. The 0-0.215 M NaCl gradient, followed by 0.215 M NaCl, eluted almost all Hex A from the column, and 0.3 M NaCl eluted all of the Hex S (Fig. 3). The activity peaks corresponding to Hex A and Hex S possessed activities toward both the synthetic substrates, 4-MUG and 4-MUGS. The Hex B peak had activity only toward 4-MUG.

The identity of the eluted activities was confirmed by Western blot (Fig. 3, inset). Only the beta -subunit was detected in the Hex B fractions; alpha - and beta -subunits were in the Hex A fractions; and only the alpha -subunit was in the 0.3 M NaCl-eluted Hex S fraction. An additional peak, of unknown identity, was eluted with 0.5 M NaCl. This fraction contained both the alpha - and beta -subunits and has previously been observed (37).

Kinetic Studies

The benign mutations, R247W and R249W, do not have a significant effect on the Km of Hex A for the synthetic substrates, although the apparent Vmax was substantially decreased (Table II). To determine if the lower apparent Vmax was the result of a change in catalytic activity of Hex A or a reduction of the alpha -subunit protein, the alpha -subunit protein levels of equal Hex A activities toward 4-MUGS were analyzed by Western blot. The levels of protein in normal and Hex A-pseudodeficient fibroblasts and the COS-7 cells transfected with normal and benign mutation-containing alpha pSVLs were similar (Fig. 4). This indicated that the benign mutations affect Hex A by reducing the alpha -subunit protein level, not by affecting Hex A's affinity toward the synthetic substrates and/or Hex A's catalytic activity.

Table II. Kinetic studies of Hex A expressed in COS-7 cells and fibroblasts

The Hex A activities were determined by incubating a reaction mix containing a similar level of DEAE column-isolated Hex A activity with 7-9 various concentrations of 4-MUG (0.133-8.133 mM) or 4-MUGS (0.133-10.66 mM) in citrate/phosphate buffer, 0.3% bovine serum albumin, pH 4.4, in 30 µl at 37 °C for 30 min (120 min for 4-MUGS). The apparent Km and Vmax values were determined using the direct linear plot method (45).

4-MUG
4-MUGS
Km Vmax Km Vmax

mM µmol/h/mg mM µmol/h/mg
WP09a 0.83  ± 0.17 (n = 2) 62.0  ± 3.6 (n = 3) 0.56  ± 0.03 (n = 2) 2.6  ± 0.22 (n = 2)
TC72/R247Wa 0.65  ± 0.10 (n = 2) 15.9  ± 1.14 (n = 2) 0.48  ± 0.11 (n = 3) 0.60  ± 0.15 (n = 3)
COS-7/R249Wb 0.60  ± 0.10 (n = 2) 10.46  ± 0.03 (n = 2) 0.50  ± 0.10 (n = 2) 1.07  ± 0.02 (n = 2)

a Hex A was isolated from normal (WP09) and Hex A pseudodeficient (TC72) fibroblast cells.
b Hex A was isolated from pCD43/alpha pSVLR249W-transfected COS-7 cells. n denotes the number of experiments. Each experiment included two or three separate sets of assays.


Fig. 4. Comparison of the alpha -subunit protein and Hex A plus S activity. Portions of fibroblast cell extracts, containing 8250 fluorescent units/90 min of activity (lanes 1-3), and of transfected COS-7 cell extracts, containing 4000 fluorescent units/30 min of activity (lanes 4-6), measured using 4-MUGS as the substrate, were separated by SDS-PAGE. Western blot analysis of the protein was done with the polyclonal anti-Hex A antibody. Lanes 1-3, 1, MCH065 (normal); lane 2, TC72 (benign R247W); lane 3, GM04863 (benign R247W); lane 4, pCD43/alpha pSVL (normal); lane 5, pCD43/C739Talpha pSVL (benign); lane 6, pCD43/C745Talpha pSVL (benign). Panels a and b indicate cell extracts from duplicate cell culture plates. Lane 7, WG1881 (Tay-Sachs disease); lane 8, GM00294 (Sandhoff disease).
[View Larger Version of this Image (45K GIF file)]

Heat Sensitivity of Benign Mutation-containing Hex A plus S

The activity of Hex A and S harboring the benign mutation, R247W or R249W, in the alpha -subunit, decreased at a rate similar to that of the normal enzyme at 45 or 50.1 °C (data not shown). The results from fibroblast and expressed Hex A plus S were shown to be similar (data not shown). Similar results were also previously reported using fibroblast extract and a treatment at 37 °C (14). Hex A plus S containing the adult onset mutation G269S was shown to be more sensitive than normal (data not shown; Ref. 25). These suggested that the benign mutations do not increase the heat sensitivity of the alpha -subunit in vitro.

Optimal pH of Benign Mutation-containing Hex A

The optimal pH for the hydrolysis of the synthetic substrate by Hex A in the cell extracts from MCH065 and TC72 fibroblasts and the COS-7 cells, transfected with pCD43/alpha pSVL or pCD43/C739Talpha pSVL or pCD43/C745Talpha pSVL, was 4.0-4.4 (data not shown).

Effect of the Benign Mutations on alpha -Subunit Processing and Stability in Vivo

To determine the effects of the benign mutations on Hex A and S in transfected COS-7 cells and on Hex A in the fibroblasts from a normal and a Hex A-pseudodeficient subject with the C739T(R247W) on one chromosome and a null mutation on the other, pulse-chase metabolic labeling was used.

The specificity of the antibody against human Hex A, which was used for immunoprecipitation, was analyzed; it was shown to recognize both the free and beta -subunit-associated forms of the alpha -subunit (data not shown). The anti-Hex B and anti-alpha -subunit antisera were confirmed to have the specificity described by Proia (34). The anti-Hex B antibody recognizes all beta -subunit monomers and beta -subunit containing dimers, while the anti-alpha -subunit antibody recognizes only the alpha -monomer.

Effect of the Benign Mutation, R247W, on Phosphorylation of the alpha -Subunit

Fibroblasts from normal, Hex A-pseudodeficient, Tay-Sachs disease and Sandoff disease subjects were pulse-labeled with 32P. The radiolabeled forms of beta -hexosaminidase were immunoprecipitated using antibodies against human Hex A, Hex B, and the alpha -subunit, separated by gradient SDS-PAGE, and detected by autoradiography. Three separate experiments showed a considerable amount of precursor alpha -subunit in the free and beta -subunit-associated forms in both normal and the C739T benign mutation-containing fibroblasts (data not shown), suggesting that Hex A with the benign mutation was normally phosphorylated. The Tay-Sachs disease sample did not show a 32P-labeled band corresponding to the alpha -subunit, and the Sandhoff disease sample did not exhibit a band corresponding to the radiolabeled beta -subunit.

Effect of the Benign Mutation, R247W, on the Secretion of Hex A

Data derived from three repeated experiments showed that the secretion of the precursor alpha -subunit from both normal and Hex A-pseudodeficient fibroblasts was enhanced by growth in the presence of NH4Cl (Fig. 5). The effect of NH4Cl on the dissociation of Hex A and the mannose 6-phosphate receptor occurs in the prelysosomal and lysosomal compartments (44). Normal secretion suggests that the benign mutation did not affect the processing and targeting of Hex A. 


Fig. 5. Effect of benign mutation on the secretion of Hex A. Fibroblasts from two cell lines, as indicated, were grown to confluence in tissue culture dishes (20 × 100 mm). Each cell line was pulse-labeled with Tran35S-label (0.3 mCi) for 3 h and chased for 20 h in the presence (+) and absence (-) of 10 mM NH4Cl. Similar amounts of protein in medium extracts were analyzed. Three antibodies, as indicated, were used for immunoprecipitation, and one-fourth of the sample was analyzed on a high porous 5-12% gradient SDS-PAGE gel. The radioactive signals were detected by fluorography. Fetal bovine serum was not used in the culture medium to avoid the hydrolysis of the secreted alpha -subunit by the proteases that may be present in the fetal bovine serum.
[View Larger Version of this Image (29K GIF file)]

Effect of Benign Mutations on the Stability of the alpha -Subunit Expressed as Hex S in COS-7 Cells

Expressed Hex S was studied because fibroblasts containing the benign mutation C745T(R249W) were not available and because the transient expression system overexpressing the alpha -subunit provided information not seen in fibroblasts. Results (Fig. 6) showed that the normal and mutant alpha -subunit precursors containing the benign mutation, C739T or C745T, or an adult onset mutation, G805A, were present at a similar level at 3 h of pulse. Benign mutation-containing alpha -subunit precursors appeared to have levels similar to or slightly higher than the wild type, and they were still present at 20 h of chase; however, the G805A mutation-containing alpha -subunit precursor disappeared by 20 h of chase. The mature alpha -subunit containing the benign mutations reached its maximum level by 8 h of chase, about 3 h later than the normal, and its level, at 20 h of chase, was significantly less than normal. The mature alpha -subunit with the G805A mutation was not detectable after 8 h of chase. These results suggested that the benign mutations produced mature alpha -subunit less stable than the wild type but more stable than the adult onset G805A mutation. The delay in the decrease of the benign mutation-containing precursor suggests the rate of conversion from the precursor alpha -subunit to its mature form is delayed. This may reflect an effect on folding or dimerization.


Fig. 6. Effect of benign mutations on the stability of the alpha -subunit as Hex S expressed in COS-7 cells. COS-7 cells were transfected with normal and mutant alpha -subunit cDNA to make Hex S and together with pRc/CMV-beta -gal to express beta -galactosidase as a measure of transfection efficiency. The transfected cells (12 × 106) from four separate electroporations were pooled and evenly dispensed into six tissue culture dishes (20 × 100 mm). The medium was changed at 24 h post-transfection, and the cells were pulse-labeled for 3 h with Tran35S-label (0.15 mCi) at 45 h post-transfection. These were chased for various intervals as indicated. Cells were harvested in 0.5 ml of lysis buffer, and cell extracts were prepared for immunoprecipitation. Two µl of cell extract was used for the protein assay, and 3 µl of cell extract was used to determine beta -galactosidase activity levels. Normalized samples (~0.7 mg of protein), based on the protein concentration and normalization factor derived from beta -galactosidase activity, were taken from each sample, and the volume was equalized to 0.8 ml with lysis buffer containing 1% bovine serum albumin. Antiserum against Hex A was used for immunoprecipitation. One quarter of the sample was analyzed on a high porous 5-12% gradient SDS-PAGE gel, and the labeled proteins were detected by fluorography. Lane 1, alpha pSVL; lane 2, C739Talpha pSVL; lane 3, C745Talpha pSVL; lane 4, G805Aalpha pSVL.
[View Larger Version of this Image (28K GIF file)]

Effect of the Benign Mutation, R247W, on the Stability of the alpha -Subunit in Hex A-pseudodeficient Fibroblasts

To test the stability of the benign mutation-containing alpha -subunit in vivo, the experiments were designed differently from those in the previous reports (13, 14). The differences are that 1) equal amounts of cell extract protein were used for immunoprecipitation, and 2) two approaches were used in immunoprecipitation. The first approach used three types of antibodies to immunoprecipitate both precursors and the mature forms of the alpha - and beta -subunit; the second method used anti-alpha -subunit antibodies in combination with anti-Hex A antibodies to immunoprecipitate only alpha -subunit. Results derived using the first method (data not shown) and the second method (Fig. 7) appeared similar. The benign mutation-containing fibroblasts, at 3 h of pulse, produced about half the level of the alpha -subunit precursor produced from normal fibroblasts. The benign mutation-containing mature alpha -subunit reached its maximal level at about 14 h of chase, similar to the normal. However, by 20 h of chase, the level of benign mutation-containing alpha -subunit was much lower than that of the normal consistent with a defect in stability.


Fig. 7. Effect of the benign mutation R247W on the stability of the alpha -subunit of Hex A in fibroblasts: immunoprecipitation with antiserum against the alpha -subunit. Fibroblasts from four cell lines, as indicated, were grown to confluence in tissue culture dishes (20 × 100 mm). Each cell line was pulse-labeled with Tran35S-label (0.3 mCi) for 3 h and chased for various intervals as indicated. The cell extracts were prepared for immunoprecipitation in 0.65 ml of lysis buffer, and 2 µl of cell extract was used for the measurement of the protein concentration. Cell extracts (480-510 µl) containing equal amounts of protein (1.20 mg) were mixed with lysis buffer containing 1% bovine serum albumin to a final volume of 600 µl. Primary and secondary immunoprecipitations were then done as described under "Experimental Procedures." One-fourth of the sample was analyzed on a high porous 5-12% gradient SDS-PAGE gel. The labeled proteins were detected by fluorography.
[View Larger Version of this Image (30K GIF file)]

Effect of Protease Inhibitors, E-64 and Leupeptin, on the Stability of the alpha -Subunit

Fibroblasts from normal and Hex A-pseudodeficient subjects were analyzed by pulse-chase metabolic labeling in the presence of 280 µM E-64 or 105 µM leupeptin. The lysosomal protease inhibitors had no apparent effect on the stability of the alpha -subunit containing the benign mutation R247W (data not shown). Leupeptin (105 µM) has successfully been used to increase the activity and protein levels of arylsulfatase A in the fibroblasts from adults with metachromatic leukodystrophy (46).


DISCUSSION

The benign mutations, C739T(R247W) and C745T(R249W), were originally identified in subjects with Hex A pseudodeficiency and in enzyme-defined Tay-Sachs disease carriers (6, 16, 17, 42). These mutations were clearly associated with enzyme deficiency, but we could not rule out the possibility that additional mutations, i.e. regulatory mutations, on the same allele were the cause of the enzyme deficiency.

Using a COS-7 expression system, the relationship between the benign mutations and Hex A pseudodeficiency was confirmed. The levels of expressed Hex S and Hex A plus S activities and their corresponding alpha -subunit protein were significantly decreased by the benign mutations C739T and C745T (Figs. 1 and 2). Hex A-pseudodeficient fibroblasts also demonstrated similar results (Table I). This is the first direct evidence that the benign mutations are the primary cause of the Hex A pseudodeficiency.

Benign mutations, C739T and C745T, were readily differentiated from the mutations associated with adult onset and other forms of GM2 gangliosidosis using transient expression (Figs. 1 and 2). Normally there is an overlap between the percent Hex A activity that is associated with various phenotypes, including pseudodeficiency, when Hex A activity is determined in serum, leukocytes, or fibroblasts (10, 13-15). Our results extended a previous study by Brown and Mahuran (25), who showed that alpha -/beta -subunit co-expression could distinguish the adult onset mutation from those associated with more severe forms of GM2 gangliosidosis. Our studies showed that the disease-causing mutations resulted in a lower percentage of Hex A plus S, compared with normal, than that previously reported for the alpha -/beta -subunit co-expression (25). This may be because 1) whole cell extracts were used, instead of immunoprecipitated enzyme, to determine Hex A plus S activity, and (2) there were differences in the ratio of alpha pSVL:pCD43, the DNA concentration, and the method used for DNA transfection.

Data derived from the current studies and previous studies suggest that the benign mutations do not cause disease. The levels of Hex A activity in fibroblasts and leukocytes of Hex A-pseudodeficient subjects with the R247W and R249W amino acid changes (10-15, 17-18) are usually slightly higher than that found in a patient with juvenile or adult onset GM2 gangliosidosis (15). Results from in vitro and in situ GM2 ganglioside hydrolysis assays (13, 14), together with the results from the current transient expression studies, suggest that the level of the enzyme associated with benign mutation is above the critical threshold required for normal GM2 ganglioside hydrolysis. Indeed, several pseudodeficient subjects are presently more than 40 years old (17-18), and patients with the adult onset mutation G805A(G269S) typically show symptoms before the age of 40 years (15, 47). However, one cannot rule out the possibility of a late onset phenotype in these subjects.

The pH optimum of Hex A was not altered by the benign mutations, indicating that the Arg247 and Arg249 residues are not required for proton transfer in the catalytic reaction and are not involved in the catalytic reaction. This is consistent with the prediction that these residues are located on the surface of the alpha -subunit molecule (48), not in the active site where the substrate binding and catalytic reaction occur.

Because the benign mutation did not alter Hex A's affinity for the synthetic substrates and there was clearly a reduction in the alpha -subunit protein level, we attempted to determine the basis of the decreased protein level. Triggs-Raine et al. (17) had previously shown the alpha -subunit cDNA could be amplified after reverse transcription of mRNA isolated from Hex A-pseudodeficient fibroblasts. This indicated that the HEXA gene with the benign mutation, C739T(R247W), was normally transcribed, because the other allele in the subject had the 1278ins4 mutation that does not produce a stable alpha -subunit mRNA (49).

Previous studies (13, 14) also showed an apparently normal amount of the precursor alpha -subunit protein in Hex A-pseudodeficient fibroblasts. This was also observed in our Western blot analysis (Fig. 2), and pulse-labeling analysis (Figs. 6 and 7). Together these results suggest that the benign mutations do not affect the alpha -subunit biosynthesis.

The benign mutation, C739T(R247W), did not affect the phosphorylation of the alpha -subunit or the secretion of Hex A (Fig. 5), indicating that 1) the benign mutations did not cause Hex A to be trapped in the endoplasmic reticulum or the Golgi apparatus, and 2) the mutant Hex A could be normally targeted to the lysosome or to the prelysosomal compartments. These results did not explain the absence of serum Hex A in pseudodeficient subjects.

Further studies using both Hex A in fibroblasts and Hex S expressed in COS-7 cells demonstrated that the benign mutations significantly decreased the stability of the mature alpha -subunit (Figs. 6 and 7). A slight delay in the precursor conversion to its mature alpha -subunit as expressed Hex S was also indicated, but this was not seen in the fibroblasts. The difference might have resulted from the effect of Hex S overexpression in COS-7 cells. These indicated that effects of the benign mutations on the alpha -subunit at the prelysosomal level may also exist.

The protease inhibitors, leupeptin and E-64, did not have an effect on the decreased level of the mature alpha -subunit by the benign mutations. This may be because the benign mutations render the alpha -subunit susceptible to a lysosomal protease(s) that cannot be inhibited by these protease inhibitors, or the mutant alpha -subunit may be susceptible to nonenzymatic factors.

The residues Arg247 and Arg249 fall in a highly conserved region of exon 7 of beta -hexosaminidase from mammals, bacteria, and yeast, suggesting that these residues are important in maintaining normal Hex A function. They have been predicted to locate on the surface of the alpha -subunit molecule and might be involved in maintaining the stability of the alpha -subunit and/or in interacting with the beta -subunit or GM2 activator protein.


FOOTNOTES

*   This work was funded in part by Medical Research Council of Canada (MRC) Grant MT-11708.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.
Dagger    Supported by a Manitoba Health Research Council (MHRC) studentship. Current address: Dept. of Pathology, University of Louisville School of Medicine, Louisville, KY 40292.
§   Supported by a Medical Research Council scholarship. To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E OW3, Canada. Tel.: 204-789-3218; Fax: 204-783-0864; E-mail: triggs{at}bldghsc.lan1.umanitoba.ca.
1   The abbreviations used are: Hex A, B, and S, beta -hexosaminidase A, B, and S, respectively; 4-MUG, 4-methylumbelliferyl-beta -N-acetylglucosaminide; 4-MUGS, 4-methylumbelliferyl-6-sulfo-beta -N-acetylglucosaminide; alpha -MEM, alpha -minimum essential medium; PBS, phosphatebuffered saline; PAGE, polyacrylamide gel electrophoresis; GM2, GM2 ganglioside, GalNAcbeta (1,4)-[N-acetylneuraminic acid (2,3)-]-Galbeta (1-4)-Glc-ceramide.

ACKNOWLEDGEMENTS

We are grateful to Dr. Hans Jacobs for critical review of the manuscript.


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