©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Immediate Interaction between the Nascent Subunits and Two Conserved Amino Acids Trp and Thr Are Needed for the Catalytic Activity of Aspartylglucosaminidase (*)

(Received for publication, June 16, 1994; and in revised form, December 28, 1994)

Aija Riikonen Ritva Tikkanen Anu Jalanko Leena Peltonen (§)

From the Department of Human Molecular Genetics, National Public Health Institute, 00300 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha- and beta-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 beta-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 alpha-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 alpha-subunit and Thr in the beta-subunit.


INTRODUCTION

Aspartylglucosaminidase (AGA, (^1)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-alpha- and 17-kDa beta-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-alpha-subunit and does not influence enzyme activity(2) . Recent data suggest that the beta-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 alpha- and 17-kDa beta-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. (^2)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 alpha- and beta-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.


MATERIALS AND METHODS

Enzyme Assay

The measurement of AGA activity was based on a colorimetric enzyme assay(12) . Cell lysate from approximately 10^5 cells was diluted to a final volume of 50 µl with 50 mM potassium phosphate buffer, pH 6.1. To this reaction 100 nmol of the synthetic substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-beta-D-glucose (Sigma) was added, and the reaction mixture was incubated at 37 °C overnight. The reaction was stopped by adding 107 µl of 0.8 M borate buffer, pH 8.8, and heating the samples at 100 °C for 3 min. 1 ml of Ehrlich's reagent (0.1 g of 4-dimethylaminobenzaldehyde, 10 ml of 17.3 M acetic acid, 50 µl of 11.6 M HCl) was added, and the reaction mixture was further incubated for 30 min at 37 °C. The absorbance of the samples was measured at 545 nm. The amount of protein in the samples was determined using the Bio-Rad microprotein assay. One catalytic enzyme unit was defined as the amount of the enzyme required to liberate 1 µmol of GlcNAc in 1 min at 37 °C.

In Vitro Mutagenesis and Construction of the Expression Plasmids

A full-length AGA cDNA in M13 mp18 (13) was used as a single-stranded template for site-directed mutagenesis, which was performed using the Sculptor in vitro mutagenesis system kit produced by Amersham (United Kingdom). The mutated AGA cDNAs were excised from the M13 vector with BamHI and further subcloned into the BamHI site of the expression vector SVpoly(14) . The correct orientation of the inserted DNA was confirmed by restriction enzyme analysis. The mutated clones were sequenced to confirm that no undesired additional mutations had been introduced. For the vaccinia/T7 polymerase expression, the wild-type AGA cDNA and the cDNAs encoding the alpha- and beta-subunits were inserted into the pGem 7z vector. DNAs coding for the alpha- and beta-subunits were prepared by polymerase chain reaction. The polymerase chain reaction-amplified signal sequence of AGA was inserted 5` to the beta-subunit. These cDNA constructs were also inserted into SVpoly vector for the COS-1 expression. The correct sequences of the subunit clones were confirmed by sequencing.

Transient Expression and Metabolic Studies of AGA cDNA Constructs

AGA cDNA constructs were transiently expressed in COS-1 cells, which were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. 3 µg of plasmid-cDNA construct was transfected into the cells grown to 60-80% confluency in 3-cm cell culture dishes. Transfection was performed using the liposome transfection method (15) (Lipofectin Reagent, Life Technologies, Inc. ). Forty-eight hours after transfection the cells were labeled with radioactive cysteine and immunoprecipitated as described previously by Riikonen et al.(16) . The precipitated proteins were separated by 14% SDS-PAGE(17) , and the radioactive bands were visualized by fluorography using the Amplify reagent (Amersham).

AGA cDNA construct and the separate constructs of alpha- and betasubunits 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.


RESULTS

Co-expression of the alpha- and beta-Subunits from Separate cDNA Constructs

The role of the alpha- and beta-subunits in the enzyme activity of AGA was studied in HeLa cells using the vaccinia/T7 polymerase expression system. Each of the cell lines transfected with the cDNA construct containing the coding sequence for either the alpha- or beta-subunit expressed a polypeptide, which in metabolic labeling experiments was of the correct size (Fig. 1) but lacked the AGA enzyme activity (Fig. 2). Cells transfected with the full-length AGA cDNA or co-transfected with the cDNA constructs of the alpha- and beta-subunits expressed both the pro-alpha- (27-kDa) and the beta-subunit (17-kDa) (Fig. 1), a polypeptide composition that is active if the heterodimeric molecule is correctly folded(2) . However, only the cells transfected with the full-length cDNA construct displayed enzyme activity, whereas the enzyme activity of the co-transfected cells did not exceed the background level, suggesting a failure in the correct folding of the heterodimeric enzyme molecule (Fig. 2). This was further indicated by the SDS-PAGE analyses of nondenatured polypeptides, which showed that the two subunits remained separate in the co-transfected cells, and no signal was observed in the position of the 42-kDa heterodimeric molecule (data not shown).


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 (alpha or beta), or they were co-transfected with the cDNA construct of the alpha- and beta-subunits (alpha+beta). 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-alpha- and beta-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 alpha-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 beta-subunit observed with the separate expression of the beta-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 betasubunit (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 alpha- and beta-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 beta-subunit could be transported to lysosomes, whereas the alpha-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 beta-subunit (Fig. 3A), whereas alpha-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 beta-subunit was close to normal, whereas the alpha-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(1) and S(2). The S(1) represents a mutated AGA polypeptide with the destroyed N-glycosylation site in beta-subunit creating subunit sizes of 27 and 14 kDa. N-linked oligosaccharides are missing in both of the subunits in S(2), producing subunit sizes of 25 and 14 kDa.



The Role of the Thr

It has been earlier suggested that the first amino acid residue of the beta-subunit, Thr, has an important role in the activity of AGA(11, 19) . To dissect the role of this residue in more detail, we mutagenized Thr to Ser by site-directed mutagenesis and expressed this mutant polypeptide transiently in COS-1 cells. The measured AGA activity of the cells expressing the mutant polypeptide was found to be reduced to the background level (Fig. 2). To verify that the lack of enzyme activity was not a consequence of intracellular degradation or altered transport of the AGA polypeptide, we monitored the processing of the mutated polypeptide by pulse-chase experiments. To compare the normally processed AGA polypeptide and polypeptide with known processing abnormalities, we expressed in COS-1 cells both wild-type AGA and the AGA polypeptide carrying the most common disease-causing mutation, AGU. The AGU double mutation (R161Q,C163S) is known to disturb the correct folding of the AGA polypeptide, resulting in the accumulation of the precursor molecule in the ER(13, 16, 20) . The metabolically labeled polypeptides were immunoprecipitated from COS cell lysates at different chase times and analyzed by SDS-PAGE. The intracellular processing of the mutated T206S polypeptide was found to follow exactly the same processing pathway as wild-type AGA. The processing of the T206S polypeptide showed no accumulation of the precursor molecule, which characteristically was observed in the case of the polypeptide carrying the AGU mutation (Fig. 4).


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-alpha- and alpha-subunits, respectively. The size of 17 kDa shows the beta-subunit. AGU represents the polypeptide containing R161Q and C163S substitutions.



In Vitro Mutagenesis of Trp Residues

A range of different group-specific chemical modifiers were tested for their effect on the catalytic activity of AGA. An efficient inactivation of enzyme activity was observed only with N-bromosuccinimide, which modifies tryptophan residues in the acidic pH range highly selectively(21) . Based on this primary observation we used site-directed mutagenesis to change the four Trp residues of AGA polypeptide, all located in the alpha-subunit, to find out which one(s) might be involved in the catalytic function. In the mutagenized constructs the Trp was replaced by the Phe residue, since this amino acid resembles the bulky Trp with an aromatic ring structure and could be expected to retain a similar structure in the polypeptide. The mutated polypeptides were then separately expressed in COS-1 cells.

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 alpha-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 alpha- and betasubunits 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.



The Role of the Trp

To analyze the role of Trp in more detail, we produced another mutagenized construct by replacing Trp with Ser. This mutation also completely inactivated the enzyme. The intracellular processing of both the inactive Trp mutants, W34F and W34S, was then monitored to ensure that the lack of enzyme activity was not a consequence of secondary events such as incomplete proteolytic activation in the ER or early intracellular degradation. The processing of the wild type and two Trp-mutagenized AGA polypeptides was followed by chasing the labeled polypeptides for 1, 3, and 7 h. Immediately after the pulse period, a detectable amount of the mutated polypeptides was normally processed into subunits, but a somewhat higher amount of the uncleaved precursor molecules was observed than in the case of the wild-type constructs. However, after chasing for 7 h, the processing of both Trp-mutated polypeptides reached the normal processing level, and no abnormal accumulation of the precursor was observed (Fig. 6). When the radioactive signals of the electrophoresed polypeptides were quantitated by scanning, the total amounts of the alpha- and beta-subunits were similar both in the cells transfected with the wild type and the Trp mutants. This indicates that although the replacement of Trp with another amino acid resulted in a small delay in the early processing step, the mutant precursor was completely cleaved into the alpha- and beta-subunits rather than degraded in the ER.

In Vitro Mutagenesis of Other Amino Acid Residues

To study the amino terminus of the alpha-subunit in more detail and to verify that the loss of the enzyme activity associated with Trp-mutagenized constructs was not a secondary consequence of misfolding of the amino-terminal part of the alphasubunit, we mutagenized two additional amino acids from this region. The first amino acid of the alpha-subunit, Ser was replaced with Thr to study whether the whole amino terminus is needed in the catalytic activity of the enzyme. Also, to study if the region near the Thr is structurally so important that the change of one amino acid may dramatically drop the enzyme activity, we also replaced Thr with Ser. When these mutagenized cDNA constructs were expressed in COS-1 cells, they were found to have wild-type enzyme activity (Fig. 2). The intracellular processing of the polypeptides was also comparable with that of the wild-type enzyme (Fig. 7).


Figure 7: SDS-PAGE analysis of processing of mutated AGA polypeptides having a mutation other than Trp in the alpha-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.




DISCUSSION

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 alpha- and beta-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-alpha-subunit and the beta-subunit faces an as yet unknown processing step. However, the heterodimer of AGA, which contains the pro-alpha/beta-subunit structure is already fully active, and the lysosomal processing of the alpha-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 alpha- or beta-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, beta-hexosaminidase, which is also a heterodimer but coded from two different genes. Co-expression of the separate subunits of beta-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 alpha- and beta-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 beta-subunit through an alpha-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 beta-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 beta-subunit is necessary for the catalytic activity of AGA, whereas the corresponding region of the alpha-subunit seems to locate few amino acids apart from the amino terminus. The change of the first amino acid of the alpha-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 alpha-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 alpha- and beta-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 alpha- and beta-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.


FOOTNOTES

*
This study was supported financially by the Academy of Finland, the Sigrid Juselius Foundation, and the Hjelt Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: National Public Health Inst., Dept. of Human Molecular Genetics, Mannerheimintie 166, 00300 Helsinki, Finland. Tel.: 358-0-474-4393; Fax: 358-0-474-4480.

(^1)
The abbreviations used are: AGA, aspartylglucosaminidase; AGU, aspartylglucosaminuria; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum.

(^2)
R. Tikkanen, J. Rouvinen, N. Kalkkinen, and L. Peltonen, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Markus Perola for producing the graphic representations and Dr. Ann-Christine Syvänen for critical comments on the manuscript.


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