From the Department of Biochemistry, Tulane
University School of Medicine, New Orleans, Louisiana 70112 and
§ Dipartimento di Biologia Cellulare e Molecolare,
Universita degli Studi di Perugia, Via del Giochetto,
06126 Perugia, Italy
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ABSTRACT |
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Tay-Sachs disease, an inborn lysosomal disease
featuring a buildup of GM2 in the brain, is caused by
a deficiency of -hexosaminidase A (Hex A) or GM2
activator. Of the two human lysosomal Hex isozymes, only Hex A, not Hex
B, cleaves GM2 in the presence of GM2
activator. In contrast, mouse Hex B has been reported to be more active
than Hex A in cleaving GM2 (Burg, J., Banerjee, A.,
Conzelmann, E., and Sandhoff, K. (1983) Hoppe Seyler's Z. Physiol. Chem. 364, 821-829). In two independent studies, mice
with the targeted disruption of the Hexa gene did not
display the severe buildup of brain GM2 or the concomitant
abnormal behavioral manifestations seen in human Tay-Sachs patients.
The results of these two studies were suggested to be attributed to the
reported GM2 degrading activity of mouse Hex B. To clarify
the specificity of mouse Hex A and Hex B and to better understand the
observed results of the mouse model of Tay-Sachs disease, we have
purified mouse liver Hex A and Hex B and also prepared the recombinant
mouse GM2 activator. Contrary to the findings of Burg
et al., we found that the specificities of mouse Hex A and
Hex B toward the catabolism of GM2 were not different from
the corresponding human Hex isozymes. Mouse Hex A, but not Hex B,
hydrolyzes GM2 in the presence of GM2
activator, whereas GM2 is refractory to mouse Hex B with or
without GM2 activator. Importantly, we found that, in
contrast to human GM2 activator, mouse GM2
activator could effectively stimulate the hydrolysis of GA2
by mouse Hex A and to a much lesser extent also by Hex B. These results
provide clear evidence on the existence of an alternative pathway for
GM2 catabolism in mice by converting GM2 to
GA2 and subsequently to lactosylceramide. They also provide the explanation for the lack of excessive GM2 accumulation
in the Hexa gene-disrupted mice.
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INTRODUCTION |
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Human tissues contain two major isoforms of lysosomal
-hexosaminidase (Hex),1
Hex A, a heterodimeric protein composed of
- and
-subunits, and
Hex B, a
-subunit homodimer (1, 2). These two isoforms have also
been reported to exist in other mammals (3). Human Hex A hydrolyzes the
GalNAc from GM2 in the presence of a specific protein
cofactor, GM2 activator (4-6). Human Hex B, on the other hand, is not able to hydrolyze GM2 with or without
GM2 activator (7-10). A deficiency of Hex A or
GM2 activator causes Tay-Sachs disease in humans, a
lysosomal storage disease characterized by an excessive buildup of
GM2 in the central nervous system (11). Burg et
al. (3) reported that, in sharp contrast to human Hex isozymes,
the partially purified Hex B prepared from several different mammalian
tissues were able to degrade GM2 and that rat Hex B degraded GM2 more effectively than the Hex A. They also
reported that the mouse activator preparation made from heat-treated
mouse kidney extract was only slightly effective in stimulating the hydrolysis of GM2 by mouse Hex A and inhibited mouse Hex B
in the same reaction. Recently, in two independent studies, mice with
the targeted disruption of the Hexa gene were found to
display neither the severe buildup of brain GM2 nor the
concomitant abnormal behavioral manifestations seen in human classical
Tay-Sachs patients (12, 13). In both studies, the mild manifestations
were attributed to the reported GM2 degrading activity of
mouse Hex B (3). Based on the fate of the radioactive GM1
fed to embryonic fibroblasts derived from Hexa
/
and
Hexb
/
mice, Sango et al. (14) proposed the
presence of an alternative pathway in mice where sialidase acts upon
GM2 to produce GA2 which can be hydrolyzed
subsequently by Hex A or Hex B.
To clarify the role of the mouse Hex A and Hex B in the catabolism of GM2 and also to understand better the observed results of the mouse models of classical Tay-Sachs disease (Type B GM2 gangliosidosis), we have purified mouse liver Hex A and Hex B. We have also prepared the recombinant mouse GM2 activator. Using the recombinant human and mouse GM2 activators, we have studied the requirement of these two protein cofactors in the hydrolysis of GM2 and GA2 by mouse Hex A and Hex B. We have also studied the cross-reactivity of human and mouse GM2 activators by studying the stimulation of mouse Hex A by human GM2 activator and of human Hex A by mouse GM2 activator.
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EXPERIMENTAL PROCEDURES |
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Materials-- GM2 was isolated from the brain of a Tay-Sachs patient (15). GA2 was prepared from GM2 by mild acid hydrolysis (16). II3NeuAcGgOse3 was prepared from GM2 using ceramide glycanase (17). Goat anti-human Hex A was a kind gift of Dr. Richard L. Proia, Section of Biochemical Genetics, Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, MD. The following were purchased from commercial sources: frozen mouse livers (Swiss-Webster strain), Pel-Freez; precoated Silica Gel 60 thin layer chromatography plates, Fractogel EMD DEAE-650(M) and Fractogel SP-650(S), Merck (Darmstadt, Germany); protein standards for molecular weight and pI, FPLC Superose 6 and Mono P columns, Sephacryl S-300-SF, Polybuffer 74, Pharmacia Biotech Inc.; phenylmethylsulfonyl fluoride, Pierce; peroxidase-conjugated rabbit anti-goat IgG, 4-chloro-1-naphthol, MUG, Coomassie Brilliant Blue R-250, Trizma base, glycine, Sigma; MUGS, Research Development Corp., Toronto, Canada; Centricon-10 (10,000 molecular weight cutoff) micro-concentrators, Amicon.
Expression of Murine GM2 Activator--
A
pBluescript vector containing a 1.1-kilobase cDNA encoding the
mouse GM2 activator (18) was used as a template to generate by polymerase chain reaction a shortened version of the encoding sequence which was homologous to the mature human GM2
activator (19). The upstream primer was
5-ATG-ATG-GAT-CCG-GTG-GCT-TCT-CCT-GGG-ATA-3
and the downstream primer
was 5
-CAG-GCA-AGC-TTG-CTG-CTG-CCA-GGT-TAT-CTG-3
. This cDNA
segment was subcloned into the pT7-7 expression vector at
BamHI and HindIII sites, and its sequence was
verified to contain the 486-base-pair DNA fragment corresponding to
amino acids 32-193 of the mouse GM2 activator (18). The
recombinant mouse GM2 activator was expressed and purified
according to the procedures described previously for the human
GM2 activator (19). The NH2-terminal amino acid
sequence of the purified mouse GM2 activator was confirmed by a pulse-liquid protein micro-sequencer equipped with an
on-line microbore phenylthiohydantoin-derivative analyzer (Applied
BioSystems).
Enzyme Assays-- Enzyme activity was determined by using fluorogenic substrates MUG and MUGS according to Potier et al. (20). The enzyme was incubated with 1.5 mM of substrate in 50 mM sodium citrate buffer, pH 5.0, in a total volume of 50 µl at 37 °C. After a set time, 1.5 ml of 0.2 M sodium borate buffer, pH 9.8, was added to the reaction mixture to stop the reaction. The released MU was determined using a Sequoia-Turner Model 450 fluorometer. One unit of enzyme activity is defined as the amount that liberates 1 µmol of MU/min at 37 °C. For glycolipid substrates GM2 and GA2, the reaction mixture contained 3 nmol of substrate in 40 µl of 10 mM sodium acetate buffer, pH 5.0. The reactions were stopped by adding 40 µl of ethanol, and the mixtures were dried under vacuum, redissolved in 20 µl of chloroform/methanol (2:1, v/v), and applied onto a thin layer chromatography plate. The plates were developed by chloroform/methanol/water (60:35:8, v/v/v), sprayed with diphenylamine reagent (21), and heated at 115 °C for 15 min to visualize glycoconjugates.
Kinetic Analysis-- Initial rate measurements and determination of kinetic parameters for the enzyme-catalyzed hydrolysis of synthetic substrates were conducted similarly to that described previously (22). The reactions were carried out in 20 mM sodium citrate buffer, pH 5.0, using 0-5.0 mM of the substrates MUG and MUGS.
Isoelectric Point Determination-- Purified mouse liver Hex A and Hex B were examined by FPLC chromatofocusing in a pH range of 7.4-3.8 using a Mono P HR 5/20 (0.5 × 20 cm) column. The starting buffer was 25 mM imidazole-HCl, pH 7.4, and the running buffer was Polybuffer 74 adjusted to pH 3.8 using HCl as described in the Pharmacia manual. After applying the sample onto the Mono P column, the column was eluted with the running buffer at 0.5 ml/min and 0.5-ml fractions were collected.
Molecular Mass Determination-- The molecular masses of purified mouse Hex A and Hex B were determined using Superose 6 FPLC gel filtration in 50 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. The column was first calibrated under the same conditions using ferritin (440,000), catalase (232,000), aldolase (158,000), ovalbumin (49,500), and chymotrypsinogen A (25,000) as molecular weight standards.
Purification of Mouse Liver Hex A and Hex B-- All operations were performed at 0-5 °C except the chromatographies on Con A-Sepharose and SP-Fractogel that were carried out at room temperature. Centrifugation was routinely carried out at 30,000 × g for 50 min using a Sorvall RC5C refrigerated centrifuge. Unless otherwise indicated, ultrafiltration was carried out with an Amicon stirred cell using a PM-10 membrane. Two hundred frozen mouse livers (391 g) were homogenized using a Polytron (Brinkmann) homogenizer with 5 volumes of cold phosphate-buffered saline (10 mM sodium phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4) containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride as protease inhibitors, followed by centrifugation. The supernatant was brought to 30% saturation with solid ammonium sulfate. After 2 h, the precipitate was removed by centrifugation, and the supernatant was further brought to 65% saturation with solid ammonium sulfate. After standing overnight, the precipitate was collected by centrifugation and resuspended in 500 ml of 10 mM sodium phosphate buffer, pH 7.0 (buffer A). The suspension was placed into several dialysis bags and dialyzed against 10 liters of buffer A overnight, changing buffer every 4 h (4 changes). This crude enzyme preparation (780 ml) was centrifuged and applied to a DEAE-Fractogel column (5 × 45 cm) equilibrated with buffer A. The column was washed overnight with buffer A at 2 ml/min, and proteins were eluted with a linear gradient of NaCl from 0 to 0.5 M in the same buffer (total volume 4 liters), and 20-ml fractions were collected. Fractions were assayed for both MUG- and MUGS-cleaving activities. Hex B, which cleaves only MUG, was eluted in the nonadsorbed fractions (Fig. 1) and was concentrated by ultrafiltration. As shown in Fig. 1, MUG-cleaving activity eluted with NaCl as a main peak with a leading shoulder. The shoulder contained very low MUGS-cleaving activity, whereas the main peak contained both MUG- and MUGS-cleaving activities. Fractions in the main peak were pooled and concentrated to make a crude mouse Hex A preparation. This preparation was applied to a Sephacryl S-300 column (5 × 90 cm) equilibrated with 50 mM sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. The column was eluted with the same buffer at 1 ml/min, and 20-ml fractions were collected. MUG- and MUGS-cleaving activities coeluted as a broad peak, and the entire peak was pooled (Fig. 2A) and concentrated to 25 ml by ultrafiltration. The concentrated Hex A was dialyzed thoroughly against buffer A overnight. The crude Hex B preparation (from DEAE-Fractogel column) was dialyzed against buffer A and applied to an SP-Fractogel column (2.5 × 17 cm) equilibrated with buffer A. The column was washed with buffer A at 2 ml/min and eluted with a linear gradient of NaCl from 0 to 0.5 M in buffer A (total volume, 500 ml) and 17-ml fractions were collected. Fractions were assayed for MUG-cleaving activity. No activity was detected in the nonadsorbed fractions. The Hex B activity eluted as a single peak starting at 0.1 M NaCl was pooled and concentrated to make an SP-Fractogel-purified mouse Hex B preparation (elution pattern not shown). This preparation was applied to a Sephacryl S-300 column and eluted under the same conditions as the DEAE-Fractogel purified Hex A (Fig. 2B).
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Western Blotting-- The purified mouse liver Hex A after SP-Fractogel chromatography was analyzed by 15% SDS-PAGE (23). The gel was electrophoretically transferred onto a nitrocellulose membrane in 20 mM Tris/150 mM glycine buffer, pH 8.0, containing 20% methanol at 18 V for 4 h using a Bio-Rad transfer apparatus. Membranes were overlaid with goat anti-human Hex A (24, 25) as the primary antibody followed by horseradish peroxidase-conjugated rabbit anti-goat IgG as the secondary antibody. For visualization, the membrane was incubated with 8 mmol of 4-chloro-1-naphthol with 0.01% hydrogen peroxide to produce a purple color. The reaction was stopped by washing the membrane with water.
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RESULTS |
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Purification and Characterization of Mouse Liver Hex A and Hex
B--
The two major Hex isozymes were resolved from the crude mouse
liver extract by DEAE-Fractogel chromatography at pH 7.0 (Fig. 1). The
acidic mouse Hex A was purified to near homogeneity using the scheme
described under "Experimental Procedures" as summarized in Table I.
The Hex A after SP-Fractogel chromatography was used for the subsequent
studies. By SDS-PAGE under nonreducing conditions, the purified Hex A
showed one broad protein band when stained by Coomassie Brilliant Blue
(Fig. 4A, lane 3).
Immunostaining with anti-human Hex A revealed two overlapping bands of
equal intensity corresponding to molecular sizes of approximately 57 and 59 kDa (Fig. 4B, lane 3). This is in agreement with the
postulated makeup of mouse Hex A, which is a heterodimer consisting of
an -subunit and a
-subunit, with molecular sizes before
posttranslational processing of 60 and 61 kDa, respectively, as deduced
from their cDNA sequences (26-28). In humans, the
-subunit is
posttranslationally processed to form two smaller polypeptides,
1
and
2, which are joined by disulfide bonds (29). Fig. 4A,
lane 2, shows that similar processing occurs in mouse Hex A,
with the appearance under reducing conditions of two overlapping bands
of about 27 and 24 kDa, and the concomitant disappearance of the 59-kDa
band. Western blot analysis was used to confirm that the protein band visualized by Coomassie Brilliant Blue staining was indeed Hex A. Goat
anti-human Hex A recognized both the nonreduced mouse Hex
- and
-subunits and the lower molecular size polypeptide chains after
reduction (Fig. 4B). The native molecular sizes of the mouse
Hex A and Hex B were determined to be 110 and 120 kDa, respectively, as
estimated using Superose 6 FPLC gel filtration. These values suggest
that the native structures of mouse Hex A and Hex B consist of dimers
as is the case for human enzymes. The isoelectric points of the two
isoforms were estimated using Mono P FPLC chromatofocusing to be
5.4-3.8 for the purified mouse Hex A and 6.3-5.8 for the mouse Hex B. When crude mouse liver extract was chromatofocused under the same
conditions, MUGS-cleaving activity was detected throughout a broader pH
range extending from pH 6.5 to 3.8.
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Expression and Characterization of Mouse GM2 Activator-- The molecular mass of the recombinant mouse GM2 activator determined by SDS-PAGE was 18.5 kDa, which is as expected from the cDNA sequence and is identical to that of the human GM2 activator. By Western blot analysis, mouse GM2 activator was recognized by the polyclonal antibodies against human GM2 activator, indicating similarities in protein structure, although with a weaker interaction than that for the human GM2 activator.
Hydrolysis of GM2 by Mouse Hex A and Hex B-- The purified mouse Hex A and Hex B were examined for their ability to hydrolyze GM2. As shown in Fig. 5A, the specificities of the mouse Hex A and Hex B toward GM2 are the same as their human counterparts. Under the same conditions, mouse Hex A effectively hydrolyzes GM2 but only in the presence of the mouse GM2 activator (Fig. 5A, lane 4, 88% hydrolysis). Similar to human Hex B, but in contrast to the previous report (3), mouse Hex B is not able to cleave GM2 in the absence of GM2 activator (Fig. 5A, lane 5) even after extended incubation (Fig. 5A, lane 7). While in the presence of GM2 activator, only a very trace of GM3 production by mouse Hex B is detected after 30 min of incubation (Fig. 5A, lane 6) or 6 h of incubation (Fig. 5A, lane 8). These results clearly indicate that mouse Hex B is similar to human Hex B with regard to the specificity for GM2 hydrolysis.
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Hydrolysis of GA2 by Mouse Hex A and Hex B--
To
understand the reported observations on the studies of mouse Hex
-chain disruption (12, 13), we also examined the ability of two
mouse Hex isozymes to hydrolyze the GalNAc from GA2 (Fig.
5B). Under our assay conditions (30 min of incubation), mouse Hex A was found to slowly hydrolyze GA2 in the
absence of mouse GM2 activator (Fig. 5B,
lane 3). We found that even though GA2 is
refractory to human Hex A in the presence of human GM2 activator (19), mouse Hex A was able to effectively hydrolyze GA2 in the presence of mouse GM2 activator
(Fig. 5B, lane 4, 45% hydrolysis). Under the
same conditions, mouse Hex B was not able to hydrolyze GA2
in the absence of mouse GM2 activator (Fig. 5B, lane 5), and no detectable hydrolysis was observed in the
presence of mouse GM2 activator (Fig. 5B,
lane 6) after 30 min of incubation. However, after extended
incubation (6 h of incubation), mouse Hex B was found to be able to
slowly hydrolyze GA2 in the presence of mouse
GM2 activator (Fig. 5B, lane 8).
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DISCUSSION |
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To understand the catabolism of GM2 in mouse, we have
purified and characterized mouse liver Hex A and Hex B and compared their properties with human Hex A and Hex B. As seen with the recombinantly expressed - and
-chains (25), the purified mouse liver Hex A was recognized by goat anti-human Hex A. Purified mouse Hex
A was determined to be composed of 57- and 59-kDa subunits by SDS-PAGE
under nonreducing conditions, and smaller polypeptides were observed in
the presence of 2-mercaptoethanol or dithiothreitol (Fig. 4A,
lane 2). Therefore, mouse Hex A has a similar subunit composition
to human Hex A, with noncovalently linked
- and
-subunits (2).
This is also the first direct evidence that one of the subunits is
composed of nonidentical cystine-linked polypeptide chains, which, by
comparison with the human enzyme, is probably the
-subunit (29).
While the isoelectric points of purified mouse Hex A and Hex B are similar to the isoelectric points of their human counterparts, the presence of mouse Hex A distributed in a wide range of isoelectric points has important consequences for purification. In the past, the separation of the mouse Hex A and Hex B isozymes has been routinely accomplished by passing a preparation over an anion exchange column equilibrated with 10 mM sodium phosphate buffer, pH 6.0-6.5. Hex B is collected in the pass-through fractions, while the retained Hex A is eluted by an NaCl gradient (3, 32). However, these reports followed the method that was originally optimized for the human Hex isozymes (1). We found that in following the previously reported methods (3), the mouse Hex B preparation that was not adsorbed to the DEAE column at pH 6.0-6.5 still contained a small amount of MUGS-cleaving activity. MUGS-cleaving activity has been correlated with the ability to hydrolyze GM2 (33). As reported by Burg et al. (3), we also found that mouse Hex B prepared by this method did contain some GM2-cleaving activity. To ascertain whether this GM2-cleaving activity was inherent in the mouse Hex B or due to contamination by Hex A, we increased the pH of the buffer solution to 7.0 for the separation of the two isozymes by anion exchange chromatography. Interestingly, the amount of MUGS-cleaving activity, as compared with the MUG-cleaving activity, decreased significantly and the resulting Hex B preparation became extremely weak in hydrolyzing GM2 (Fig. 5A), as was observed with human Hex B (11).
From the binding behavior of mouse Hex A to DEAE-Fractogel and also because of its acidic pI, the retention of the enzyme by the SP-Fractogel column at pH 7.0 (Fig. 3) was totally unexpected. This suggests that interactions other than ionic may be involved. This chromatography step was very effective for removing contaminating proteins. Because the Hex B preparation contained other proteins not adsorbed to DEAE-Fractogel at pH 7.0, it is not surprising that the SP-Fractogel chromatography was not as effective for purifying Hex B as for Hex A. Based on the DEAE-Fractogel chromatography, we estimated that approximately 90% of the total MUG-cleaving activity present in the crude mouse liver extract was Hex A and 7% was Hex B. This is in agreement with previous reports of the level of the two isozymes in mouse liver tissues (32). Because the amount of Hex B in mouse liver is very low compared with Hex A it was not practical to purify Hex B to homogeneity as done for Hex A. However, the final Hex B preparation is free from contaminating glycosidases and proved to be suitable for the studies presented.
The recombinant human and mouse GM2 activators were expressed using the shortened version of cDNAs which encode only the mature activator proteins. The cDNA for human GM2 activator encodes for a protein of 193 amino acids that consists of a signal peptide (23 amino acids), a propeptide (8 amino acids), and a mature protein (162 amino acids). The signal and the propeptides are excised proteolytically to form the mature GM2 activator protein (5). In the full-length cDNA encoding for the mouse GM2 activator, the predicted cleavage site is between positions 19 and 20 of the deduced amino acid sequence (34). This site is very close to the cleavage site (positions 23 and 24) of the human sequence (5). Although there is no direct evidence that the first 31 amino acids in the mouse sequence contains a signal peptide and a propeptide, the mouse sequence shows a hydropathy profile similar to that of the human sequence (18). In addition, the recombinant mouse GM2 activator and the native human protein were found to have the same specific activity toward the hydrolysis of GM2, indicating that the mature form of mouse GM2 activator is very likely to start from amino acid 32 as in the case of humans.
As seen with the human Hex isozymes, mouse Hex A hydrolyzes
GM2, with the requirement of the GM2 activator,
whereas mouse Hex B has only a trace of activity to cleave
GM2 with or without GM2 activator. To our
surprise, in contrast to human Hex isozymes, mouse Hex A was also able
to effectively hydrolyze GA2 in the presence of mouse
GM2 activator (Fig. 5B, lane 4). We
were not able to detect the hydrolysis of GA2 by Hex B
without GM2 activator, but when the activator is present,
some hydrolysis of GA2 could be seen after extended
incubation (Fig. 5B, lane 6). These results provide the
explanation for the observations made in mice with disrupted
-subunit gene. Mice defective in Hex A but not Hex B, because of the
disrupted
-subunit were found to show relatively little buildup of
GM2 or GA2 with no behavioral abnormalities, as
compared with humans with defective
-subunits. (12, 13). The fact
that mouse Hex B cannot hydrolyze GM2 but can act on GA2 suggests that in mice GM2 can be converted
to GA2 that serves as a substrate for mouse Hex B. We have
shown previously that clostridial sialidase can effectively convert
GM2 to GA2 in the presence of human
GM2 activator (35). Our results complement the recent
pathobiological findings of the three mouse models of human Tay-Sachs
disease, types B, O, and AB of GM2 gangliosidosis. The
mouse models of type B (Hexa
/
) and O
(Hexb
/
) were generated by targeted disruption of Hex A
(
-subunit) (12, 13) or Hex B (subunit) (36) genes encoding Hex A
(
) and Hex B (
). The model of type AB GM2
gangliosidosis (Gm2a
/
) (GM2 activator deficiency) was produced by targeted disruption of Gm2a gene
(37). Unlike human type B GM2 gangliosidosis, the
Hexa
/
mice were asymptomatic (12, 13), while
Hexb
/
mice (36) were severely affected as in the case
of human type O GM2 gangliosidosis. The Hexb
/
mice accumulated more GM2 and GA2 in the
brain than the Hexa
/
mice. The Gm2a
/
mice (37) showed a phenotype which is intermediate to those of
Hexa
/
(12, 13) and Hexb
/
(36) with
storage of an excess amount of GM2 and a low amount of
GA2. From these three murine models of Tay-Sachs disease,
it has been proposed that Hexa
/
mice escape the disease
through partial catabolism of GM2 via GA2 by
the combined action of sialidase and Hex B (14). The pathogenesis of
Gm2a
/
mice also suggested a role for the
GM2 activator in GA2 degradation in mice
(37).
Our results provide the explanation for the results generated by the above three mouse models. We have demonstrated the ability of mouse Hex A to participate in the catabolism of GA2 and a very weak activity of Hex B toward the degradation of GM2. We have also shown the ability of mouse GM2 activator to stimulate the hydrolysis of GA2 by mouse Hex A and to a lesser extent by mouse Hex B. We have also examined the species specificity of the interactions between the mouse and human Hex isozymes and the activators. Previously, crude activator preparations from other mammalian species (3) and purified mullet roe GM2 activator (38) were found to activate the hydrolysis of GM2 by human Hex A. We have shown here that purified recombinant mouse GM2 activator can effectively stimulate the hydrolysis of GM2 and GA2 by human Hex A. In reverse, human GM2 activator was not effective in stimulating the hydrolysis of GM2 or GA2 by mouse Hex A.
Although the mouse GM2 activator is 73.5% identical to the human protein, it also appears that the mouse activator does not share the specificity to the characteristic branched trisaccharide epitope of GM2 (19) but assists Hex A to hydrolyze GA2 as well. The observation that mouse GM2 activator can stimulate the hydrolysis of GM2 by both human and mouse Hex A, while human GM2 activator can only stimulate the hydrolysis of GM2 by human Hex A but not mouse Hex A, provides strong evidence that the GM2 activator proteins must somehow interact with Hex A. Similarly, the observation that the mouse GM2 activator can stimulate the hydrolysis of both GM2 and GA2 by human Hex A, but the human GM2 activator can only stimulate the hydrolysis of GM2 by human Hex A, shows that the GM2 activators of these two species may have different specificities for the two glycolipids.
Biochemical analysis of enzyme systems is an important complement to molecular and genetic studies in the effort to fully understand the roles of Hex isozymes in mouse. Despite the biochemical similarities between human and mouse Hex isozymes and GM2 activator proteins, the catabolic pathways for GM2 in mouse and human are clearly not identical. Therefore, the murine model for Type B Tay-Sachs disease does not truly reflect its counterpart in man.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS 09626.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.
¶ To whom correspondence should be addressed: Department of Biochemistry SL 43, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112. Tel.: 504-584-2459; Fax: 504-584-2739; E-mail: yli{at}tmcpop.tmc.tulane.edu.
1
The abbreviations used are: Hex,
-N-acetylhexosaminidase; MU, 4-methylumbelliferyl; MUG,
4-methylumbelliferyl-
-GlcNAc; MUGS, 4-methylumbelliferyl-
-GlcNAc-6-S04; GM1,
Gal
1
3GalNAc
1
4(NeuAc
2
3)Gal
1
4Glc
1-1
Cer; GM2,
GalNAc
1
4(NeuAc
2
3)Gal
1
4Glc
1-1
Cer;
GA2, GalNAc
1
4Gal
1
4Glc
1-1
Cer; GM3, NeuAc
2
3Gal
1
4Glc
1-1
Cer;
II3NeuAcGgOse3, the oligosaccharide derived
from GM2; FPLC, fast protein liquid chromatography; SP,
sulfopropyl; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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