Aspartylglycosaminuria in a Non-Finnish Patient Caused by a Donor ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemietry and Molecular Biology, Inc.

Vol. 267, No. 5, Issue of ‘February 15, pp. 3196-3199,1992 Printed in U S.A.

Aspartylglycosaminuria in a Non-Finnish Patient Caused by a Donor Splice Mutation in the Glycoasparaginase Gene*

(Received for publication, April 15,1991)

Ilkka Mononen$Qll$$, Nora HeisterkampII , Vesa KaartinenQ**$$, Tarja Mononen$ $$, Julian C. Williams$, and John Groffenll From the .$Division of Medical Genetics and the IlSection of Molecular Diagnosis, Department of Pathology, Children’s Hospital of Los Angeles, Los Angeles, California 90054-0700, the §Department of Clinical Chemistry, Kuopio University Central Hospital, SF-70210, Kuopw, Finland, the **Department of Chemistry, University of Kuopw, SF-70200, Kuopio, Finland, and the #$Center for Diagnostic Bwtechnology, A. I. Virtanen Institute, SF-70210, Kuopw, Finland

Aspartylglycosaminuria is a lysosomal storage disease caused by deficient activity of glycoasparaginase (EC 3.6.1.26), and it occurs with a high frequency among Finns. We have recently shown that the molecular defect in all Finnish aspartylglycosaminuria patients examined to date consists of two single base changes in the heavy chain of glycoasparaginase (Mon- onen, I., Heisterkamp, N., Kaartinen, V., Williams, J. C., Yates, J. R., 111, Griffin, P. R., Hood, L. E., and Groffen, J. (1991) Proc. Natl. Acad. Sci U. S. A. 88, 2941-2946). This is the first report on the identifica- tion of the molecular defect causing aspartylglycosaminuria in a patient of non-Finnish origin. Total RNA from fibroblasts of a black American aspartylglycosaminuria patient was isolated, first-strand cDNA was synthesized, and the cDNA encoding glycoasparaginase was amplified by the polymerase chain reaction. The patient’s mRNA nucleotide sequence was different from the normal sequence by a deletion of 134 nucleotides at positions 807-940. Nucleotide sequence analysis of the normal glycoasparaginase gene demonstrated that the deletion corresponded precisely to a 134-base pair exon. Moreover, analysis of the splice sites demonstrated a single base change, G to T, that altered the donor splice site of the exon deleted in the patient’s mRNA. This change led to an exon-skipping event resulting in a frame shift and generation of a stop codon.

Glycoasparaginase (N4-(/3-N-acetylglucosaminyl)-L-aspa- raginase, aspartylglucosaminidase, asparatylglycosylaminase) is a lysosomal enzyme that catalyzes hydrolysis of the linkage between N-acetylglucosamine and asparagine in glycopro- teins. Native glycoasparaginase is an oligomer composed of two nonidentical subunits: a heavy subunit (MI 24,000) and a light subunit ( M , 19,000). The active human glycoasparaginase has a molecular weight of 88,000, and it appears to be composed of two heavy and two light subunits (1). Both subunits are encoded as a single polypeptide chain that is

* This work was financially supported by the Academy of Finland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M83247.

TI To whom correspondence should be addressed: Dept. of Clinical Chemistry, Kuopio University Hospital, SF-70210 Kuopio, Finland. Tel.: 011-358-71-173-180; Fax: 011-358-71-173-179.

posttranslationally cleaved and glycosylated (2,3). Deficiency in the activity of glycoasparaginase causes a lysosomal storage disease, aspartylglycosaminuria (McKusick 20840) in hu- mans, which is inherited as an autosomal recessive trait (4). Characteristic findings in aspartylglycosaminuria include pro- gressive mental retardation and accumulation of large amounts of glycoasparagines in body fluids and tissues.

Aspartylglycosaminuria is concentrated in the Finnish population (4, 5). We recently reported (3) on two single base changes six nucleotides apart from one another in the mRNA of glycoasparaginase of all eight Finnish aspartylglycosaminuria patients studied. These changes resulted in replacement of two amino acids, arginine by glutamine (R161Q) and cysteine by serine (C163S), respectively, in the heavy chain of glycoasparaginase. The finding suggests molecular homogeneity in the aspartylglycosaminuria alleles in the Finnish aspartylglycosaminuria patients. Independently, Ikonen et al. (6) described the presence of the cysteine to serine substitution in all their 20 Finnish aspartylglycosaminuria patients, but they found the replacement of arginine by glutamine only in three of those patients (6). Isolated cases of aspartylglycosaminuria of non-Finnish origin have been reported (4). This is the first report on the molecular defect in a non-Finnish aspartylglycosaminuria patient. The clinical findings of this black American patient, which were reportedly similar to those of the Finnish cases, have been described earlier (7).

MATERIALS AND METHODS

The fibroblast cell line was from a black American male with aspartylglycosaminuria originally reported by Hreidarsson et al. (7). It was obtained from the National Institute of General Medical Sciences, Human Mutant Cell Repository, Coriell Institute for Med- ical Research, Camden, NJ (GM03560). The K562 human myeloge- nous leukemia cell line (GM5372) was from the same source. We maintained the cells under standard culture conditions before use. We selected control cell lines from our own collection. Commercial material was purchased from standard suppliers as described else- where (3). Sources for nonstandard materials will be indicated below as appropriate.

Enzyme Assays-Glycoasparaginase activity was measured by high-performance liquid chromatography (8).

Polymerase C h i n Reaction of Glycoasparagiwe cDNA-We isolated total RNA from cultured fibroblasts of controls and the Amer- ican aspartylglycosaminuria patient and from leukocytes of two Fin- nish aspartylglycosaminuria patients. We annealed an oligonucleotide corresponding to glycoasparaginase cDNA residues 1047-1068 (Fig. 1, amplimer B) (3). We prepared first-strand cDNA by using Moloney murine leukemia virus reverse transcriptase (Bethesda Research Lab- oratories). The second amplimer was annealed to cDNA residues 80- 101 from the beginning of the coding sequence (Fig. 1, amplimer A ) (3). Both amplimers contained a nucleotide substitution to create a

3196

Splice Mutation in Glycoasparaginase Gene 3197

SalI site to facilitate subcloning. The PCR' mixture contained 2 pg of total RNA, 20 mM Tris-HCI, pH 8.3, 50 pmol of each amplimer, 0.2 mM of each dNTP, 2.5 mM MgCI, 50 mM KCI, 100 pg/ml nuclease- free BSA, and 2.0 units of TaqI DNA polymerase in a total volume of 100 pl. The reaction mixture was incubated for 2 min at 94 "C to denature the cDNA, annealed for 90 s at 55 "C, and extended for 2 min at 72 "C. A new amplification cycle was started by denaturing the DNA. We performed PCR by using a programmable heat block (Perkin-Elmer Cetus Instruments) for 35-40 cycles of amplification.

Polymerase Chain Reaction on Genomic DNA-The sequencing primer S3 (3) and its reverse complement contained within the deleted region in the patient's mRNA were annealed to DNA isolated from a genomic clone of human glycoasparaginase subcloned into pUC 18. The insert size of the clone was 15 kb and it was isolated from a human library constructed from a partial MboI digest in EMBL 3. The size and nucleotide sequence of the exon and short regions of the nucleotide sequence of the bordering introns were determined. Based on the obtained intron sequences we synthesized two amplimers, one (I) annealing upstream of the exon and the other one (11) downstream of the exon concerned. The nucleotide sequences of the amplimers were I, 5'-TGAGACACTAAGCTTCACCTC-3'; and 11, 5"GGAAGTTGACACAAATACAAAA-3'. The former amplimer contained a nucleotide substitution to result in a Hind111 site and the latter one to result in a HincII site. Amplification of DNA between these two ,amplimers was as described for PCR of the cDNA.

DNA Sequence Analysis-The PCR amplification products were extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and digested with SalI (cDNA) or HindIII/HincII (genomic DNA). After purification on low melting point agarose (International Biotechnologies, Inc.) and phenol/sodium acetate extraction (9), DNA fragments were subcloned into SalI- or HindIII/HincII-digested M13 mp18 or M13 mp19 vector DNA, respectively. DNA sequence analysis was carried out by the dideoxynucleotide chain termination method according to Sanger et al. (10) with appropriate sequencing primers (3) and %-labeled dATP using Sequenase (United States Biochemical Corp.). We sequenced multiple clones for statistical reliability and to exclude artifacts due to polymerase errors during the DNA amplification.

Southern Blot Analysis-High molecular weight DNA was isolated from cultured fibroblasts of the patient and controls. Samples (5 pg) of DNA were digested with EcoRI, BamHI, ApaI, BglII, SstI, PstI, and HincII. The DNA fragments were separated by electrophoresis through agarose and, after alkaline denaturation and neutralization, transferred to nylon membranes (Nytran, Schleicher & Schuell). Resulting filters were hybridized with a 304-bp probe (corresponding to nucleotides 752-1056) generated by digestion of the glycoasparaginase cDNA with PstI and SalI. The probe was labeled by primer extension.

RESULTS

An earlier report demonstrated that the patient had clinical symptoms typical of aspartylglycosaminuria and a severe deficiency of glycoasparaginase activity in fibroblasts (3-496 of normal) assayed by a colorimetric reaction (7). Glycoaspar- aginase assays by HPLC (8) showed activity that was less than 1% of normal in the patient's fibroblasts, indicating a significant defect in the glycoasparaginase gene.

PCR amplification of the mRNA of the patient's glycoasparaginase gene was performed to generate enough copies of the cDNA for nucleotide sequencing. This was accomplished with a pair of amplimers (Fig. 1, A and B) that allows amplification of virtually the entire coding region of the gene (3). Upon electrophoresis, the size of the PCR product of the American patient was about 900 bp (Fig. 2 A , lane 1 ), whereas no product of the expected size (about 1 kb) was observed. PCR followed by gel electrophoresis using different amplimers confirmed the presence of a deletion in the mRNA (data not shown) and eliminated the possibility that the deletion was the result of a PCR artifact. The PCR product of the patient's mRNA was approximately 100 bp smaller than that of the

' The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); HPLC, high-performance liquid chromatography.

I A - A

5' 3'

EcoRl B

k b ~ ~ * ~ ~ ~ ~ ~ ' ' ' ~ 0.5 1 .o

FIG. 1. The structure of glycoasparaginase cDNA and location of PCR amplimers. The bored area represents the coding sequences of glycoasparaginase, and the lines represent portions of 5'- and 3"untranslated regions. An open box indicates the signal peptide; E4 and represent the coding sequences of the heavy chain and the light chain, respectively. The locations of amplimers labeled A and B are shown above and below the cDNA. The blnck bar labeled with an open triangle indicates the deleted region shown in Fig. 4A. The location of the EcoRI restriction site in the normal glycoasparaginase cDNA is shown.

A M Control 1 2 3

0

FIG. 2. Ethidium bromide-stained agarose gel (1%) of normal controls and aspartylglycosaminuria patients. Lune M , molecular size markers (HaeIII-digested aX174RF DNA); lane 1, American patient with aspartylglycosaminuria; lanes 2 and 3, Finnish patients with aspartylglycosaminuria. A, the PCR amplification products of glycoasparaginase mRNA. B, electrophoretic mobility of DNA fragments after digestion of the PCR products shown in A with EcoRI.

corresponding DNA fragments of normal controls or Finnish aspartylglycosaminuria patients (Fig. 2 A ; controls, lanes 2 and 3) (3). As the size of the observed PCR product was 900 bp, whereas no PCR product corresponding to the normal expected product was seen, this data suggested that the mRNAs of both alleles contained a similar deletion. However, we cannot exclude the possibility that in this patient only one allele is transcribed or, alternatively, that the transcript from one allele is unstable and is rapidly degraded. Digestion with the restriction endonuclease EcoRI resulted in a 171-bp fragment from normal controls and the Finnish patients' cDNA (Fig. 2B; controls, lunes 2 and 3). This fragment was not found in the American patient's cDNA (Fig. 2B, lane I), indicating that the restriction site for the enzyme located close to the 3'-end of the coding sequence of the light chain of glycoasparaginase (Fig. 1) was absent. In addition, the cDNA of the American patient lacked the new restriction enzyme recognition site for EcoRI located in the coding sequence of the heavy chain of glycoasparaginase in the cDNA of the Finnish patients, whose 830-bp fragment was further cleaved to two DNA fragments of about 400 bp (Fig. 2 B ) .

The PCR product of mRNA of the American patient's glycoasparaginase gene was subcloned and sequenced, and a deletion of 134 nucleotides was found in the middle of the coding region of the light chain (Fig. 1). This mutated region includes the sequence from position 795 to position 822 and is shown in Fig. 3. The deletion from position 807 to position 940 in the normal sequence (Fig. 4A) resulted in a frame shift at the mutated region. This caused a change of five amino acids and a stop codon four amino acids to the 3'-end of the

3198 Splice Mutation in Glycoasparaginase Gene a

”+ 8 E. b

FIG. 3. DNA sequence of the region containing the mutation in the PCR product of the American aspartylglycosaminuria patient. The open triangle indicates the location of the deletion within the mRNA.

A

B ?Q Iqo ‘Do 9 *? ?

~ T O & T A T A H Q A T O C O C T r C C m M ~ ~ ~ T m ~ T M Q D I L Y R F L P R C C L O -

mTrC**c t?

FIG. 4. Amino acid sequence of the mutated region in the American aspartylglycosaminuria patient. Nucleotides are numbered above the sequence starting from the beginning of the coding region. The deduced amino acid sequences are shown below the nucleotide sequences (3). A, the normal nucleotide and amino acid sequence of glycoasparaginase cDNA (nucleotides 778-963). The underlined sequence is deleted in the mutated sequence. The potentially N-glycosylated asparagine residue is indicated in boldface. B, the mutated nucleotide and amino acid sequence (nucleotides 778- 831). An open trMngfe indicates the location of the deletion within the mRNA shown in A .

mutation (Figs. 3 and 4B). Thus the deduced size of the encoded light chain polypeptide was 68 amino acids instead of the normal 141 amino acids. The deletion removed 2 cysteines and a potentially glycosylated asparagine residue from the light chain of glycoasparaginase. The mutation did not create any novel restriction enzyme sites. The two point mutations typical of Finnish aspartylglycosaminuria patients were not present in the cDNA of the American patient (3).

To determine the origin of the deletion in the glycoasparaginase mRNA of this patient, we analyzed the patient’s genomic DNA by Southern blot analysis, using several different restriction enzymes and a 304-bp cDNA probe containing the sequence deleted in the patient’s mRNA including the EcoRI site. This analysis indicated that no deletion had occurred. Although an EcoRI site was absent from the patient’s mRNA (Fig. l), two EcoRI fragments of approximately 8 and 3 kb hybridize to the probe, both in the patient’s and in the control’s DNA (data not shown), strongly arguing against any major deletion in this region of the gene.

Nucleotide sequence analysis of the normal genomic glycoasparaginase gene demonstrated that the 134-bp deletion corresponded precisely to the nucleotide sequence of one exon encoding nucleotides 807-940 in the glycoasparaginase

mRNA (Fig. 5). PCR amplification of high molecular weight DNA from the patient and a control using amplimers corresponding to intron sequences bordering this exon resulted in both cases in a DNA product of about 200 bp (data not shown). Nucleotide sequence analysis of these DNA products demonstrated that the acceptor junction of the intron located upstream of the exon was identical in patient and control (Fig. 5). However, in the patient’s glycoasparaginase gene, the first nucleotide of the splice donor was altered from the consensus nucleotide G to T (Fig. 6). This nucleotide change generated a new restriction site for Mae11 (ACGT) and MseI (TTAA). Apparently, this splicing mutation causes the exon encoding nucleotides 807-940 in mRNA to be skipped, resulting in direct joining of the preceding and following exons.

DISCUSSION

This is the first report on the molecular defect in the glycoasparaginase gene of a non-Finnish aspartylglycosaminuria patient. The black American patient and the Finnish aspartylglycosaminuria patients were reportedly similarly clinically affected (7). This is consistent with a complete lack of glycoasparaginase activity in the patient’s fibroblasts. The patient’s glycoasparaginase mRNA produced a single PCR product that had a normal nucleotide sequence in the heavy chain, as compared with the two single base changes typical of Finnish aspartylglycosaminuria patients (3). In contrast, this patient’s mRNA had a 134-bp deletion that spans 31% of the total length of the coding region of the light chain and is located in the middle of the polypeptide. Due to the newly formed stop codon five amino acids after the deletion, the length of the encoded light chain in the patient is only 48% of that of normal. Additionally, the deletion removes 2 cysteines and an asparagine residue that may carry an N-glycosidic carbohydrate chain. The native glycoasparaginase contains 15% carbohydrate, and the monosaccharide analysis suggests the presence of both complex type and high mannose type N-glycans and, thus, there must be at least two N-glycosylation sites in the protein (1). The nucleotide sequence of the coding region indicates the presence of two potential N-glycosylation sites, one in each chain (2,3). Both

- l n w o n l r E x o n - Trlnuon- c” Exon

5’ a a t c a g C T A C C A A G C ...... G A A G T T A C G g t a a g t t 3’ Normal

I 5’ a a t c a g C T A C C A A G C ...... G A A G T T A C G t t a a g t t 3’ Mutant

I I 807 940

FIG. 5. Partial nucleotide sequence of the exonlintron boundary of the exon corresponding to nucleotides 807-940 of the human glycoasparaginase mRNA (top). The mutated sequence causing aspartylglycosaminuria in the black American patient is shown in the lower part of the figure.

Normal Mutant

FIG. 6. DNA sequence of the region containing the mutation in the patient. The sequence data from a normal and the black American aspartylglycosaminuria patient are shown. Note the G to T change at the first nucleotide of the intron (splice donor site) of the patient.

Splice Mutation in Glycoasparaginase Gene 3199

of these contain a peptide sequence known to be commonly glycosylated (11). The N-glycosylation site of the light chain is deleted in the glycoasparaginase gene encoding the mutated protein in this non-Finnish patient.

Sequence analyses of exon-intron boundaries have shown a clear sequence conservation (12). The consensus sequences of

the donor and acceptor site are 5’-CAGJGTGAGT-3’ and

5’-YYYYYPCAGJGT-3’, respectively, where Y is a pyrimi-

dine nucleoside and P is any phosphoric residue (13). In the mutated glycoasparaginase mRNA, the normal sequence bor- ders the deletion at AG (5’ of the deletion), and the normal sequence beyond the deletion starts with GT (3’ of the deletion). These nucleotide sequences located immediately upstream and downstream of the deletion suggested that an entire exon might have been skipped. The complete exon- intron structure of the glycoasparaginase gene, which has an approximate size of 12 kb (14), is currently not known. To further analyze the molecular defect in the glycoasparaginase gene of this patient, we initially analyzed the gene by Southern blotting. In comparison with the normal gene, no alterations of the size of the hybridizing bands were observed with any of the enzymes tested, indicating that both alleles lack dele- tions or gross rearrangements. Therefore, we decided to analyze the genomic structure of the patient’s gene. Sequence analysis of the cloned genomic DNA sequences of the gene confirmed our hypothesis that the deleted sequences originate from one exon. Moreover, sequence analysis of exon/intron boundaries of this exon identified a mutation of the splice donor site of this exon. We conclude that the molecular basis of aspartylglycosaminuria in the black American patient is a G to T change in the first nucleotide of the intron downstream of the exon missing in the mRNA. Within the consensus splice sequence, the G at intron position 1 is obligatory (13), and its mutation has been shown to abolish normal splicing. Other examples of exon skipping due to mutated donor splice junction include &thalassemia (15), phenylketonuria (16), Ehlers-Danlos syndrome affecting human type I procollagen (17), and a variant of human albumin (18).

The single PCR product indicated that the patient’s mRNA contained a deletion of 134 bp in both alleles of the glycoasparaginase gene. If this is correct, it might have been caused by independent random mutations or parental consanguinity. On the other hand, we cannot rule out the possibility that the PCR product containing the deletion is obtained from one allele and that the other allele did not produce a (stable) RNA or the amplimers did not anneal to it. The homogeneity of aspartylglycosaminuria alleles in the Finnish population is due to a high carrier frequency (1:30) of the defective gene in the population (19). As far as we are aware, the present aspartylglycosaminuria case is the only one diagnosed to date among black Americans.

The deletion of one exon in the mRNA as described above localizes a third region in the protein structure of glycoasparaginase that is important for its normal enzymatic activity. The replacement of arginine by glutamine (R161Q) and cysteine by serine (C163S) located close to the carboxyl-terminal end of the heavy chain of glycoasparaginase results in a complete deficiency of glycoasparaginase activity (3). Another functionally important structure is located at the amino-

A A

G

terminal end of the light chain that covalently binds diazoox- onorvaline (an irreversible inhibitor of the enzyme) and has similarities to peptide sequences of bacterial L-asparaginases (1). The splice site mutation described here results in forma- tion of a light chain that has half the size of the normal polypeptide. The putative absence of the N-glycosidic carbohydrate chain from the mutated protein may have additional effects. Glycosylation of proteins alters conformation and protects sensitive sites of the protein to retard proteolysis (20). In the presence of a specific inhibitor of N-glycosylation, tunicamycin, several cellular proteins were degraded more rapidly. Proteinase inhibitors could partially retard the en- hanced cellular degradation of nonglycosylated proteins, sug- gesting that specific cleavage sites are unmasked in the absence of asparagine-linked carbohydrate chains (20). If this applies to glycoasparaginase, proteolytic enzymes might de- grade more rapidly the mutated, nonglycosylated light chain than the normal one.

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