(Received for publication, December 5, 1996)
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
In a previous study the photoactivable affinity
probe,
3-azi-1-[([6-3H]2-acetamido-2-deoxy-1--D-galactopyranosyl)thio]-butane,
was used to identify the active site of
-hexosaminidase B, a
-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,
-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
-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
-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
-subunits were properly
assembled. They could also participate in the formation of functional
-hexosaminidase A activity as indicated by
activator-dependent GM2 ganglioside degradation
activity produced by co-expression of the mutant
-subunits with the
-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
-hexosaminidase B-mediated catalysis.
The human lysosomal -hexosaminidases (EC 3.2.1.52) are composed
of two structurally related subunits,
and
(for reviews see
Refs. 1 and 2). After synthesis, the precursor subunits dimerize and
form three isozymes:
-hexosaminidase A (
),
-hexosaminidase B (
), and
-hexosaminidase S (
). 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 -hexosaminidases degrade glycoproteins, glycolipids, and
proteoglycans through the release of terminal
-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
and
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
-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
-hexosaminidase A. The
release of the terminal N-acetylgalactosamine residue from the ganglioside is accomplished via the
-chain active site (5, 7,
8). However, the
-subunit contributes essential functions in this
reaction, possibly in promoting the interaction with the activator
protein (9).
Mutations in the HEXA (-subunit) and HEXB
(
-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 -subunit, implicating this amino
acid in the architecture of the active site. To determine if Glu-355
has a role in
-hexosaminidase B-mediated catalysis, we altered this
residue by site-directed mutagenesis. The mutant
-subunits lost
catalytic activity but retained their ability to bind a substrate
analogue, form homo- and heterodimers, function in the context of
-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
-hexosaminidase B.
The mutations were made with the
Sculptor in vitro mutagenesis system from Amersham Corp.
(RPN 1526) using the human -subunit cDNA that had been cloned
into M13mp18 (13). The mutant oligonucleotides (antisense) were as
follows: E355A, CCCAACATTTAAATTCCAC
ATCTCCTCCCAAATG; E355Q, CCCAACATTTAAATTCCAC
ATCTCCTCCCAAATG; E355D,
CCCAACATTTAAATTCCAC
ATCTCCTCCCAAATG; E355W,
CCCAACATTTAAATTCCAC
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 ConstructsClonal 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 -subunits rather than by
enzymatic activity (9). The recombinant baculovirus containing
wild-type
-subunit was described previously (14). Production of
expression media by infection of insect cells (High Five and SF21) and
co-expression of the mutant
-subunit cDNAs with the
-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).
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 ChromatographyThe affinity gel was prepared as
described by Izumi and Suzuki (16) using
2-acetamido-N-(-aminocaproyl)-2-deoxy-
-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
-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).
Disuccinimidyl suberate (DSS, 10 mM) was freshly prepared in dimethyl sulfoxide. -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.
The codon specifying Glu-355 in the -hexosaminidase
-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
-subunit protein by Western blotting (Fig. 1).
Abundant
-hexosaminidase activity was detected in medium from insect
cells infected with virus expressing the unaltered
-subunit
(Glu-355). In contrast, expression media containing the Glu-355 mutant
-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
-hexosaminidase B
after normalization to the amount of
-subunit protein present.
The expression media were passed through miniaffinity columns
containing the ligand,
2-acetamido-N-(-aminocaproyl)-2-deoxy-
-D-glucopranosylamine (Fig. 2). This is the classical affinity ligand used for
purification of
-hexosaminidase via its substrate binding site (16,
19, 20). After washing the columns, elution was carried out with the
high affinity competitive inhibitor,
-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
-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
-subunit (20), was specifically eluted from the affinity column that
had been loaded with expression medium containing
-hexosaminidase B
(Glu-355 (E355), Fig. 2A). Similar results were
obtained with the expression media obtained by infection with virus
carrying the mutant
-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
-subunit by virtue of its reactivity with antiserum
prepared against
-hexosaminidase B (Fig. 2B). No
comparable peptide was found in the medium from cells infected with
wild-type virus (control).
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.
|
The mutant subunits were tested to ensure their structural and
functional integrity in light of their catalytic deficiency. First,
mutant -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
-hexosaminidase B (Glu-355) in their capacity to be
cross-linked by DSS.
Next, we tested whether the catalytically deficient mutant -subunits
could function in the context of the heterodimer
-hexosaminidase A
in activator-dependent GM2 ganglioside
degradation. The
-subunit was co-expressed with each
-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
-chains, in concert with
the
-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
- and
-subunits (Fig. 4).
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 -subunit precursors
(Fig. 5, Pulse) that were converted to the mature, lysosomal
forms (
a-chain, Mr 29,000 and
b-chains, Mr 24,000) upon chase
in a manner indistinguishable from the unaltered
-subunit (Glu-355)
(Fig. 5, Chase) (13).
Using [3H]ATB-GalNAc as a photoaffinity label for
the substrate binding site of -hexosaminidase B, Liessem et
al. (11) showed a specific incorporation into Glu-355 of the
-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
-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
-subunit were normal, indicating that the mutations did not
adversely affect the folding and quaternary structure of the
protein.
The mutant -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
-subunits were indistinguishable from wild-type
-subunits in
their capacity to be chemically cross-linked. Activator-dependent GM2 ganglioside
degradation, a functional assay for heterodimer formation with the
-subunit, was also found to be unimpaired. Since ganglioside
degradation is mediated through the
-subunit active site, it would
be expected that a defective
active site should not affect
hydrolysis. However, the presence of the
-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-(-aminocaproyl)-2-deoxy-
-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
-hexosaminidase B. Due to the high degree of sequence
similarity between the
- and
-subunits and the functional
similarities of their active sites, it is expected that
-subunit
Glu-323, the counterpart of
-subunit Glu-355, would also be involved
in catalysis mediated by the
-subunit. Mutagenesis of
-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 -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
-subunit, the equivalent of Glu-355 in the human
-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
-hexosaminidase B.
Previously, two other amino acids, Arg-211 and Asp-196, were suggested
to be involved in the -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
-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 -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
-subunits
with Glu-355 altered to any of four other amino acids display minimal
-hexosaminidase B activity even though other properties of the
-subunit, including formation of the substrate binding site,
subunit assembly, activator-dependent GM2
degradation by
-hexosaminidase A, and intracellular transport, remain intact.
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.
After this manuscript was accepted for
publication, Fernandes et al. (26) reported that mutagenesis
of Glu-323 of the -subunit, the equivalent of Glu-355 of the
-subunit, impairs catalytic activity, supporting our conclusions.