Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3 ketothiolase deficiency

Molecular cloning and sequence of the

complementary DNA encoding human

mitochondrial acetoacetyl-coenzyme A thiolase

and study of the variant enzymes in cultured

fibroblasts from patients with 3-ketothiolase

deficiency.

T Fukao, … , T Osumi, T Hashimoto

J Clin Invest.

1990;

86(6)

:2086-2092.

https://doi.org/10.1172/JCI114946

.

Complementary DNAs encoding the precursor of human hepatic mitochondrial

acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these

clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid

precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue

leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt).

The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was

analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as

that of the control. However, the amounts of T2 mRNA differed: the content was reduced in

two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and

4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the

fibroblasts from these patients are present in the mitochondria. These results suggest that

different mechanisms are involved in the enzyme defects in the four patients.

Research Article

Find the latest version:

Molecular Cloning

and

Sequence

of the

Complementary

DNA

Encoding

Human

Mitochondrial Acetoacetyl-Coenzyme

A

Thiolase and

Study

of the

Variant

Enzymes

in

Cultured

Fibroblasts from Patients with 3-Ketothiolase

Deficiency

Toshiyuki Fukao,*SeijiYamaguchi,* Masatsugu Kano,*TadaoOrii,*YukioFujiki,* Takashi

Osumi,$

and TakashiHashimoto

*Department ofPediatrics, Gifu UniversitySchoolofMedicine, Gifu, Gifu 500, Japan;tDivisionofMolecularCellBiology, Meiji Institute of

Heafth

Science, Odawara, Kanagawa 250, Japan;and§Department ofBiochemistry,

Shinshu UniversitySchoolofMedicine, Matsumoto, Nagano 390, Japan

Abstract

Complementary

DNAs

encoding

the precursor of human

he-patic

mitochondrial

acetoacetyl-CoA

thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were

1,518

bases inlength when overlapped, and

en-coded

the

427-amino acid

precursor of

this

enzyme

(45,199

molwt). This amino acid sequence included a 33-residue leader

peptide moiety and

a

394-amino acid

subunit of the mature enzyme

(41,385

mol

wt).

The T2gene expression in

fibroblasts

from four

patients with 3-ketothiolase deficiency

was analyzed

by Northern

blotting.

The T2 mRNA in all

four cell

lines had

the same 1.7 kb as that

of

the control.However, the amounts of T2 mRNA

differed:

the content was reduced in two cell

lines

(cases

1and

3),

whereas

it

was

within

anormal range in others

(cases

2 and

4). Pulse labeling followed by

subcellular

fraction-ation revealed that the T2

proteins

in the

fibroblasts from

these

patients

arepresent in the

mitochondria.

These results suggest that

different mechanisms

are

involved

in the enzyme

defects in

the

four

patients. (J. Clin.

Invest.

1990.

86:2086-2092.) Key words: intracellular localization * ,-ketothiolase

deficiency

- Northern

blotting

*

organic

acidemia- pulse

la-beling

Introduction

3-Ketothiolase

deficiency

(3KTD'

McKusick

20375)

isan

in-born

error

of

isoleucine

and ketone

body

catabolism. It is

characterized by intermittent ketoacidosis

episodes

and uri-nary

excretion

of

organic

acidssuch as

2-methyl-3-hydroxy-butyrate, 2-methyl-acetoacetate,

and

tiglylglycine.

Since

Daum et

al.

(1) reported

this disorderin

1971,

over 10 cases

have been

documented

(2-12).

Mammalian tissues have at

least

four

thiolases;

mitochondrial

3-ketoacyl-CoA

thiolase

(T

1),

acetoacetyl-CoA thiolase

(T2), peroxisomal

3-ketoacyl-CoA thiolase

(PT),

and

cytosolic acetoacetyl-CoA

thiolase

AddressreprintrequeststoDr.Fukao, Department ofPediatrics,Gifu University School ofMedicine,40Tsukasa-Machi,Gifu500, Japan.

Receivedfor publication IMay1990andin revised

form

24

July

1990.

1.Abbreviations used in this paper: CT, cytosolic acetoacetyl-CoA thiolase;PT,peroxisomal3-ketoacyl-CoA thiolase;T1, mitochondrial 3-ketoacyl-CoAthiolase; T2, mitochondrial acetoacetyl-CoAthiolase;

3KTD, 3-ketothiolasedeficiency.

(CT) (13-15).

The

etiology of

3KTD has beenattributed to a defect in T2 because K+ activation of acetoacetyl-CoA thio-lase, a

characteristic

feature of T2 (7), is absent in tissue from

patients

with

this disorder

(6, 9, 10, 12). T2 alone seems to be

responsible

for the cleavage of 2-methylacetoacetyl-CoA, an

intermediate

of isoleucine catabolism (7, 16). The term

"3KTD" is

commonly used for T2 deficiency, although

defi-ciencies of

PTand CT have also been reported

(17,

18).

Previously,

we reported that 3KTD

in

a

patient

was due to a

defect

in the T2

biosynthesis (19).

Subsequently, we demon-strated

using

the pulse-chase

experiments

that, in the fibro-blasts from five 3KTD

patients (including

the

first

one), the T2

proteins of

thesepatients were synthesized but they

differed

in

stability

and

in

mobility in

SDS-PAGE (20, 21),

providing

evidence for heterogeneity

in the molecular

defects

(21). To

better

understand 3KTD at the molecular level, we

first

cloned cDNA

for

rat T2

(22).

We now report the molecular

cloning

andnucleotide se-quence

of

cDNA

for

human T2 and the geneexpression of T2 in fibroblastsfrom four patients with 3KTD.

Methods

Patients.Case 1isa Japanese boy who presented withepisodic

ketoac-idosis (10) and case 2 isaSpanish girlwho repeatedly experienced

severeketoacidosis attacks (9). Case 3 isaLaotianboy who was well

developed but had several episodes ofketoacidosis (9). Case 4 is a boy

with normaldevelopment,despiteepisodic ketoacidosis. His father is

alsoapatient but withoutsymptomsofthis deficiency (7). The

diag-nosis of 3KTD inthefour patientswas based on theresults of urinary

organic acid analyses bygaschromatography-massspectrometry, and

of theenzyme assayoftheirfibroblasts(7, 9, 10). Thefourcelllines werewellcharacterized,at theprotein level. The amount, biosynthesis,

and degradation ofT2 protein in the fibroblasts were analyzed by

immunoblotandpulse-chase experiments (19-21). These results are

listed later in TableII togetherwiththe results obtained in the present work.

Screeningof thecDNA library. TheXgtl1 cDNA library

con-structedfromhumanhepatic poly(A)RNA withrandom primers was

kindly providedby Dr. M.Mori.The cDNA library wasscreened with theprobe, RT2-6, a full-lengthcDNA ofthe rat T2(22), using the

plaquehybridizationmethod (23).

Subcloning andDNA sequencing. Inserts of cDNA clones were subclonedtoplasmavectorspTZ (U.S.BiochemicalCorp.,Cleveland,

OH)orpHSG(TakaraShuzo, Kyoto, Japan).Appropriate

fragments

forsequencingwereobtained bytreatment with nucleaseBa131 or

restriction endonucleases(TakaraShuzo).DNA sequenceswere

ana-lyzedonthedouble-strandplasmidDNAbythedideoxychain termi-nationmethodwiththe Klenowfragment(Takara Shuzo)or

Sequen-ase(U. S. Biochemical Corp.)(24, 25).

Northern blotanalysis. Skin fibroblasts were growninEagle's min-imumessential medium containing 10% fetal calfserum. The cells wereharvested 3-4 dafter achieving confluency.Total RNA was pre-J.Clin.Invest.

©TheAmerican Society for Clinical Investigation, Inc.

pared

from fibroblasts

by

acid

guanidium

thiocyanate-phenol-chloro-formextraction

(26).

Total RNAwasdenatured in

formamide/form-aldehyde

and

electrophoresed

ina_{1.0% agarose}

_gel

containing

formal-dehyde (27).

RNAwastransferredtoasheet of

Hybond-N

(Amersham

Corp.,

Arlington

Heights,

IL) by

capillary blotting.

Prehybridization,

hybridization,

and

washing

werecarriedout

using

theconditions

rec-ommended in the manufacturer's

protocol.

Theinsert of HT2-3(see

Fig.

1)was used as a

probe.

In orderto

_provide

standardsfor the

amountof

applied

RNAs,

thesamemembranewas

rehybridized

witha

,B-actin probe (28). Messenger RNA was quantitated by scanning

den-sitometry

of the

autoradiograms.

Protein

analyses.

The human enzymewas

purified

froman

post-mortemliver

using

the

procedure

previously

usedfor thepurification

ofratliver enzyme

(22).

Two

peaks

of T2

activity

onthe

phosphocel-lulose column

chromatography,

isoenzymes

A and B(15,

29),

were

separately

purified.

The

purified proteins

were

S-carboxymethylated.

Theamino-terminal amino acid sequence wasdetermined by

auto-mated Edman

degradation

with a

_protein

sequencer

(model 470A,

Applied Biosystems, Inc.,

Foster

City, CA).

Amino acid

analysis

was

performed

withan aminoacid

analyzer (model 6300,

Beckman In-struments,Inc.,Palo

Alto, CA)

after

hydrolysis

of the

protein

with 5.7

NHCIat 110°Cfor24, 48,and 72h,

respectively.

Pulse

labeling

andsubcellular

fractionation offibroblasts.

Fibro-blastswere

pulse-labeled

asdescribed

(21).

Subcellularfractionation of thefibroblastswasdone

according

tothe methodofMackalletal.

(30),

withsomemodification.

Briefly,

theharvested cellsweresuspendedin 0.25 M

_sucrose/20

mM

_{potassium phosphate,}

pH 7.5, and 1 mM

EDTA and

digitonin

wasaddedto afinalconcentration of 1 mg/ml.

Thesolutionwas

_kept

onicewaterfor 1min,thenwas

_centrifuged

for 5 minat

_8,000

g. The

pellets

wererinsed with the samebuffer and

resuspended

inbuffer

containing

1

mg/ml

digitonin.

One-third

vol-umeof 80mMTris

HCI, pH 7.5,

0.4%

SDS,

0.4%TritonX-

100,

and 4

mM EDTA was added to the supernatant and the

pellet

frac-tion.

Immunoprecipitation

and

fluorography

wereperformed, as

de-scribed (2 1).

Results

Isolation

of

cDNA

clones

for

human

T2. The

Xgt

11 cDNA

library

wasscreened

using

as a

probe

RT2-6

(22),

a

full-length

cDNA forthe ratT2. After

screening

4.0

X

105 recombinant

plaques,

13

independent

positive

clones

were

obtained (Fig. 1).

All cDNA

inserts

were

subcloned

to

plasmid

vectorsand were

sequenced

for the entire

regions

of inserts.

Sequence

analysis

of

the

cDNA.The

nucleotide

sequence

of

the cDNA

for the

human

T2

is

summarized in

Fig. 2,

together

with

the

deduced

amino acid

sequence. A

1,28

1-base open

reading frame,

starting

atthe

first ATG triplet (positions

1-3), encoded427

amino acids.

The

amino-terminal 20-amino acid

sequence

of

the

purified

enzyme

determined by

Edman

degra-dation, perfectly

matched the sequence

(Fig.

2).

Hence, the presequence

of

the

thiolase

precursor

probably consists of

33

amino acid residues. The relative of molecular

mass

of

the precursor

and

the mature

subunit of this

enzyme was calcu-latedtobe

45,199

and

41,385 D, respectively.

The

combined

cDNA sequence

included

76 bp of the

5'-noncoding region

and 161

bp of

the

3'-noncoding

region. A

putative

polyadenylation signal

and

poly(A) tail

wasnot

found

in the cloned sequence. This may be due to the use of the

library

constructed with cDNAs

synthesized using

random

primers.

The

3'-noncoding regions

ofrat andhuman cDNAs werewell conserved.IntheratcDNA sequence (22), a typical

polyadenylation signal

AATAAA was present 15 bp down-stream

of

the

site

that

corresponds

tothe 3' end

of

the human cDNA.

ATG Hindlil

RT2cDNA

ATG Hindlil Hindill

TAG_I

TAG

HT2cDNA HT2-1 I

-ix 100

bp

HT2-2 _,A.

' v

HT2-3 L I

HT2-4 B

,L v d

HT2-5

x I

HT2-6 1

Figure1.Restrictionmapandsequencingstrategyfor the cDNAs of T2. Partialrestrictionmapsofrat(RT2)and human(HT2)cDNAs

areshownin theupperscheme.ATGs, TAGs,andsolid boxes

indi-cateinitiator methioninecodons,stopcodons,and thecoding

re-gions

forpresequences,respectively. 13 human cDNA clones from HT2-1toHT2-13wereanalyzed,and cDNAsfrom HT2-1toHT2-6

areshown. Thehorizontalarrowsunder eachclone indicate the di-rection andextentofsequencing.

When the deduced

amino acid

sequences

of human

andrat T2 were

compared (Fig. 3),

these sequences share 84% of identical residues.Incontrast, sequence

homologies

of human

T2tootherrat

thiolases,

TI andPT

(31, 32)

are40 and 33%,

respectively.

The subunit of themature enzyme

consisted

of

394 amino acids in both ratandhuman T2. The nucleotide

sequences ofcDNAs for rat and human

T2, including

the

noncoding regions,

are 82% identical. In

vitro

_{transcription/}

translation of

full-length

human cDNA was

performed

ac-cording

tothe methodasdescribed

(22).

The

polypeptide

gen-eratedwas

immunoprecipitated

with

anti-[rat T2]IgG,

which cannot cross-react with human

T1, PT,

and CT

(data

not

shown).

Thus,

thehuman cDNA clones shown here encodeT2 and the first ATG codon is the true initiation methionine

codon. Several base substitutions which led to amino acid

changes

werefound among 13

independent

cDNA

clones,

as shown in

Fig.

2:C320toA

(Ala

to

Glu)

and T483to A

(Tyr

to

stop

codon)

in

HT2-3,

G'018

toA

(Val

to

Met),

G1036

toA(Asp to

Asn),

G'

38toT

(Ala

to

Thr),

and A'234toT

(Ile

to

Phe)

in

HT2-2,

andC793 to A

(Pro

to

Thr)

in

HT2-5. However,

there were no

substitutions

common to more than two cDNA

clones. These

substitutions

may

possibly

be

artifacts occurring

during

the cDNA

synthesis

and

cloning,

but sequence

hetero-geneity

would havetobe excluded.

Protein

analyses.

Two

peaks

of the

thiolase activity

on a

phosphocellulose

column

chromatography

(termed

isoen-zymesAand

B)

were

separately purified.

The

purified

isoen-zymes A and B were

indistinguishable

in

mobility

in SDS-PAGE

(data

not

shown).

The

amino-terminal

sequences of

0HE40. OHao0-:Oco O<H OOHr 0<H OHco OE-4 OHF4 0<-: 00 0<

00<04) --t OH-q OO r- E0-4co 'HCOi 00HF4co T I-A0H 0<* 'IO0a) C710 00H -It r- 0< 000<.9 aO'0< 0'0 > 00u> 00u> "-40< C.IO cqE-l0 CY)E-4 )O

-HH4r- <4 E4 EO -4HCo H.-4Hj--IH'-F< -Hco -0 0

-EH4CO 0 r <0) O <C) H4co OHri 04 OH

HE-0 > EH 0< 0<c 0<4 0> 00 < 0< < <

E-W I i <4co HCa<oCto 0 ~ O~ H-4 H H

HCaHco " OH-I > F4 OH Ha)0w- 0 H

0< 0 > 0<-4 0< 14 0 -1. 00 H~ 00 0

<n-4 E04p u0O H Hp4 H4 _HCID) < u

ON 0O,I 0O 1 C0<0) OH) 00a : Ic0 0<H :)ur-OH 0) 00u

HF-4 u u 0< 0> HP4 0CH u0 00u 0 0

<Cl E-aH HaQ) 0 H-- <) H co OHi 0 H

00u0 000 0> 00<04 0 00 00> 0H 0C < OH 0<

H " -4 U 0oo 0 -Tt<H

0CO

.OH~O J ) 00000_(A OC OH _{u0 CNH} F

00 > 00Ha) ON _H4coa OOHur- I Cl) - ) -0p " % y _{E- CY}

eH -I

0 uH -<a

HC4

4a

94C

H 40 -Ca

HE

Ha) -40 4 U) u 4

OHco

-i <-4P 01- OH 0a H H

H4F4P4 0< <<-. HHE4 00 0 0 <0 H 0 H

Lr)Hco "-4HE4ca -. _.OH e_CFOr-lC_{'co Lr) 1-P}

_-4Cl)

_r-O)

CYIOHr-a'o

Lr)H

-o4

OH 0u> o <-4a,U< ON H > E-04E-4 4Ha)

-04E-i

En -4 H -: -%4 -) -I

HCa

P~

OH. Hal3 01- Ha HH Ha)4i Ha 0 04

H-0>c <H 00- 0 ) OP- <H <H <~

<aH

H

<H>

--00) H0 <0S HCa4 -CCa 0u0H4 04 0

HC4

a En <0). 01 H

coOH

OH OH> CD H H

0>CT -1 0< OP-i 0<- 0<-- 00 00 00 HH4 0 <

0<0) OH4 0) 0 c00 4 OHHr OHH O-)HaQ) OHH 0<4 OHa 00) 00C)

-O 0Ou '-0 CCa<CN<H 00 <H~- -4< H 0DHj. %O<- 04 H-4H 00OU0- -ItH OHE-r-<o 0000C) 00u0~ o'HH4 ON-C0 OH P -0u4-i --4 C-1 ~C-JO 0) Cy)<

01-4 <cF4 ) HCa 0P- ~-4 l r) -~a) -01-IP -4 -<4 > -A

-0P~~~~~~~~~~~ 14 O> OP-i 0<4 U0PL 0004

-C0

<14

F4 Ha-) < Cl) HO)En 0~

OUP-i <Hl HP-i0 0 04 00 0 0 H

H-4Co 0a) Q <0A ) F4H r- H HE-4 H H

00 0> 0<00C 00)_{~ 00<.4} 0 0H 00u0 000 0<) H 00u COH

c Cy'<0) Lr) UCo -0U00 r-HaQ Y)e 0) oON Lr) : -00)( r- 0 cn O'HE

>N r_ 000Hr- a'01-P ON -HH1C0 _{0<0)1C EHa) c-I} a-4 ) CnU CnH

r-i HP00) HO 0 co H P 4- H

HCaco a) H- a) OHr- 0a) Ha4) Ha)

-0 > <00 -4<o1 OP- H <4 H0E0 F4H C4<z 0 0

0 000E- 000 001- i 0Or-0< 00o-0H 0<0E- 0< 0<-I 0E- OH

'.00 10 '-0 c--I 00 a) -tO OO OO 0NIH a) 00 I- t Ca 00 OONI4C

0- 0 _O:H HO0 -<

0l

0 HOM E-<00 0>- <H E-4 <

0 0 Ha)~~~~~~~u Ha14 Ha)u04 u-4 uu u H _O-:4 H-i O

0 H- 0~ HI-i 01P-i 00) H <04 0

<4 0 Ha) 0 0 0a4) < O 01-P H <0) Ha4) <H 0a H

H H Op- < <0 >%

00

E0

P00

a"

H

00

H00 0>

O

4U OuP Ou )urIC

0 00 00

Or-

<

u

I-HI-iQ 0u

1)c

nC

HP- HCa

HI HOO

-0

U V uPI O 00

41-i

00)

I OOH 0 H OOH 0, 00

an rH)00

_CY)0<0)4

u-:

OOH 0<0) 00 1

C4

uco0Ha44)NIa)C)H-

OHi

H

HH1

I I-4 ) _rH-HE4( F-4_{0a) E-OC}E-4 Q) _{-40LIu 0) 'u a)i -H a)}

< H 00 00-4riFqPF H00 04i u uU

O4 01-i4 Oa)) HC Ha)O C HH4 Ha)4 ua) Ha) Oa)u OHH HCa

c--JO₁₀ -I4000oC:₀₁ OH_-<CP-i> -OH'.0< 0CNI0CN<Hr C-I00HE4r.-T0) c' -go,EHa) -tH0Oa)t '.00U4.1Ha) Lr)HCONICaH _000u4-iLOHaF,) -It'OHH c-0HO

0 0< EHH- 00 00 -C4E<H-1 <Hc < u > <94 0<

< OCa HI-i Ha4) H- a) H- > u>O u Ca 0 N F-HO) OP -Ca 0 OH- 0aQ) HH" HH OH OH--OH OHl OH _<4H <0) O

H_{0 OHi}O % 000 00 ~co - <Ca <0) H >. H- E4Q 0~ Ha)

u

0Pi01- OHr Ha) OH -~ur-OH HHr- <Hr- H

H 0 0 0<-C OP-4 0<-4 <940< 00 H 00 H PL

-0C 001-iP . 0 _<-i OHa) 0EI4HN 04-i 0<-4co 0HE1$ 0 0)CDI:

40c0a 0'

F

H

C`r40,-4-HH

r

C<

~0H) CyHa

LfO

-c r 4L)uu~0 0 00 O -Ca0 01- <ca -Caq0 OH <0 .

< 01-i E-HCa 01i a) _OH~ Ha4) OH Hca 01- <0) <0)

OH-H 0< ₀

>E-P < H0 0< 4

HP

0 0>

0P4$4 uHC

H- Ha4) <-. O 4 OH~ EHHCa Oa) HaQ) 0 4 -4 01-i OH

<4 Ho<00 HCa H~ 0) <-Cac Hca OH- I-Pi OH- <ca

-4H OHa-() 00 CDOHl OH4 a) 0<>. 0OOH--O" O H ca 00) a() OHE- Ca OOHr-4OI -t NIO1-) 00<<9 -tu40< C00 1- I'D0 CNIO<-. 000u<4 -It10> 0H00ic '.00> c-4u-lO 0000

10₀ <Ca_OHr-ci _HHr4a) E-H-iP C7-JO 0) NIOH1- Cle) O H)E0)( -THEPC- LO<t4>. Lc-HP- '-OHaq) %O.0P u() >.

Qal <H O. <0) OH <0) HHr- 01-i

0 0<9 <H 01.u> 00 HOu 0<.4 00 0< <H9 E4HH

H -CaH

utk

HCai <H 01-

OHE4P

f0c

0 0 ur.O)-: 0 ca <U<) E-0) r. 0 C0~ <Ca

-< 0Ou0- 000u 0<H: _0u-4C0< 000U 0<H: -4 0)I.I Ha Ha 00<

u-L(rO -OHU c--O CY) OH- 0<0 LO) <) -HO& c-P-u0 cnC4) >N a0. Lc-Iu 0) - 0 10 E-HCa HCa -Ca -O 1-c--I> CY)O 1-i Cy)I< ) -,tOHr- -TE4-Ha) Lr) H

> > > L4 uPL << u u '.O

EO4HCa O OHDr- 0 HCa-CO a) H0r) _I11 ua ) F

H-_H- OH_0< Ha)₀₄ H_{0 >}ca H-_O (L)OIP OHr HHr- O% HCa _<0)t4( >. u

0P4 'V40< < HO 0> 0<-4 ~ 4 c

<Z

<<k

00

H0

<H-

0)

.

Ha)

4 (i

Ico-k

0 Ha)_04-i 01-i~~~~~~~₀₀₀$ Hr-a) HH 01 <0)>1> r4~

-RT2: 1 MAALA-VLH--GVVRRPLLRGLLQEVRCLGRSYASKPTLNDVVIVSATRTPIGSFLGSLA

** ** * **** * ** * ***i ***** ******************

HT2: 1 MAVLAALLRSGARSRSPLLRRLVQEIRYVERSYVSKPTLKEVVIVSATRTPIGSFLGSLS

RT2: 58 SQPATKLGTIAIQGAIEKAGIPKEEVKEVYMGNVIQGGEGQAPTRQATLGAGLPIATPCT ****** ******************* ***** ************ ******* **** HT2: 61 LLPATKLGSIAIQGAIEKAGIPKEEVKEAYMGNVLQGGEGQAPTRQAVLGAGLPISTPCT

RT2: 118 TVNKVCASGMKAIMMASQSLMCGHQDVMVAGGMESMSNVPYVMSRGATPYGGVKLEDLIV

* ***************************************** ** ************* HT2: 121 TINKVCASGMKAIMMASQSLMCGHQDVMVAGGMESMSNVPYVMNRGSTPYGGVKLEDLIV

RT2: 178 KDGLTDVYNKIHMGNCAENTAKKLSISREEQDKYAIGSYTRSKEAWDAGKFANEIMPITI

************** .********* * * *** *** ****** ** **** ** * *

HT2: 181 KDGLTDVYNKIHMGSCAENTAKKLNIARNEQDAYAINSYTRSKAAWEAGKFGNEVIPVTV

RT2: 238 SVKGKPDVVVKEDEEYKRVDFSKVPKLKTVFQKENGTVTAANASTLNDGAAAVVLMTAEA

**** *********************************************** ***** * HT2: 241 TVKGQPDVVVKEDEEYKRVDFSKVPKLKTVFQKENGTVTAANASTLNDGAAALVLMTADA

RT2: 298 AQRLKVKPLARIAAFADAAVDPIDFPLAPAYAVPKVLKYAGLKKEDIAMWEVNEAFSVVV * ** * ***** ******* ***** ** ** *** ***************** **

HT2: 301 AKRLNVTPLARIVAFADAAVEPIDFPIAPVYAASMVLKDVGLKKEDIAMWEVNEAFSLVV

RT2:

HT2:

358 LANIKMLEIDPQKVNVHGGAVSLGHPIGMSGARIVVHLAHALKQGEFGLASICNGGGGAS *************** ****************** ** ******* *************

361 LANIKMLEIDPQKVNINGGAVSLGHPIGMSGARIVGHLTHALKQGEYGLASICNGGGGAS

RT2: 418 AVLIEKL * ** ** HT2: 421 AMLIQKL

indicated by

underlining).

Amino acid

compositions

ofthe

two

isoenzymes

werealso

indistinguishable.

The

composition

was

also

compatible

with that calculated from the deduced

sequence(Table I).

Expression

of

the

thiolase

gene

in

_fibroblasts.

Expression

of

T2 gene in

fibroblasts

from the controls and from the

patients

wasanalyzed

by Northern

blotting, using

the cDNA insert of

HT2-3

as a

probe

(Fig.

4

A).

A

single

band forT2 mRNA was

detected

intwo

controls

and in all four

patients.

The size ofthe

T2 mRNAs was 1.7 kb and was

indistinguishable.

However,

in

cases 1 and

3, the radioactive

signal

wasvery weak and the

band

was

detected

only

aftera

long

exposure. In orderto esti-mate

the

amount of RNAs

applied,

thesamemembranewas

rehybridized with

a

f3-actin

probe.

The

intensities

of

signals

for (3-actin band in cases 1 and 3

samples

were not

significantly

smaller

than others. Itis apparent,

therefore,

that theamount

of

T2 mRNA in the fibroblasts fromcases 1 and 3wasreduced

whereas that

for

cases 2

and

4 wasnot.

In order to

confirm

the reduced

quantity

of

T2 mRNA present

in

cases 1 and

3,

T2 mRNA

abundance

was

quanti-tated

by densitometric

scanning

of

autoradiograms, using 1, 3,

and

9

Jg

of each of the

total RNA

samples

fromfour controls and

four

patients.

Typical

results

are shown in

Fig.

4B.

Pa-Figure3.Comparison oftheamino acidsequencesof humanand ratT2. Thestandard one-letteramino acid ab-breviationsareused. RT2 and HT2

representthesequencesofthe rat and

humanenzymes,respectively. Gaps(-) wereinsertedtoachievemaximum

ho-mology. Identicalresiduesareindicated bystars.Sequencesarenumbered,

be-ginning with the initiator methionines.

Sequencingdataof theratenzyme are

from Fukaoetal. (22).

tients' RNAs were prepared separately from three fibroblast cultures of different passages andweresimultaneously electro-phoresed

and blotted

on a

Hybond-N membrane.

The

ratio

of

intensity

of the band of

T2 mRNA to

that of

,3-actin

was 0.55±0.32 (n = 4), in case of the four controls. The ratio of cases1,2, 3,and 4 was

0.085+0.014,

0.50±0.18,0.009±0.013, and

0.42±0.02, respectively.

These results indicate that the amountof T2 mRNA is lower in cases 1 and 3 than in the controls but is within a normal range in cases 2 and 4.

Subcellular

fractionation

after pulse labeling

of

the

fibro-blasts. Subcellular fractionation was thendonetodetermine whetheror not the pulse-labeled T2

of

the

fibroblasts from

the

patients

were

transported

into

the mitochondria

or were

de-graded

in

cytosol.

The

labeled

T2 was recovered in the

particle

fraction,

whereas lactate

dehydrogenase,

a

cytosolic

enzyme, was present

in

thecytosol,

without exception (Fig.

5). The T2

proteins

can

therefore

be

translocated

into

anorganelle,

pre-sumably

into the

mitochondria.

Discussion

The cDNA

for human T2

which

we

cloned

was

1,518 bp

in

length,

including

a 1,281-base open

reading

frame encoding

Figure2.Nucleotidesequenceofthe cDNAforhuman T2 and deducedprimarystructureofthe enzyme.Nucleotidesarenumberedinthe di-rection 5'to3',beginningatthefirst residue oftheinitiator ATGtriplet.Thenucleotidesonthe

5'-side

from residue 1areindicatedbynegative numbers. Thededucedamino acidsequenceisshown under thenucleotidesequence. Theunderlined amino acidsequencecompletely

matchedtheamino-terminalsequenceof thepurifiedenzyme,determined byEdmandegradation.The stop codonis representedbyTER. Base

variationsfoundamongdifferentclones are asfollows:Awasfoundatthepositions -22, -6, 33,and93 in HT2-3, HT2-9, HT2-13,and

HT2-1,respectively.Thesesubstitutionsledto noamino acid changes. Positions320 and483wereoccupied byAinHT2-3 instead ofC and

T,respectively.Asaresult,AlaandTyrweresubstituted byGlu and stop codon in HT2-3. HT2-2containedA atpositions1018 and 1036,T

C

1

42C3

A

B

Thiolase

1

3

Controls

3

: -28S

. ...

. -. 0:

..

_.:...

._. ?_ fI _

4

Patients

*.-S ,smifibL .,_biu

*# _

IF||wl

3

.il

..1

.

Figure 4.Northern blotanalysisofthethiolasemRNAinfibroblasts fromcontrolsandpatients.(A) 50

,g

oftotal RNA extractedfrom fi-broblastsweredenatured and electrophoresed ina1.0% agarose-form-aldehyde gel. TheRNA wastransferredto aHybond-Nmembrane andhybridized with the 32P-labeled HT2-3 insert. Thesame

mem-branewasrehybridized with,B-actin probe. The filterwaswashed to afinal stringency of 0.2X SSCat65°C.The celllinesareidentified with thecasenumbers at the topofeach lane. (B) RNAsfrom four controls andfour patientswereanalyzed. Patients'RNAs were

pre-paredseparately from threefibroblastculturesof differentpassages andelectrophoresedatthesametimefollowed by blottingon a Hy-bond-N membrane.The amountof appliedRNAis indicatedatthe topofthefigure. The filterwaswashedto afinalstringencyofIX

SSC, 0.1% SDSat65°C and exposedto apreflashed X-ray film.A

typicalblotting pattern ofeachpatient isshown.

the entire

precursor

for this

enzyme. This cDNA willserve as a

pertinent

tool

for

further studies

on the

molecular basis of

heterogeneity

in

3KTD.

Ithas been proposed that thereare two

isoenzymes

of T2

since this

enzymewas

separated

intotwomain

peaks

on phos-phocellulose column chromatography

during

the process

of

purification (13, 29).

Weindeedconfirmed the presence oftwo

peaks of

T2

activity.

The

existence

of these

isoenzymes might

cause

confusion

in the

study

of 3KTD.

However,

Huth

(33)

has

proposed

that these

isoenzymes

are

produced

by

post-translational

modification

such as coenzyme A-mediated

transformation,

basedonthe facts that

isoenzyme

A was

spon-taneously transformed

in

vitro

to

isoenzyme

B,

and that the rechromatography

of

coenzyme A-treated

isoenzyme

B

clearly

demonstrated the

conversion

of

isoenzyme

B to A.

Also,

we

could

not

distinguish

two

purified

preparations

of

isoenzymes

A and B

from

rat

(22),

nor those

from

human

tissue, with regard

toseveral parameters

including

the

amino-terminal amino acidsequence, amino acidcomposition,and

electrophoretic mobility

ofthe subunits.

Only

one cDNA clone had a nucleotide substitution causing an amino acid

change among therat 18 cDNAclones,atposition 698 (Thr233

to

Met) (22),

butnosuch

substitution

wasnoted inthe human cDNA clones. These results support the concept of Huth (33). We examined by Northern blotting the expression of T2 mRNAin

fibroblasts from four

patients who have been well characterizedattheprotein level ( 19-21). The resultsarelisted in Table IItogether

with

a summary

of

the previous studies done atthe

protein

level. The present results

provide

further

information

onthe molecularbasis of defects in T2 biosynthe-sis of these

patients.

T2 mRNAs detected in all cell lineswere

thesamesizeasthosefrom the controls. However, the amount of themRNA wasreduced in thetwocell

lines,

cases 1 and3,

particularly

in the latter. The amount in the other two cell

lines,

cases2 and 4, was within a normal range. We previously considered thatcases3 and4

belong

to asamegroup since the

Thiolase

,/-actin

.-

acti

_n

RI

I

l

18S

Table L Amino AcidComposition ofT2

Numberof residuespersubunit

Direct analysis

Predictedfromthe Amino acid Isoenzyme A Isoenzyme B cDNAsequence

Asp 33.8 34.2 15

Asn J 19

Thr 19.7 20.6 21

Ser 20.7 22.5 24 Glu 35.9 35.4 22

Gln LL. 12

Pro 19.0 18.9 18 Gly 38.7 37.5 38 Ala 48.0 47.9 47

Cys 7.3* 6.2* 5 Val 40.6 40.4 39 Met 13.9 14.3 16 Ile 25.1 25.1 25 Leu 32.8 32.1 31 Tyr 7.2 7.5 9 Phe 7.3 7.4 7

Lys 28.7 29.1 30 His 5.3 5.4 5 Arg 9.9 9.5 9 Trp ND$ NDt 2 * Determinedas

S-carboxymethylcysteine.

Not determined.

pulse-labeled

T2

proteins could be detected after

a72-h chase, in

either

case

(21). However, Northern blot analysis revealed

that

they differed

in

expression

of

T2

mRNA.

In case 1, the T2 mRNA was

moderately

decreased. The

pulse-labeled T2 had

a slower

electrophoretic mobility

than

did the

normal mature T2

subunit. The

synthesized

T2 was

unstable.

Fractionation of

fibroblasts after

pulse

labeling

re-vealed that the

variant

T2

protein is translocated into

mito-chondria

and

degraded there.

In case

2,

the

amount

of

T2 mRNA was

within

a

normal

range;

however,

a

smaller

amount

of

normal-sized T2

protein

was

detected in the mitochondrial

C

1

2

3

4

IPs~-l P

p

_S7

_pI

_IS

s

P

_IS

_II

PI-'SlPIS'

_s

_IF-T

T20

~ I SI

lPSIPSi

IlPS

LDH v-; X

-I

Figure5.Subcellularfractionation after pulselabeling.The pulse-la-beledfibroblastswerefractionated into supernatant(cytosolic frac-tion, S) and pellets (particle fracfrac-tion, P),asdescribed in Methods. Eachfractionwassubjectedtoimmunoprecipitationwith anti-mito-chondrial acetoacetyl-CoA thiolase (T2)antibodyand anti-lactate dehydrogenase(LDH)antibody followed bySDS-PAGE and fluo-rography. C, controlfibroblasts;1-4, the cell lines ofcases1-4,

re-spectively. Arrowheads indicate the bands for T2orLDH.

fraction,

andwas unstable. Hence the mutation ofT2 gene mustbe located in the region

coding

for the mature T2 pro-tein. In case 3, the amountof T2 mRNAwasmarkedly de-creased, and theamountofT2protein detected was alsosmall.

The mobility in SDS-PAGE and

stability

of the T2 were indis-tinguishable from those of controls. Thecauseofanenzyme defect in this patientseems tobeagene mutationaffecting the splicing, or inefficient

transcription.

Middleton et al.

(9)

re-ported that some residual T2

activity,

- _7%ofthe

_control,

_was

detected in fibroblasts from this

patient.

Taken

together,

the T2 protein in case 3 might be functional.Incase4, the amount ofT2mRNA was within a normal range, and the pulse-labeled T2 was normal in size but

unstable.

T2

protein

was

smaller

in amount than that of the controls and

degraded

considerably

at a 72-h chase. Apoint mutation in the

protein coding

region may be

considered

asthe cause

of the

enzyme

defect

in case

4.

The mutation sites in the

coding region

in cases 2 and 4 pa-tients

presumably

differ because themutant

protein

in case2

degraded more rapidlythan didthat from case 4.

Thus,

the molecular basis of the disease appears to

be

different

in all

four

patients.

Table IL Summary

of

Heterogeneityin3KTD

Pulse-chaseexperiment

Pulse Immunoblot

Amountof Northem T2

Case blot mRNA* CRM* Size Chasestability CRM activityl

kD

Control N +++++ 41 Stable +++++ +

Case I Moderatelyreduced + 42 Unstable -

-Case2 N + 41 Unstable

Case 3 Markedlyreduced + 41 Stable +

Case4 N + 41

_Relatively

unstable +

Acknowledgments

The authorsthankDr. M. Mori,Institute for MedicalGenetics,

Ku-mamotoUniversityMedicalSchool,Japan for thegiftof the human cDNA library; Dr. R. B. H. Schutgens, Department ofPediatrics, University Hospital, Amsterdam,TheNetherlands;Dr. L.Sweetman,

Department ofPediatrics, UniversityofCalifornia, Berkeley;andDr. N.Sakura, Department ofPediatrics,HiroshimaUniversity School of

Medicine,Japan forprovidingthecelllines;and M.Oharaforhelpful

comments.

This studywassupportedinpartbygrantsfor thepromotionof science research(01570522)from theMinistryofEducation, Science,

andCulture ofJapan,andforstudiesoninherited disorders(630104)

from theMinistryof Health andWelfare,Japan, and bygrant 2-11-19 from the National Center of Neurology andPsychiatryof theMinistry

of Health andWelfare, Japan.

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