Proc. Natl. Acad. Sci. USAVol. 84, pp. 8623-8627, December 1987Medical Sciences

Human aldolase A deficiency associated with a hemolytic anemia:Thermolabile aldolase due to a single base mutation

(DNA sequencing/expression vector/hereditary disease)

HIROYUKI KISHI*t, TSUNEHIRO MUKAI*, AKIRA HIRONOt, HISAICHI Fundi, SHIRO MIWAt,AND KATSUJI HORI*§*Department of Biochemistry, Saga Medical School, Nabeshima, Saga 840-01, Japan; and tDepartment of Internal Medicine, Institute of Medical Science,Tokyo University, Tokyo 108, Japan

Communicated by Charles C. Richardson, July 20, 1987

ABSTRACT Fructose-1,6-bisphosphate aldolase A (fruc-tose-bisphosphate aldolase; EC 4.1.2.13) deficiency is anautosomal recessive disorder associated with hereditary hemo-lytic anemia. To clarify the molecular mechanism of thedeficiency at the nucleotide level, we have cloned aldolase AcDNA from a patient's poly(A)+ RNA that was expressed incultured lymphoblastoid cells. Nucleotide analysis of the pa-tient's aldolase A cDNA showed a substitution of a singlenucleotide (adenine to guanine) at position 386 in a codingregion. As a result, the 128th amino acid, aspartic acid, wasreplaced with glycine (GAT to GGT). Furthermore, change ofthe second letter of the aspartic acid codon extinguished a FokI restriction site (GGATG to GGGTG). Southern blot analysisof the genomic DNA showed the patient carried a homozygousmutation inherited from his parents. When compared withnormal human aldolase A, the patient's enzyme from eryth-rocytes and from cultured lymphoblastoid cells was found to behighly thermolabile, suggesting that this mutation causes afunctional defect of the enzyme. To further examine thispossibility, the thermal stability of aldolase A of the patient andof a normal control, expressed in Escherichia coli usingexpression plasmids, was determined. The results of E. coliexpression of the mutated aldolase A enzyme confirmed thethermolabile nature of the abnormal enzyme. The Asp-128 isconserved in aldolase A, B, and C of eukaryotes, including aninsect, DrosophUa, suggesting that the Asp-128 of the aldolaseA protein is likely to be an amino acid residue with a crucial rolein maintaining the correct spatial structure or in performingthe catalytic function of the enzyme.

Fructose-1,6-bisphosphate aldolase (fructose-bisphosphatealdolase; EC 4.1.2.13) is a glycolytic enzyme that is com-posed of three distinct isozymic forms, aldolases A, B, andC (1). This enzyme has been studied extensively with respectto tissue distribution, changes during development, andcarcinogenesis (1). It seems, therefore, that this enzyme isuseful for studying molecular mechanisms ofgene expressionand also for understanding the evolution of the gene. Inaddition, elucidation of the molecular mechanism of aldolasedeficiency is helpful for understanding the regulation ofaldolase expression.Genes for aldolases A and B have been cloned and

characterized in rat (2-4), human (5-7), and other animals (8,9). These results now permit us to study in detail themolecular basis of hereditary diseases caused by humanaldolase deficiency. In inherited deficiency ofaldolase in manthere is a fructose intolerance, which is due to a liver aldolaseB deficiency (10). Recently, another type of clinical entity,erythrocyte aldolase deficiency associated with hereditary

hemolytic anemia, has been described (11, 12). Two cases(one kindred) of three were found in Japan. In these caseserythrocyte aldolase activity was very low and thermolabile,suggesting that the mutation is on the structural gene asopposed to being a mutation of gene regulation.

In the present paper we examine a case of erythrocytealdolase deficiency and report that by comparing the nucle-otide sequence of the patient's erythrocyte aldolase A cDNAwith that of a normal control, an A-G transversion was foundto occur in the codon for the 128th amino acid, aspartic acid(GAU). This results in the production of an enzyme havingglycine (GGU) instead of aspartic acid. We also discuss thecharacteristics of the altered enzyme expressed in Esche-richia coli.

MATERIALS AND METHODSMaterials. Reagents for measuring aldolase activity were

obtained from Boehringer Mannheim. The cDNA synthesissystem was from Amersham; the nitrocellulose filter wasfrom Schleicher & Schuell; the nylon filter was from NewEngland Nuclear; and [a-32P]dCTP (3704 Ci/mmol; 1 Ci = 37GBq) was from ICN. Restriction enzymes and the otherenzymes were from Nippon Gene and Takara Shuzo.

Cell Cultures. A lymphoblastoid cell line was establishedfrom a patient (Y.K.) with erythrocyte aldolase A deficiency(12) and from a normal volunteer by transforming the periph-eral blood lymphocytes with Epstein-Barr virus. These celllines were used in these studies because aldolase expressedin cultured lymphoblastoid cells was confirmed to be aldolaseA (data not shown), the same type of enzyme as thatexpressed in erythrocytes (13).

Preparations of Aldolase A and Enzyme Assay. For elec-trophoresis, aldolase was partially purified from erythrocytesas follows. Cells that were briefly washed to remove the buffycoat were disrupted and the lysates were passed throughCM-Sephadex C-50 and fractionated by ammonium sulfate toremove hemoglobin. Aldolase activity was determined bytwo methods: activity staining (zymogram) and spectroscop-ic methods. Electrophoresis on cellulose polyacetate stripsand staining for aldolase activity were carried out as de-scribed by Susor et al. (14). Aldolase activity in the eryth-rocyte lysates was determined spectrophotometrically asdescribed (15). The human aldolase activity expressed in E.coli was determined by the two methods described above inthe presence of 5 mM EDTA to inhibit E. coli aldolaseactivity (16). To determine thermal stability of aldolase, celllysates were incubated for 30 min at various temperatures inthe presence of proteinase inhibitors-leupeptin, pepstatin,

Abbreviation: PhMeSO2F, phenylmethylsulfonyl fluoride.tPresent address: Medical Institute of Bioregulation, Kyushu Uni-versity, Fukuoka 812, Japan.§To whom reprint requests should be addressed.

8623

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8624 Medical Sciences: Kishi et al.

N-a-tosyllysine chloromethyl ketone, and phenylmethylsul-fonyl fluoride (PhMeSO2F)-and then assayed for the re-maining activity at 30'C. The remaining activity in the heatedsample is expressed as a percentage of the activity in thenontreated sample.

Construction and Isolation of cDNA Clones and DNA Se-quencing. Poly(A)+ RNA was prepared from the lymphoblas-toid cell line. Construction of a double-stranded cDNA andits ligation to XgtlO DNA was carried out according to Gublerand Hoffman (17) and Huynh et al. (18), respectively. ThecDNA fragments cleaved with restriction enzymes weresubcloned in pUC13 plasmid. DNA sequencing was per-formed by the modified method ofdideoxy chain-termination(19).

Construction of Patient Aldolase A Expression Plasmids andTheir Expressions in E. coli. pHAA47, an original E. coliexpression plasmid of human aldolase A, was constructedfrom pIN-I1, an E. coli expression vector containing lpppromoter and lacUV5 promoter-operator (20), pUC13 DNA,and normal human aldolase A cDNA pHAAL116-3 (6), aswill be described elsewhere (I. Takahashi, Y. Takasaki, M.Sakakibara, T.M., and K.H., unpublished data). An E. coliexpression plasmid carrying the patient aldolase A cDNAfragment was constructed by substituting a restriction frag-ment of the patient aldolase A cDNA for the same restrictionfragment of aldolase A cDNA in pHAA47 as follows. First,pHAA47 was digested with Acc I to remove a 312-base-pair(bp)-long Acc I DNA fragment that was cleaved at the Acc Isites located at the 325th and 637th positions from the ATGinitiation codon. Then, the same Acc I fragment from thepatient aldolase A cDNAs (pHAdA 5-2) that contained thesubstituted base at the 386th position from an initiation codon(see Results) was ligated to the large fragment of E. coliexpression plasmid pHAA47 at the Acc I sites in the sensedirection and transfected into E. coli strain JM83 (see Fig. 4A,pHAdA524 and pHAdA526). The Acc I fragment ofpHAdA526 was again replaced with that of normal humanaldolase A cDNA to construct pHAA471 and pHAA473. Thetranisformants thus obtained were cultured overnight at 370C,suspended in buffer (25 mM Tris HCI, pH 7.5/1 mM EDTA/1mM PhMeSO2F), and sonicated to prepare cell lysates. Thelysates were then used for enzyme assays as describedabove.

RESULTSThermolabile Aldolase Activity in a Patient with Hemolytic

Anemia. Two cases (one kindred) of erythrocyte aldolasedeficiency associated with hereditary hemolytic anemia havebeen reported in a Japanese family (12). The activity ofaldolase in the patient's erythrocytes was shown to be about5% of that of normal and was thermolabile (12). To determine

A

4-

the isozymic form of the aldolase, the enzyme was subjectedto zymography using the crude lysates of erythrocytes orlymphoblastoid cell lines from the patient and a healthyvolunteer along with an authentic human aldolase A prepa-ration. Aldolases in the normal control erythrocytes and thelymphoblastoid cell line migrated toward the anode alongwith the authentic enzyme (data not shown), indicating thatthe enzymes expressed in these human cells are of type A.Aldolase A activities in erythrocytes ofthe patient (Y.K.) anda normal control were 0.12 and 2.99 units/g of Hb, respec-tively, supporting previous observation (12). In the lympho-blastoid cell line of the patient and the normal control thealdolase A activities were 53.2 x 103 and 154.8 x 103 units/gof protein, respectively. These results indicate that thealdolase activity in the patient's lymphoblastoid cells is alsosignificantly lower than that of normal lymphoblastoid cells.To examine the kinetics ofthe enzyme activity in the cell linesof the patient and the normal control, the thermal stability ofthe aldolase was measured in cell lysates that were incubatedfor 30 min at various temperatures as described above (seeMaterials and Methods). Fig. 1 shows that the aldolase oferythrocytes and lymphoblastoid cells of the normal controlretained almost all of its original activity after incubation at55TC, whereas the patient's aldolase in these cells was almostcompletely lost under the same conditions. These resultsindicate that the aldolase A expressed in the patient's cells ismuch less stable than that in normal cells and also thataldolase A in the lymphoblastoid cell lines has the samedefect as in the erythrocytes. The temperature sensitivity ofthe patient's aldolase A suggested to us that the mutationprobably occurred in the coding region ofthe aldolase A geneand that sequence analysis of the gene may be informative.A Single Base Mutation Associated with Disappearance of a

Restriction Site and an Amino Acid Substitution. cDNA cloneswere constructed from poly(A)+ RNA of the patient's lym-phoblastoid cell line using XgtlO as a vector and werescreened using a rat aldolase A cDNA fragment as a probe(21). Ten clones positive for aldolase A were obtained from20,000 plaques of cDNA library. One of the clones thatappeared to carry the entire length ofcDNA (pHAdA5-2) wascompletely sequenced. The nucleotide sequence in the cod-ing region of pHAdA5-2 was the same as that of normalaldolase A cDNA (6) except for one nucleotide. In thepatient's cDNA clone, the 386th base from the ATG startcodon, adenine, was replaced with guanine (Fig. 2). As aresult, the 128th amino acid, aspartic acid (GAT), wasreplaced with glycine (GGT).There are four Fok I recognition sites (GGATG) in normal

human aldolase A cDNA that produce internal fragments of33, 120, and 144 bp. One of the recognition sequences islocated at the site including the 128th aspartic acid codon(GAT). Therefore, this single base change that occurs in the

B

50

1.1040 45 50 55

Incubation Temperature (*C)

FIG. 1. Effect of temperatureon aldolase activity in the pa-tient's erythrocytes (A) or a lym-phoblastoid cell line (B). Extractsfrom erythrocytes or the lympho-blastoid cell line were incubated atthe indicated temperature for 30min and cooled on ice and then thealdolase activity was measured. oand A, activity of the extract froma normal control; a, activity of theextract from the patient.

Proc. Natl. Acad. Sci. USA 84 (1987)

11

Proc. Natl. Acad. Sci. USA 84 (1987) 8625

129 GLy

128 Asp

127 Lou

126 Gty

ATC A-G AT C7 'GAT C_

FIG. 2. Comparison of the nucleotide sequence of the patient'sand normal aldolase A. The nucleotide sequence was determinedfrom a cloned cDNA. The amino acid sequence shown was deducedfrom the nucleotide sequence as described (6). A' represents a partof the sequence of normal aldolase A cDNA. A- represents thecorresponding sequence of the patient's aldolase A cDNA. Thesubstituted nucleotides are circled. The arrowhead shows the sub-stituted guanine.

patient's cDNA should be accompanied by the disappearanceof this site and, therefore, results in the disappearance of oneof the Fok I fragments. In fact, the 120-bp Fok I fragmentdisappeared in the patient's cDNA (data not shown), indi-cating that there was a base change in the GGATG of a FokI site.To verify that the base change in the patient's aldolase A

cDNA was a consequence of the nucleotide substitution inthe patient's genomic DNA, high molecular weight DNA(extracted from the patient's lymphoblastoid cell line) was

digested with Fok I, separated on agarose gel, transferred toa nylon filter, and probed with the Fok I-Sau3A fragment ofthe normal human aldolase A cDNA (6) (Fig. 3A). If the FokI site of the genomic DNA (corresponding to that of cDNA)was lost, then a 1-kilobase (kb) fragment should appear and

A B

Probe(cDNA)

7 910a1112 Exon

a' d a

a -

b'-C-

1 2 3

d.

d- ~ ~ ~ :

FIG. 3. Southern blot analysis ofaldolase A gene in chromosomalDNA. (A) Fok I map of the relevant region of the human genomicDNA (shown by open and closed circles) (T.M., H. Yatsuki, K. Joh,Y. Arai, and K.H., unpublished results). The closed circle underexon 8 corresponds to the Fok I site not present in the patient'scDNA. (B) High molecular weight DNA was extracted from lym-phoblastoid cell lines. DNA was digested with Fok I, fractionated ona 2% agarose gel, transferred to a nylon filter, and hybridized witha 32P-labeled Fok I-Sau3A cDNA fragment as indicated. a, a', andd correspond to the DNA fragments denoted by the same letters inthe Fok I map (A); b and c correspond to bands derived from analdolase A pseudogene (data not shown). DNA was obtained from anormal lymphoblastoid cell line (10 ,ug, lane 1), the patient'slymphoblastoid cell line (10 ,ug, lane 2), and the cloned humanaldolase A gene, pHAAR10 (95 ng, lane 3). We observe only a faint"a" band in lanes 1 and 2 because a smaller amount of the genesequence was applied as compared with that in lane 3 (<0.001 withrespect to the aldolase A gene sequence) and, in addition, becausethe smaller hybridizing region was shared with the probe.

a 0.3-kb fragment should disappear. As shown in Fig. 3B, the0.3-kb fragment (labeled d in Fig. 3) completely disappearedand, instead, a 1-kb fragment (labeled a' in Fig. 3) appearedin the patient's DNA, whereas DNA from a normal lympho-blastoid cell line and genomic DNA cloned by us (T.M., H.Yatsuki, K. Joh, Y. Arai, and K.H., unpublished data) gavea 0.3-kb fragment (Fig. 3B). The leukocyte DNA from thepatient also gave essentially the same result (data not shown).Thermal Stability of the Patient's Aldolase A Expressed in E.

coli. To examine whether the adenine to guanine nucleotidesubstitution in genomic DNA resulted in the production ofthermolabile aldolase A in the patient's erythrocytes,aldolase A cDNAs, prepared from normal control and pa-tient's lymphoblastoid cell lines, were inserted into an E. coliexpression vector that contained an lpp and lacUVS promot-er-operator (20). The expression vector was then transfectedinto E. coli strain JM83. The human aldolases, encoded for bynormal and patient cDNAs and expressed in E. coli, werethen used for a comparison of enzyme thermal stability (Fig.4). Normal aldolase A retained about 70% of its activity afterheat treatment at 500C (pHAA47), whereas the patient'saldolase A entirely lost its activity even at 40TC (pHAdA524and pHAdA526). Thermal stability ofthe aldolases expressedin E. coli was quite similar to that of the enzymes fromerythrocytes and lymphoblastoid cells, although the enzymessynthesized in E. coli were less stable.When the Acc I fragment of pHAdA526 was substituted

with the corresponding Acc I fragment of pHAA47, a normalaldolase A cDNA, the enzyme encoded for by the reconsti-tuted plasmid (pHAA471 and pHAA473) displayed the ther-mal stability of the enzyme encoded for by the pHAA47.These results indicated that a single base change of adenineto guanine at the 386th position was responsible for heatlability of the enzyme but that the remaining portion of theexpression plasmid DNA was normal.

DISCUSSIONThree cases (two kindreds) of aldolase deficiency associatedwith congenital nonspherocytic hemolytic anemia have beendescribed (11, 12). The present study presents an analysis ofone of these cases that demonstrated the aldolase A defect atthe nucleotide level. The nucleotide sequence analysis of thepatient's aldolase A cDNA showed a substitution of the 386thbase (adenine by guanine) in the coding region and theresultant replacement of aspartic acid, the 128th amino acid,by glycine. Using a system expressing human aldolase A inE. coli, the characteristic of temperature sensitivity of thealdolase A was reproduced in E. coli (Fig. 4). We clearlydemonstrated that glycine instead ofaspartic acid at the 128thposition caused the aldolase A to be thermolabile.Although there are many instances of inherited diseases in

which the mutation of nucleotide sequence is identified (22),it is generally difficult to assess whether, if more than onemutation exists in a sequence, a particular mutation onlyrepresents a DNA polymorphism or causes a functionaldefect. However, our use of the E. coli expression vector inthis study has proved to be very useful in surveying formutations in coding sequences of genes that may influencethe function of proteins. In addition, this method enables usto purify proteins from E. coli extract that contains theproduct encoded by E. coli expression plasmid and further tocharacterize the proteins biochemically.Our study proves that the patient's aldolase A gene carries

a homozygous mutation in the coding region of the genomesince in Southern blotting analysis cleavage at the relevantFok I site did not occur in the patient's genomic DNA. Thepatient's parents carry the same mutation and are heterozy-gous for the mutant gene since they are phenotypicallynormal and have aldolase activities in erythrocytes that are

Medical Sciences: Kishi et A

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8626 Medical Sciences: Kishi et al.

A

LpptLac G

/Ampr_--a- pHAdA526 1

pHAdA524

B

0J'+o

Noo

40 45 50 55Incubation Temperature (°C)

intermediate between normal and affected levels (12). Theseobservations, together with the evidence that the aldolase Agene (ALDOA) is on chromosome 16 (23), support the ideathat this hereditary disorder has an autosomal recessive modeof inheritance.

Vertebrate aldolases have three isozymic forms: A, B, andC. The amino acid sequences of aldolase A and aldolase B arehighly conserved (24). The 128th amino acid, aspartic acid,found in normal human aldolase A is conserved in all aldolaseisozymes so far examined, including human, rat, and rabbitA, human, rat, and chicken B, and rat and mouse C (25) andeven in Drosophila aldolase (26). One of the reasons that thesubstitution of aspartic acid by glycine causes the thermola-bility of the enzyme may be due to the loss of a negativecharge that is indispensable for retaining its conformationalstability, especially at high temperature. It is thus possiblethat the aspartic acid fulfills an important role in the functionof aldolase either by maintaining its structural stability or asa regulatory site.The patient's aldolase activity in erythrocytes was as low

as about 5% of that of the normal control and wasthermolabile. In the patient's lymphoblastoid cell line theactivity was about 30% of that of the normal control and alsowas thermolabile. We cannot explain at present why theremaining aldolase activities in lymphoblastoid cell line arehigher than those in erythrocytes. Since the level of aldolaseA mRNA in the patient's lymphoblastoid cell line is usuallyhigher than that in the normal control (data not shown), it israther likely that large quantities of aldolase mRNA in thecultured cells partially compensate for its low activity,although we cannot eliminate other possibilities. Furtherstudies are necessary to answer this question, includingdetermination of the relative amount of aldolase mRNA inreticulocytes or lymphocytes of the patient.We are grateful to Dr. M. Inouye of the State University of New

York at Stony Brook for providing E. coli expression vector pIN-Ill,

FIG. 4. Effect of temperatureon aldolase A activity expressedin E. coli. (A) Construction ofplasmid DNA containing normalor the patient's cDNA. Aldolase AcDNA was inserted into theexpression vector (pHAA47);then the Acc I fragment was re-placed with that of the patient'scDNA (pHAdA524, pHAdA526).Finally, the Acc I fragment wasreplaced with that of a normalcontrol (pHAA471, pHA473).Ampr, ampicillin resistant. (B)Aldolase activity after treatmentat the indicated temperatures. E.coli strain JM83 transformed withthe above plasmid DNA was cul-tured overnight at 370C. The ex-tracts of the transformants wereincubated for 30 min at varyingtemperatures and the aldolase ac-tivities in the extracts were mea-sured. The full activities were 7.76nmol/min for pHAA47 (o), 11.74nmol/min for pHAA471 (A), 9.41nmol/min for pHAA473 (o), 3.62nmol/min for pHAdA524 (e), and7.50 nmol/min for pHAdA526 (A).

our colleagues for helpful discussions and comments, and Dr. MarkBogart for reviewing the manuscript. This investigation was sup-ported in part by Special Project Research Grant 60127010 (InbornErrors of Metabolism) from the Ministry of Education, Science andCulture of Japan.

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