(Received for publication, June 16, 1994; and in revised form, December 28, 1994)
From the
Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric
lysosomal hydrolase involved in the degradation of glycoproteins. The
synthesized precursor polypeptide of AGA is rapidly activated in the
endoplasmic reticulum by proteolysis into two subunits. Expression of
the - and
-subunits of AGA in separate cDNA constructs showed
that independently folded subunits totally lack enzyme activity, and
even when co-expressed in vitro they fail to produce an active
heterodimer of the enzyme. Both of the subunits are required for the
enzyme activity, and the immediate interaction of the subunits in the
endoplasmic reticulum is necessary for the correct folding of the
dimeric enzyme molecule. The specific amino acid residues essential for
the active site of the AGA enzyme were further analyzed by
site-directed mutagenesis and in vitro expression of
mutagenized constructs. Replacement of Thr
, the most
amino-terminal residue of the
-subunit, with Ser resulted in a
complete loss of enzyme activity without influencing intracellular
processing or transport of the mutant polypeptide to the lysosomes.
Analogously, replacement of the most amino-terminal tryptophan,
Trp
with Phe or Ser in the
-subunit, resulted in a
totally inactive enzyme without influencing the intracellular
processing or stability of the polypeptide. These results suggest that
the catalytic center of this amidase is formed by the interaction of
the amino-terminal parts of two subunits and requires both Trp
in the
-subunit and Thr
in the
-subunit.
Aspartylglucosaminidase (AGA, ()EC 3.5.1.26) is a
lysosomal hydrolase that catalyzes one of the final steps in the
degradation of glycoproteins. AGA hydrolyzes the N-glycosidic
linkage between asparagine and N-acetylglucosamine and
requires the presence of both free carboxyl and amino groups on the
asparagine, whereas the requirements of the carbohydrate chain are less
strict(1) . Human AGA is synthesized as an inactive 42-kDa
precursor, which is processed into two subunits immediately after
removal of the signal peptide. This rapid proteolytic cleavage into the
27-kDa pro-
- and 17-kDa
-subunits is also the activation step
of the enzyme and takes place in the endoplasmic reticulum(2) .
The dimeric AGA molecule is transported to the lysosomes, most probably
via the mannose 6-phosphate receptor pathway, although recent evidence
also suggests the existence of an alternative transport
pathway(3) . The third maturation step of the AGA polypeptide
takes place in the lysosomes and involves a specific removal of 10
amino acids from the C-terminal end of the pro-
-subunit and does
not influence enzyme activity(2) . Recent data suggest that the
-subunit is also processed in lysosomes, but the nature of this
processing step is so far unknown(3) . The mature AGA enzyme is
a heterodimer consisting of 24-kDa
- and 17-kDa
-subunits,
which are both heterogeneously glycosylated and associated by
noncovalent interactions(2) . Lack of aspartylglucosaminidase
activity in humans results in a lysosomal storage disease,
aspartylglucosaminuria (AGU), characterized by psychomotor retardation
starting in early childhood and mild connective tissue
abnormalities(4, 5, 6) .
AGA is widely
distributed in mammalian tissues and has been purified from a variety
of sources(7, 8, 9) . Although an efficient
purification procedure has been established using human
leukocytes(10, 11) , heterogeneous glycosylation of
the subunits has complicated crystallization trials of the enzyme. ()Consequently, as a complementary strategy, other
approaches must be taken to determine the functionally essential
domains of the polypeptide chain. Characterization of the active site
is important for understanding the function of this unique lysosomal
amidase in molecular detail, and this knowledge might be of use in the
treatment of AGU patients. Here we have used in vitro expression of mutagenized AGA cDNA constructs to analyze the role
of the subunits and specific amino acid residues of AGA. These results
emphasize the necessity of the immediate interaction of
- and
-subunits in the endoplasmic reticulum (ER) and stress the
significance of specific amino acids in the amino-terminal parts of the
subunits for the formation of the catalytic center of this lysosomal
amidase.
AGA
cDNA construct and the separate constructs of - and
subunits
were transfected into HeLa cells 1 h post-infection with vaccinia/T7
virus, essentially as described by Ausubel et
al.(18) . Sixteen hours after transfection the cells were
labeled with radioactive cysteine for 4 h. The immunoprecipitated
proteins were analyzed as above.
Figure 1:
SDS-PAGE analysis of the subunits
translated from separate mRNAs. Vaccinia-infected HeLa cells were
transfected with the full-length AGA cDNA construct (wt) or
cDNA of the separate subunits ( or
), or
they were co-transfected with the cDNA construct of the
- and
-subunits (
+
). The cells expressing
different polypeptides were pulse-labeled with
[
S]cysteine for 4 h and immunoprecipitated from
the cell lysates. The immunoprecipitated polypeptides were then
analyzed on 14% SDS-PAGE under reducing conditions. The size of 42 kDa
indicates the precursor of the AGA when translated from continuous
mRNA. 27 and 17 kDa are the sizes of the pro-
- and
-subunits,
respectively.
Figure 2: The specific AGA activities from different expression studies. A colorimetric assay was used to measure AGA activity from the lysates of COS-1 cells and HeLa-cells (indicated with an asterisk) expressing different AGA cDNA constructs. One catalytic unit was defined as the amount of enzyme required to liberate 1 µmol of GlcNAc in 1 min at 37 °C from the synthetic substrate.
The two characteristic
processing steps of the AGA polypeptide, the activation cleavage
occurring in the ER and the trimming of 10 amino acids from the
carboxyl terminus of the -subunit taking place in the lysosomes,
facilitated monitoring of the intracellular maturation and transport of
the AGA polypeptides. In this expression system, the final lysosomal
processing of the enzyme was not observed even in the case of wild-type
AGA. This is probably because of high overexpression of AGA, which
leads to accumulation of the polypeptides in the ER and largely blocks
the secretory pathway. A smaller form of the
-subunit observed
with the separate expression of the
-subunit most probably
represents a nonglycosylated form of this subunit (Fig. 1),
based on the size of the polypeptide corresponding exactly to the size
of the nonglycosylated
subunit (3) and on the fact that
the relative amount of the smaller polypeptide did not increase during
long chase times, indicating that the polypeptide could not represent a
processing or a degradation product.
To study the intracellular
stability and folding of the - and
-subunits, we also
expressed the separate cDNA constructs transiently in COS-1 cells.
Metabolic labeling of the transfected cells showed that both of the
subunits were expressed separately but that the intracellular stability
of these polypeptides was reduced. The polypeptides of the different
subunits were gradually degraded, and 24 h after the radioactive pulse
the signals of the polypeptides were no longer detectable. However,
both of the subunits were fully glycosylated, which was demonstrated by
expressing the AGA cDNA constructs containing mutated N-glycosylation site in one or both of the
subunits(3) . The lysosomal processing step of the glycosylated
subunits was not observed, indicating that the individual polypeptides
were not transported to the lysosomes (Fig. 3). However,
immunofluorescence analysis of the transfected cells suggested that a
minor portion of the
-subunit could be transported to lysosomes,
whereas the
-subunit remained in the ER (data not shown). The
conformation of the separately expressed polypeptides was studied by
immunoprecipitation with different antibodies. Antibody against the
native AGA enzyme efficiently precipitated the separately expressed
-subunit (Fig. 3A), whereas
-subunit could be
precipitated only with the antibody against the denatured form of the
subunit (Fig. 3B). These findings suggest that the
conformation of the
-subunit was close to normal, whereas the
-subunit failed to attain the conformation of the native enzyme.
Figure 3:
SDS-PAGE analysis of the separately
expressed subunits in COS-1 cells. COS-1 cells transfected with the
cDNA of separate subunits were pulse-labeled with
[S]cysteine for 1 h and then chased for 1, 3, 6,
and 24 h. Intracellular stability and folding was studied by
immunoprecipitating the polypeptides with the polyclonal antiserum
against native AGA (A), and with the antiserum against the
denatured form of AGA (B). Glycosylation of the separately
expressed subunits was detected using two standards, S
and
S
. The S
represents a mutated AGA polypeptide
with the destroyed N-glycosylation site in
-subunit
creating subunit sizes of 27 and 14 kDa. N-linked
oligosaccharides are missing in both of the subunits in S
,
producing subunit sizes of 25 and 14 kDa.
Figure 4:
SDS-PAGE analysis of the intracellular
processing of wild-type AGA and mutated polypeptides. COS-1 cells
transfected with different AGA cDNA constructs were pulse-labeled with
[S]cysteine for 1 h and chased for 1 and 5 h.
The labeled cells were lysed, and the proteins were immunoprecipitated
using a rabbit polyclonal antiserum against purified AGA. The samples
were then analyzed on 14% SDS-PAGE under reducing conditions. The size
of 42 kDa indicates the precursor of the AGA polypeptides, and 27 and
24 kDa are the sizes of the pro-
- and
-subunits,
respectively. The size of 17 kDa shows the
-subunit. AGU
represents the polypeptide containing R161Q and C163S
substitutions.
The replacement of the Trp residue at position 34 resulted in a
totally inactive enzyme, whereas the other three polypeptides mutated
at different Trp residues (W44F, W158F, and W168F) had an enzyme
activity comparable with that of the wild-type AGA (Fig. 2). The
intracellular processing and transport of different Trp-mutagenized AGA
polypeptides were monitored using metabolic labeling followed by
immunoprecipitation of the labeled polypeptides. When the radiolabeled
polypeptides of the wild-type AGA were immunoprecipitated and analyzed
by SDS-PAGE after a 1-h chase period, the activation cleavage into two
subunits was already completed. After chasing for 3 h, the processing
of the -subunit into the mature, 24-kDa lysosomal form could be
observed, and this lysosomal processing step was completed after
chasing the cells for 6-7 h. In the case of three Trp-mutagenized
polypeptides with wild-type enzyme activity (W44F, W158F, and W168F),
the proteolytic maturation was comparable with that of wild-type AGA (Fig. 5), whereas in the case of the mutant polypeptide carrying
the W34F substitution the first processing step into
- and
subunits did occur but was somewhat delayed (Fig. 6).
Figure 5:
Intracellular processing of different
Trp-mutated AGA polypeptides with wild-type enzyme activity. The COS-1
cells expressing different tryptophan-mutated AGA polypeptides were
pulse-labeled with [S]cysteine for 1 h and
chased for 1 and 6 h. The immunoprecipitated proteins were analyzed on
14% SDS-PAGE under reducing conditions.
Figure 6:
Intracellular processing of the
Trp-mutated AGA polypeptides with reduced enzyme activity.
Cells expressing wild-type AGA and both of the Trp
-mutated
polypeptides were pulse-labeled with [
S]cysteine
for 1 h and chased for 1, 3, and 7 h. The radiolabeled AGA polypeptides
were immunoprecipitated from the cell lysates, and the samples were
analyzed on 14% SDS-PAGE. Anti-AGA represents an expression construct,
in which the AGA cDNA is cloned in antisense
orientation.
Figure 7:
SDS-PAGE analysis of processing of mutated
AGA polypeptides having a mutation other than Trp in the -subunit.
COS-1 cells expressing S24T or T33S mutated polypeptides were
pulse-labeled with [
S]cysteine for 1 h and
chased for 1, 3, and 7 h. The immunoprecipitated samples were analyzed
on 14% SDS-PAGE under reducing conditions.
Lack of AGA enzyme activity leads to AGU disease, which is
the most common disorder associated with the failure of the degradation
of Asn-linked glycoproteins in lysosomes. Although the intracellular
maturation and the proteolytic processing of AGA have earlier been
described by us(2) , the events leading to the formation of the
catalytic center of AGA have not been well characterized. In this study
we have used site-directed mutagenesis and in vitro expression
of specific cDNA constructs as an approach to clarify the roles of
- and
-subunits of AGA for the enzyme activity and to
localize key amino acid residues, essential for the biological function
of this enzyme.
The activation of AGA requires an early proteolytic
cleavage of the precursor molecule. The maturation of the enzyme is
continued in the lysosomes, where 10 amino acids are removed from the
C-terminal end of the pro--subunit and the
-subunit faces an
as yet unknown processing step. However, the heterodimer of AGA, which
contains the pro-
/
-subunit structure is already fully active,
and the lysosomal processing of the
-subunit does not influence
the enzyme activity(2) . We studied whether the translation of
subunits from a continuous mRNA is a prerequisite for the formation of
the dimeric enzyme structure and to what extent the separate subunits
contribute to the enzyme activity. In vitro expression of
separate cDNAs of the
- or
-subunits both in vaccinia/T7
polymerase expression system and in COS-1 cells showed that neither of
the subunits alone can be active. Furthermore, co-expression of the
subunit constructs showed that independent folding of the two subunits
prevents formation of the catalytically active dimeric molecule. This
contrasts, for example, with another lysosomal enzyme,
-hexosaminidase, which is also a heterodimer but coded from two
different genes. Co-expression of the separate subunits of
-hexosaminidase results in a fully active enzyme molecule (22) unlike in the case of AGA. This suggests that the
formation of the correct quaternary structure of the AGA enzyme
requires immediate interaction and coordinated folding of the newly
translated and proteolytically released
- and
-subunits into
a dimeric enzyme molecule.
Kaartinen et al.(11) have reported the inhibitory effect of the chemical
inhibitor 5-diazo-4-oxo-L-norvaline on the enzyme activity of
AGA. 5-Diazo-4-oxo-L-norvaline is bound to the hydroxyl group
of the first Thr of the -subunit through an
-ketone ether
linkage, which was shown to effectively inhibit AGA activity. This
inhibition was protected by a natural substrate, providing evidence
that the most amino-terminal residue of the
-subunit,
Thr
, is located near or at the active site of the AGA
enzyme. However, recent data by Fisher et al.(19) have suggested that the substitution of Thr
by Ala interferes with the correct processing of the AGA
precursor into the subunits. These observations encouraged us to
investigate the role of Thr
in more detail in our
expression system. As expected, the expression of the Thr-mutagenized
AGA cDNA in COS-1 cells revealed drastically reduced enzyme activity,
not exceeding the background level. Furthermore, when the intracellular
transport and the proteolytic maturation of the mutated polypeptide
were monitored in pulse-chase experiments, no difference was observed
between the wild-type AGA and the mutant polypeptide. To confirm that
our expression system did not favor the processing of the mutant
polypeptides, we also expressed the polypeptide carrying the
AGU
mutation, known to result in the accumulation of the
precursor molecule in the ER and defective proteolytic processing of
the polypeptide(16, 20) . The processing of the
AGU
polypeptide into subunits was completely blocked, but
contrary to previous data (19) we could not observe any
aberration from the normal maturation pathway in the AGA polypeptides
carrying the amino acid change at position 206. The observation that
the replacement of Thr
by Ser inactivates the enzyme but
does not influence the normal processing or transport of the
polypeptide provides further evidence that Thr
is located
close to or at the active site of the AGA enzyme and most probably
participates in the catalytic mechanism of the enzyme.
The first
amino acid of the -subunit is necessary for the catalytic activity
of AGA, whereas the corresponding region of the
-subunit seems to
locate few amino acids apart from the amino terminus. The change of the
first amino acid of the
-subunit did not have any influence on
either the enzyme activity or the intracellular processing. The
significance of Trp residues for the catalytic function of the AGA
enzyme was first suggested when the chemical modifier N-bromosuccinimide was found to effectively inhibit the
activity of the enzyme. All four Trp residues of AGA are located in the
-subunit, but none of them are adjacent to the proteolytic
cleavage sites involved in the intracellular processing of the
polypeptide chain (2) . Cells expressing the polypeptides
carrying an amino acid other than Trp at position 34 were found to
totally lack AGA activity, whereas the change of any other Trp did not
influence enzyme activity.
Several lines of evidence indicate that
Trp truly represents an active site residue. First, the
normal processing (although with some delay) of the mutated
polypeptides was observed, and the lysosomal maturation of the enzyme
was found to occur normally. Second, the relative amount of the
Trp
-mutagenized subunits was equal to that of wild-type
AGA, indicating that the disappearance of the precursor molecules was
not a consequence of intracellular degradation in the ER. Third, the
region near the Trp
does not seem to be structurally
sensitive for other amino acid changes since the replacement of
Thr
with Ser did not affect activity or the maturation of
the enzyme. If the observed decrease in the processing rate of the
Trp
-mutagenized precursor polypeptides resulted in a loss
of enzyme activity, the activity should be restored when the normal
processing of the enzyme is established. All of these data together
strongly suggest that Trp
has a role in substrate binding
and/or stabilizing the structure of the catalytic center of the AGA
enzyme. This is also supported by the general observation that the
binding site grooves of several enzymes interacting with substrates
that contain oligosaccharides are stabilized by stacking interactions
of aromatic residues and most often by Trp(23) .
The data
provided here emphasize the importance of the amino-terminal parts of
both the - and
-subunits of AGA for the formation of the
active site. These regions of AGA subunits are phylogenetically well
conserved, and the highly conserved amino acids include Trp
and Thr
(24) . The significance of the
amino-terminal parts of the subunits is further supported by the fact
that none of the naturally occurring AGU mutations have been reported
in these regions. Disease mutations typically disturb the folding and
stability of the AGA enzyme but seemingly skip the active
site(25) . We conclude that during the formation of the higher
order structure of the AGA enzyme, the
- and
-subunits fold
in the ER in a coordinated and interactive manner, which brings the
amino-terminal parts of the subunits into the immediate vicinity of
each other to form the catalytic center of this amidase.