GENOMICS

11,206-211

(1991)

ln Vitro Mutagenesis Helps to Unravel the Biological of Aspartylglucosaminuria Mutation ls~0 Uuwvwu,

ELINA IKONEN, *sl NINA ENOMAA,*

Consequences

t AND LEENA PELTONEN*

*Laboratory of Molecular Genetics, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland; and tOrion Corporation, Laboratory of Molecular Genetics, Valimotie 7, SF-00380 Helsinki, Finland ReceivedApril17,

1991;revised

INTRODUCTION

Aspartylglucosaminuria (AGU) is a recessively inherited inborn error of metabolism caused by a rare amidase deficiency. Defective activity of aspartylglucosaminidase (AGA) results in the accumulation of uncleaved glycoasparagines in enlarged lysosomes in all tissues and consequent secretion of these unprocessedmetabolites of glycoprotein degradation in the urine (Pollitt et al., 1968; Maury, 1980). AGU patients have been reported with the prevalence of 1:20,000 in the genetically isolated population of Finland, but single cases have been found also in several other populations (Pollitt et al, 1968; Borud and Torp, 1976; Gehler et al., 1981; Hreidarsson et al., 1983; Aula et al., 1986; Chitayat et al., 1988). The clinical picture of AGU includes mild to severe mental retardation manifesting from the age of 2 years, charcorrespondence

should

be addressed.

0888-7543/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

15, 1991

acteristic coarse facial features, and mild connective tissue abnormalities (Autio, 1972). All patients demonstrate strikingly (>90%) decreased activity of AGA in isolated lymphoblasts or cultured fibroblasts (Aula et al, 1973). Normal expression of AGA is typical of housekeeping-type enzymes with low but detectable activity in all analyzed human tissues (Aula et al., 1982). We have purified the human enzyme to homogeneity from liver and isolated leukocytes (Baumann et al., 1989; Halila et al., 1991). It is a hydrophilic, about 50-kDa glycoprotein with acidic p1 and high thermal stability. Post-translationally, the AGA polypeptide is cleaved into two subunits, which are heterogeneous in their glycosylation (Halila et al, 1991). We have also recently isolated and characterized the 2.1-kb cDNA coding for the 346 amino acid long human AGA polypeptide. In the AGA-cDNA of an AGU patient we have identified two mutations (Ikonen et al., 1991). These mutations were always found coupled and present in 98% of AGU alleles in the Finnish population, and absent in non-AGU controls (Syvlnen et al., submitted for publication). Both nucleotide changes, located only 5 bp apart, result in amino acid substitutions: a G,, transition to A changes Arg,,, to Gln and G, to C Cys,, to Ser in the amino-terminal larger subunit of AGA. Computerbased analyses of the mutated polypeptide chains predicted that the Cys --+ Ser change results in a significant increase in the expected flexibility of the AGA protein, whereas the predictable consequence of the Arg to Gln change was a slight decrease in pl from 5.81 to 5.65 (Ikonen et aZ., 1991). In this study we expressed in vitro mutagenized constructs of the AGA-cDNA in COS-7 cells, and we provide evidence that the cysteine mutation alone causes the loss of AGA activity. Further, we demonstrate that this cysteine participates in an intrapolypeptide chain disulfide bond, which is missing in the purified AGA enzyme from an AGU patient. We conclude that

Aspartylglucosaminuria (AGU) is a lysosomal storage disease resulting in severe mental retardation. We have recently reported that mutations in the aspartylglucosaminidase (AGA) locus are responsible for this disease. About 90% of reported AGU cases are found in Finland, and we have shown that the vast majority (98%) of AGU alleles in this isolated population contain two point mutations located 6 bp apart. We expressed these Arg,,, + Gln and separately in vitro and demonCY%ss + Ser mutations strated that deficient enzyme activity is caused by the CY%3s + Ser mutation, whereas the Arg,,, + Gln substitution represents a rare polymorphism. Further analyses of in vitro expressed AGA proteins and the enzyme purified from an AGU patient revealed that Cys,, participates in an S-S bridge. The absence of this covalent cross-link in the mutated protein most probably results in disturbed folding of the polypeptide chain and a consequent decrease in its intracellular stability. 0 ieoi Academic b, 1~.

1 To whom

May

206

CYSTEINE

MUTATION

IN

the abolishment of a disulfide cross-link disturbs the folding of the AGA polypeptide, which results in deficient enzyme activity and the biological consequences of aspartylglucosaminuria. MATERIALS

Construction of Mutant Pkfsmids

AND

METHODS

AGA-cDNA

Expression

A cDNA library from fibroblast poly(A)+ RNA of an AGU patient was prepared in the h ZAP vector according to Gubler and Hoffman (1983) using reagents from Pharmacia and Stratagene. The cloned AGU-cDNA identified by screening the library with the AGA-cDNA as hybridization probe was excised together with the Bluescript phagemid sequences from the X ZAP vector using an fl helper phage (R408, Stratagene). The AGU-cDNA insert extending from nucleotides 13 to 1108 of the full-length AGA-cDNA (see Ikonen et al., 1991) was removed from Bluescript as a To.&BamHI fragment and a synthetic BamHITuqI linker was ligated to its 5’ end (see Fig. 1). The glutamine residue at position 161 of the AGU-cDNA was exchanged for the normal arginine and the serine at position 163 for cysteine in separate clones by oligonucleotide-directed site-specific mutagenesis of the AGU-cDNA in M13mp18 single-stranded DNA using the phosphorothioate DNA selection method (Taylor et al., 1985). The oligonucleotides of the linker and for the mutagenesis (see Fig. 1) were synthesized on a Applied Biosystems Model 381A DNA synthesizer using cyanoethyl phosphoramidite chemistry and purified by polyacrylamide gel electrophoresis (PAGE). The mutant clones were identified after PCR amplification of the phage DNA using the solidphase minisequencing method which allows the detection of the Arg + Gln and Cys + Ser changes separately (Syvanen et al., submitted for publication). The AGU-cDNA clone and the three mutated clones were further subcloned into the BamHI site of a derivative of the mammalian expression vector pCD-X, in which the SV 40 early promoter is located immediately upstream of the cloning site (Okayama and Berg, 1983). The clones with the insert in the correct orientation were identified by restriction enzyme mapping. The induced base changes and correct sequence at all other positions were confirmed by sequencing the clones in the expression vector using the dideoxynucleotide chain termination method (Sanger et al., 1977). Transfection and Metabolic Labeling of COS-7 Cells COS-7 cells (ATCC, CRL 1651) were maintained in Dulbecco’s modified Eagle’s medium supplemented with antibiotics and 10% fetal calf serum. The cells,

ASPARTYLGLUCOSAMINURIA

207

seeded 1 day prior to transfection at a confluence of 10’ cells/lO-cm petri dish, were transfected with 20 c(g of the pCD-X plasmid DNA constructs using the liposome transfection method (Felgner et aZ., 1987) (Lipofectin Reagent, BRL). The equal transfection efficiency of the different constructs was confirmed by analyses of four independent transfection experiments and cotransfections of the AGA and AGU constructs with a pCAT control plasmid (Promega). Forty-eight hours after the transfection the cells were either harvested directly or labeled with 200 &i/dish of [35S]cysteine (Amersham) for an additional 5 h in a cysteine-free medium. The cells were harvested by trypsinization and homogenized into 20 mM sodium phosphate buffer, pH 7.4, containing 1% Triton X-100 and lysed by freeze-thawing. The CAT assay was performed under standard assay conditions (Gorman et al, 1982). Analysis of Radiolabeled Proteins Radiolabeled AGA proteins encoded by the AGAcDNA constructs were immunoprecipitated from the COS cell extracts using antiserum prepared against purified AGA protein (Halila et al., 1991) and Staphylococcus aureus cells (Immuno-Precipitin, BRL) to precipitate the antigen-antibody complexes (Proia et al, 1984). The precipitated immunocomplexes were solubilized in either the presence or the absence of 50 mM dithiotreitol as reducing agent and the labeled AGA polypeptides were separated in a 12% SDSpolyacrylamide gel (Laemmli, 1970). The separated 3SS-labeled proteins were visualized by fluorography using the Amplify reagent (Amersham). Enzyme Assay and Protein Analyses The AGA activity assay is based on calorimetric measurement of N-acetylglucosamine released by the enzyme from the synthetic substrate 2-acetamido-lB-(L-aspartamido)-1,2-dideoxy-&D-glucose (AADG) (Makino et al., 1966; Reissig et al., 1955). The assay was carried out using 200 nmol of the substrate AADG (Sigma Chemical Co.), a suitable amount of cell extract, and 50 mM potassium phosphate buffer, pH 6.1, in a final volume of 50 ~1 by incubation at 37°C for 4 h. The reaction was stopped by adding 150 ~1 of 0.8 M borate buffer, pH 9.2, and heating the samples at 100°C for 3 min. The liberated N-acetylglucosamine was measured according to Reissig et al. (1955). One unit of enzyme activity was defined as the amount of enzyme that cleaves 1 pmol of N-acetylglucosamine-Asn linkages/min. The measured enzyme activity was correlated to the total amount of protein in the cell lysates, which was determined using the microassay version of the Bio-Rad protein assay. The cell extracts were analyzed by Western blot (Towbin

208

IKONEN

et al., 1979). The proteins were transferred to a nitrocellulose membrane (Amersham Hybond-C extra) by electroblotting and stained with Ponceau S (Salinovich and Montelaro, 1986). The antisera, which have been described earlier (Halila et al., 1991), were used at I:200 to 1:600 dilutions in the immunostaining (ProtoBlot Western Blot AP System, Promega). Purijkation

of the AGU Enzyme

The AGA enzyme was purified from 5 g of autopsy liver of an AGU patient. The purification steps included concanavalin A affinity chromatography, BioGel P-100 gel filtration, and chromatofocusing with Mono P fplc, which were performed according to Halila et al. (1991). The minimal residual activity of the AGU protein and specific antibodies raised against the AGA polypeptide were used to monitor the purification. RESULTS cDNA

Constructs

AND

DISCUSSION

Used for in Vitro Expression

Four constructs of the AGA-cDNA were used in transient in vitro expression for studying the relationship between the enzyme activity and protein structure. One of the constructs encoded the normal aspartylglucosaminidase, and one was the AGAcDNA of an AGU patient with the two nucleotide changes. To investigate the effect of the two mutations of the AGU allele on the biological function of the AGA enzyme, one of the constructs contained only the mutation causing the Arg,,, + Gln and the other only the mutation causing the Cys,,, + Ser substitution. To serve as a control for the background expression of AGA, the COS cells were transfected with the AGA-cDNA in antisense orientation. A derivative of the mammalian expression vector pCD-X (Okayama and Berg, 1983) was used as a vector in transfection of COS cells with the AGA-cDNA constructs. A stretch of 11 nucleotides directly 5’ of the AUG initiation codon was modified from normal sequence to create a suitable cloning site and to improve the sequence context of the translation initiation codon (Kozak, 1987). A detailed description of the mutagenized cDNA constructs is given in Fig. 1. AGA Activity

Expressed

by the Different

Constructs

With the construct containing the normal AGAcDNA, the AGA activity of the COS cells increased 20-fold (the mean of four transfections) over the endogenous activity of the COS cells. The AGU-cDNA construct with both the nucleotide changes resulted in no increase in the AGA activity above the background level. The construct containing only the Cys

ET

AL.

+ Ser mutation did not increase the AGA activity over the background, whereas a 13-fold increase in the AGA activity was measured using the construct encoding only the Arg + Gln change (see Table 1). Since the residual enzyme activity expressed by the Cys + Ser construct as well as by the AGU-cDNA was undistinguishable from the background, these in vitro analyses unequivocally demonstrated that the CYSl63 --) Ser mutation alone is sufhcient to abolish the AGA activity. In contrast, the Arg,,, + Gln substitution most probably represents a rare polymorphism tightly linked to the AGU mutation due to the extremely short distance between these two base changes, and enriched along with it in this genetic isolate. The extremely low enzyme activities detected in the experiments with both the Cys + Ser and AGU constructs could in fact reflect the situation in vivo, as the AGA activities measured in the cultured cells of AGU patients are often at the detection level of the calorimetric assay (Aula et al., 1973). Actually the experiment described here suggests that once the gene therapy of AGU is feasible, the deficient AGA activity will be restored by correcting only one of the mutations found in the AGU-cDNA. In Vitro Expression

of the AGA Proteins

To analyze further the biochemical background of the differences in enzymatic activity of COS cells transfected with the different constructs, we performed immunoprecipitations and Western blot analyses. The sequence data obtained from our full-length AGA-cDNA clone revealed that the corresponding 346 amino acid polypeptide chain contains 11 cysteine residues, 9 of which remain in the AGA polypeptide after the cleavage by signal peptidase (Ikonen et al., 1991). Because of this high number of cysteine residues in AGA, we used [3SS]cysteine for biosynthetic labeling of the proteins in the transfected cells. As shown in Fig. 2, the autoradiography of the immunoprecipitated polypeptides revealed, in addition to the minor about 50-kDa uncleaved AGA polypeptide, major signals of post-translationally cleaved 24and 17-kDa subunits. The identity of these biosynthetically labeled immunoprecipitated polypeptide chains of AGA was confirmed by Western blot analysis of total cell lysates using specific antisera raised against each subunit (Fig. 2). The metabolic labeling for 5 h also enabled rough quantitation of the expressed AGA subunits. In vitro expression of the AGU-cDNA and the construct with only the Cys + Ser mutation resulted in a signal clearly exceeding the intrinsic AGA production in COS cells, but the AGA-cDNA and the construct with the Arg + Gln mutation were expressed at a

CYSTEINE 5’

MUTATION

linker

I 1

IN

mutagenesis region I I G->A G-k-C 402 480

I 13

209

ASPARTYLGLUCOSAMINURIA

1041

1108

////////////I------------////////// Taq

AGU-

5’

I

161 Arg->Gln

163 Cys->Ser

BamH

I

TCTTCGGTGGTCAGGGGGCG...

cDNA:

linker: S’GCTAG CGATC BamH

mutagenesis

3'

GZGCGCGGAAGT I

Taq

oligonucleotides: Gln

AGU-

I

Ser

ATTGGCTTGCT’CBG’AAT

cDNA:

CAGCCAAATT Ser

Cys,,,->Ser

-mutant:

Arg,,,->Gln

-mutant:

AGA-

GCTTGCT CT

cDNA:

GCTTGCT

AAT

CAGC

AAT

CAGCCAAATT

AAT

CAGCCAAAT

FIG. 1. Schematic presentation of the cDNA constructs used for in vitro mutagenesis. The AGA-cDNA is indicated hy black boxes (coding area) and a dashed line (noncoding region). The Bluescript polylinker is marked ///, the detailed structure of the modified 5’linker is shown below the AGA-cDNA, and the 11 modified nucleotides are marked gray (see text). The sequence information of the oligonucleotides used for in vitro mutagenesis is given in the lower part of the figure.

level still significantly higher. This difference is not caused by different efficiency of the antibodies to precipitate normal and mutated proteins, since similar data were obtained when the AGA subunits were visualized with specific antisera directly from cell lysates in Western analyses (data not shown). Although direct conclusions from these in vitro expression studies cannot be drawn for AGU cells in viuo, this quantitative difference in the observed protein expression levels controlled by the same promoter in equally efficient transfections may suggest enhanced intracellular degradation of the synthesized AGA polypeptide chains containing the Cys + Ser mutation. It is possible that most of the improperly folded AGU protein does not leave the endoplasmic reticulum.

Both subunits of AGA are differentially glycosylated, resulting in two isoforms of each subunit (Tollersrud and Aronson, 1989; Halila et al., 1991). Figure

% i =F 14-c

TABLE

Aspartylglucosaminidase (AGA) Activity from COS Cells Transfected with the AGA-cDNA Constructs

AGA Arg,,, + CysIs3 + AGU Antisense

Assayed Different

AG A activity (milliunitjmg protein)

Construct

Gln Ser

.-

1

4.92 3.11 0.24 0.22 0.24

SDS-PAGE and Western analyses of the AGA polypeptides immunoprecipitated from COS cells transfected with different constructs. The [36S]cysteine-labeled AGA polypeptides are visualized as five bands: noncleaved about 50-kDa AGA and two proteolytically processed subunits both demonstrating two isoforms. The typical pattern of the isoforms in the purified human enzyme is shown on the left for comparison. In vitro expressed isoforms of the higher molecular weight subunit demonstrate migration different from that in the purified human AGA in all constructs (see text) and isoforms of the lower molecular weight subunit are merged together. The identity of both subunits is confirmed with subunit specific antibodies in Western blot analysis shown on the right.

210

IKONEN

ET

AL.

2 visualizes these typical forms of both AGA subunits in the SDS-PAGE of the immunoprecipitate. Similar to its effect on the purified human AGA (Halila et al., 1991) IV-glycosidase F digestion of these immunoprecipitated polypeptide chains resulted in single forms of both subunits (data not shown). Our data demonstrate that post-translational glycosylation of the AGA polypeptide chains takes place in COS cells and is sufficient for normal AGA activity. However, the slight difference in the migration of the higher molecular weight AGA subunit compared to that of the enzyme isolated from human sources suggests some differences in the trimming of the oligosaccharide chains (see Fig. 2).

The Deficient S-S Bridge in the AGU Protein Previous analyses of human and rat AGA proteins have demonstrated that some of the cysteine residues participate in disulfide cross-links. The number and location of these disulfide bridges are unknown, but they do not link the subunits (Baumann et al., 1989; Tollersrud and Aronson, 1989; Halila et aZ., 1991). Disulfide linkages between cysteine residues are known to stabilize the secondary structure of proteins by reducing the number of conformations accessible to the unfolded protein and consequently reducing the entropy of unfolding (Kauzmann, 1959; Pakula and Sauer, 1989). To determine whether the mutated CY.Q~ participates in the S-S bridges, we analyzed the crude lysates of 35S-labeled cells after expression of the different constructs using Western blotting and omitting the pretreatment of the samples with SDS or reducing agent. This experiment revealed a size difference between the migration of the normal AGA and that of the AGU (or Cys-mutated) protein, suggesting that the Cys mutation interferes with disulfide bridge formation. To confirm this, we purified AGA protein from the autopsy liver of an AGU patient carrying the Cys (and Arg) mutation. Western blot analysis of the partially purified AGA proteins also revealed a difference in the migration of the normal and the AGU polypeptide chains under nonreducing conditions, whereas the reduction of intrapolypeptide S-S bridges with dithiotreitol resulted in identical migration of the polypeptide chains (Fig. 3). From these data we conclude that the Cys,, residue participates in the formation of a covalent disulfide cross-link and is a target for a destabilizing mutation. The in vitro expression analyses of this AGU mutation, designated as AGU,,, have narrowed the gap between the established mutation and the disease phenotype. This gap in knowledge still remains for many inherited diseases despite the detailed characterization of the mutations at the nucleotide level.

-D-l-r

+D-l-r

FIG. 3. The reduction experiment demonstrating the missing S-S bridge in the AGA protein purified from an AGU individual. Purified normal AGA enzyme (C) and purified AGU enzyme (A) were analyzed on PAGE without pretreatment with SDS in the presence (+DTT) or absence (-DTT) of a reducing agent, dithiotreitol. Only the uncleaved AGA polypeptide is visualized in these circumstances.

ACKNOWLEDGMENTS The authors thank Ms. Seija Ukkonen and Ms. Ritva Timonen for intelligent technical assistance, Dr. Ritva Halila for help and advice in protein analyses, Dr. Ilkka Julkunen for valuable advice in designing the constructs, and Ms. Helena Rantanen for professional secretarial work. The mants from The Academv of Finland and Rinnekoti Foundation ak gratefully acknowled&.

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