Identification of the promoter region and gene expression for human acid alpha glucosidase

Vol.

176,

No.

May

15, 1991

BtOCHEMlCAL

3, 1991

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BIOPHYSICAL

RESEARCH

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Stephanie

Tzall

1509-1515

and Fmnk Ma?&niuk*

New York University Medical Center, hparbent of Medicine 550 First Avenue, New York, NY 10016 Received

March

20,

1991

SUMMARY: Genetic deficiency of acid alpha gluaxsidase (GAA) results in glycogen storage disease type II. A CCNA containing the complete coding region was constructedandcloned intotheexpxessionvector@V2 andwastransiently transfected into an sV40 imortalized CAA deficient hmen fibrcblast cell line which has undetectable levels of CAA enzyme activity and does not express GAA mFuK Transfected cells had 4.9% of normal human fibmblast enzyme activity. Additionally a 5' 1.8 kb genomic fragment was ligated to the 5' end of the GAA cDNA construct and cloned into pUC19. Transient and stable transfection also resulted in expressed GAA enzyme activity in deficient fibroblast cells, indicating that the gentic fragment has GAA p?xxnoter function. 0 1991 Rcademlc Press,

1°C.

Acid alpha glucosidase enzyme that

hydrolyzes

GAA, glyccgen that

varies

slowly

storage

or acid maltase

glycqen disease

from a rapidly

progressive

to yield type II,

fatal

glucose

infantile

of enzyme activity,

accmulation

of glycogen

in

tissues.

In the adult

is limited

to skeletal

to residual with antibody,

enzym

muscle (2-6). activity,

and abnornkalities

We have previously human GAA (16,17) genetic

onset form, enzyme activity

cloned

presence

Cells

abnormlitiesofnif@JAardDN?+.

weakness,

muscle is variable

of

disease

and mssive

as well

as other

and involvement

frompatientsareheterogenecusas

or absence 0fproteincmssIeacting

of post-translational and detemnimd

the

and have used the cDJA to deteznune

heterogeneity

deficiency

form is characterized

muscle

and skeletal

Genetic

(Pcanpe's di sease)toa

The infantile

low levels

is a lysosomal

heterogeneous

disorder

by extremly

cardiac

(1).

is a clinically

onset myopathy.

adult

(GAA) (JZC 3.2.1.3)

among GAA deficient

patients

Approximatelyhalfof

p recessing

(7-15).

sequence of the &&JA for that

there

is extensive

as detected

by gross

infantileonsetpatimts

*Towhomallcorreqmx&anceshouldbeaddressed. 0006-291x/91

$1.50

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176,

lack

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BIOCHEMICAL

GM mRNA, while

and/or

amounts

(18,19).

been determined, junctions

many adult

(20).

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onset patients

The organization

including

the

Tenrestriction

nu&er

RESEARCH

exhibit

mR?A of altered

of the stmctuml and sizes

of

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gene for GAA has

exons and intron-exon

fra~tleqthpolymorpkisms

have been

identified

(16,21-24).

determined

(17,25).

This

hcxnologyto

Splbindingsites,

(RFLPS) forGAA

The sequence

region

contains

size

5'

several

butnoC.AATorTATAbox

to

exon

GC rich

1 has been regions

with

(17).

Wehavenawconstructedafulllengthcodingregionc~clonedinthe expressionvectorpSV2 brtalized levels

andusedthisplasnidto

GAA deficient

human fibmblastcell

of GAA enzyme activity

region,weisolatedagenomic contains

a GC rich

genrnnic fragment

construct

line

and no mRNA. To grossly

region

site

transiently

including

at least

localize

to the polyadenylation and stably

expressed

site

the pmnnoter 5' -which

two Spl birding

onto the 5' end of the full

ansV40

which has undetectable

fragmentthatincludedexononeand

was ligated

frnn the A'II; start

tmnsientlytransfect

length

sites.

coding region

and the poly A tail.

GAA enzyme activity

This

This

in deficient

cells.

RNA, INA and cell

lines

F@?A, cDNA and genomic CNA were isolated or synthesized as described Amplification and purification of plasmid constructs and previously (17). Southern and Northern analysis were done by ~TAx&xI nukhcds (26). Norml fibroblastcell line GM5758 and normal lyqhoid cell line GM3202 wereusedas controls in Southern andNorthern analyses. Recipient cells for transfection were an SV40 immortalized human fibmblast cell line @I4912 (an infantile onset patient; NIH Human Genetic Mutant Cell Repository, Garden, NJ) which exhibited no enzyme activity for GAA and no GAA mRNA. The cell line was utilized for transfection experiments at passages Tll to T95. DNA-mediated

transformation

and enzyme assay

SV40 immortalized hunan fibroblast cell line GM4912 was transiently transfected as previously described (27). Briefly, cells were plated at 0.4 X lo6 cells/lOOrm~ petri dish 24 hours before addition of DNA. Calcium @osphate was carried out with plasmids precipitated transient gene expression containing constructs in either forward or reverse orientation (40 micmgrams per petri dish) by the method of Graham and van der Eb (28) as modified by washedaMias.sayed Wigler et al. (29). After 48 hours, cellswereharvested, for GAA activity using the artificial substrate 4-methyl~lliferyl-alpha-Dglucoside as previously described (30). GAA expression was also visualized by starch gel electrophoresis followed by staining for GAA (30). Stable cotransformation was carried out as previously described (31) with l-2 1510

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microgranspm-~ with the nemycin

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and 40 mkrqram ofplamid, analog G-418 (Geneticin
Constructionofthe

RESEARCH

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amlthe~genewas

selected

fulllenothccdimregionclXA

AcDNAcontainingthecmpletemding regionwasconstructed by ligating a 3.2 kb cDNA fragmnt digested at an S&II site (bp 318) aMi an S@iI site in the 3' non-coding region (bp 3150) to a 0.45 kb 5' fragmant digested with NcoI at the AT3 start site (bp 1) and with SstII (bp 318) (17). -11 linkers were added and the construct cloned into lzUC18. The axxstruct was resequd by the Sanger dideoq chain termination method (32) through the 5' NcoI site, and the SstII restriction site, all of whi& wexe the ATG start signal, Theconstructwas recloned intotheSV4O+as&expressionvector, lrlaintained. pSV2 (gift of Dr. S. H. Orkin.) and named GAA-pSV2-forward. As a control, the insert was also ligated in the reverse orientation (GAA-pSK+reverse). Isolation

of the promoter

r&on

and liaation

onto the c&ins

reuion

In order to grossly locate the promter region, we isolated a 1.8 kb XhoI/AatII genmic fragmentwhichcontainedthe St untranslated186bpe.xon1, plus an additional 5' 1.5 kb segment. AfteradditionofHiMIII linkers, this fragmnt was ligated to the ATG start site on the 5' end of the full length GAA cDNA including the poly (A) addition site and poly (A) tail, and cloned into plJcl9 (GAA-pmter-forward). As a control, the reverse orientation was also isolated (GAA-promoter-reverse).

Cells

transiently

(0.11 U/g)

transfectd

of norm1 fibroblast

with

the GAA-@V2-forward

activity

(2.26 U/g),

with the GAA-pSV2-reverse showed no detectable to

determine

electrophoresed

activityby

if

the &acts

staining

increase

in GAA enzyme activity

of transfected asdescribed

activity

cells

inmaterials

on star&

while cells (Table 1).

o.ll+o.o7(n=11)

GAA-psv2-met-se

< 0.003

GAA-Pmmote~Fomanl

0.040 0.018

GAA-hnmoter-Rarerse

< 0.003

Normal fibmblast

Ql 08399

(u=5)

Eqmassionofthe

2.2eO.25 (~4)

a pies of 4-methyl~lliferyl-alFSla-Dglucoside hydrolyzed/min/g protein at 37OC_+SD. 1511

% Nomel 4.9 < 0.0 1.8 1.2

0-1

In order

gel armdvisualized

Table 1. Transient Ekpression 0fGAA in sV40 linmrtalized HmanGAAtkficient Fibroblasts

GA?+-W-Forward

transfected

was indeed GAA, we

andmethods.

EZmymeActivitya

cC@?Ahad 4.9%

< 0.0 100.0

GAA

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BIOCHEMICAL

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GAA-pSV2-forward could

be visualized

cmigratingwiththeextracts

GAA-promoter-forward

cm

reverse cDNAconstruct further

replicated

nemycin

with

the next

1.5

U/g

fibroblasts no&

E'coFU and HiMI

gencm follow&

were

1). stable

gene

picked

into

were assay&

lA).

by Northern

equal tothatseen

activities

for

(W)

with

These levels cells

e.

GAA

appeared

DJA from this

and was

colony rang&i

as observed

were in the rarqe

The

to be stable

of this

confluemcy,

(0.7-2.0

The colony

to keunstablewithtime.

The enzyme activity

frcan

with

normal

of enzyme activity

frcm

U/g).

colony

(W) seven days @passage.

of copies of the plasmid (releasing

analysis.

(Fig.

These colonies

activity

increased

fibroblast

the nmber

by Southern

highest

which

We next extracted estimated

M-pm

(Table

colonies

(ES) appear&

analysis.

(13).

control

the

we utilized

resistant

with the highest

enzyme activity

expanded for further to

activity

with the

u/g) of normal

(0.03 with

region,

plates.

and the four colonies

colony

1.5%

kansfected

promotor

transfect&

in~t~ialsandmethods.?heenzymeactivityperwlony

with the highest

0.5

the

microtiter

activityasdescribed varied

cells

as a band

(not shown).

transiently

showed nodeteckableenzym

analyze

24-well

cells

COMMUNICATIONS

gel electrcphoresis

exhibited

while

RESEARCH

fibrublasts

construct

Twenty-four

expression.

by starch

GAA deficient

enzyme activity,

To

BIOPHYSICAL

frmnorml

SV40 immortalized

fibrcblast

AND

the coding

in this

region)

We

colony by digestion

or with FmRI

alone,

with

follmed

~~wereapproximatelyfivetotencapiespresentper We additionally analysis.

estimated

ccpy nuker

GAA nWNA was of normal

fromanormalfibroblastcellline

by mRNA isolation

size and approximately (Fig.

US).

DISUJSSION

Wehavepreviouslydeteminedthe GAA and identified sequeme

ad

an intron

demonstrated

deficient

that

transient

human fibroblast

the coding gene cell

fortheccdingregionofhman

separates

from the second exon containing We have now ligated

(17).

sequeme

the

the initiation region

into

expression

in

line

as detected 1512

5'

untranslated

leader

of translation

the expmion

(AYE)

vector

an SV40 innmrtalized by an increase

pSV2 GAA

in enzyme

Vol.

176,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

-23

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B

- 9.6

- 6.6 -44 -23 1234667

3

2

1

Fiu. 1 A Scuthexn analysis of gencmic DNA from the stable cotransfonnantD6 prabe utilized for to estimate the GAA-promter-forward copy rumba. hybridizationwas theGAAccdingr@on. Lam 1: CNA frmnomal fitmblast Lanes 2 and 3: CNA frm stable cell line (CN5758) digested with EcoRI. u&.ransfomantD6digestedwithEccRI inlane 2, aMwithhalftheCNAinlane 3. Digestion with EccRI n&es a siqle cut at the 3' end of the plaanidconstict. Onemjor site of integrationcanbe seenas averydensebamlof approximately 9 kb. Lane4:DNAfnmnormalfibrcblastoelllinedigested with EcaRI a& Hi&III. Lanes 5,6 and 7: DNAfmn stable cotzamformantD6 digffi~witi EknRI andHindII1 in lane 5, with lanes 6 a& 7 1:lti 1:4 dilutions of lane 5. AnEcoFU/Hi.rSUII digestion releasesthe czdim~regian, seenasaverydensebandatapproximtely3 kb, dermnsbattingasinglemajor site of integration. We estimtethatthexeare approximtely five to ten copies inthisbard. analysis of URNA fztm stable cmtransf0rmarrtD6~rlc& Ficr. 1 B Northern Probe utilized for hybridization was the GAA coding region. Iane cell lines. lmhoid cell line (GN3202). lane 2: mRliA fran norml 1: mRNA frm normal fibmblast cell line (CN5758). Iane3:MNAfrmstable cotransformantD6. Hybridization for all samples was to the sqazcbd 3.6kbGAAbarL Theanmunt 0fmRNA fromD6 (Iane 3) wasapproximtelyequal tothatfmnthenoxmal fibroblastline (lane 2). activity

5% of normal)

(approximately

starch

and visualization

We tested the deficient

gel.

humm cell

by el~oresis

line

for its

in

ability

to take

up at-d express the &g gene, alX3wefouIXIthatthiscelllinetakesupand qresses

DNA approximately

(unpublished obsemation).

one fifth

to one tenth

merefore

that

of muse 3T3 cells

the apparentlmlevelof

qression

is

a functionofthehostcellline. We have also demnstrated

that

with a GC rich area and at least&o TheGAAprcmoterresulted

of transcription,

analysis

fragment 5' to axon 1

SplbimIir~~siteshaspzmmter

function.

inlowertransientgeneexpzessionccprcparedtothe

This may be due to the akseme of the 32 bp 58 of the An; start

P=P-ter. site

a 1.8 kb genmic

of

stable

which was not cotransformants

inclukd

reveald

copies per gencme were presentatonemajor enzyme activity

qua1 to that

obseme the expect&

ratio

okerved

in the construct. that

approximately

integration

site,

in normal fibroblasts.

However, five

to ten

with express& We did not

of estimated mpy number versus expressed enzym 1513

Vol.

176,

level,

No.

BIOCHEMICAL

3. 1991

that

is,

f&&lasts.

AND

we did not observe expression This

would suggest

that

prcsnoter sequences but my be lacking for

the

interest, activity (13).

higher the with

Thismay

or that nongenetic

the

enzyme activity stable

that

the

obsexved

RESEARCH

five

in

with

will

in

GAA specific

activity

When the regulatory

that

of the nomal

fragment

does contain

fibrcblast

cell

increase

in

nornbal fibroblast

irdicatethatsomeregulatoryorenhaxer increase

COMMUNICATIONS

or -elementsneeded

showed the

is observed

times

gerxxnic

regulatory

cotransfomant

confluency

factors.

these questions

BIOPHYSICAL

lines.

Of

GAA specific cell

lines

elertentsampresent with

or enhaxer

confluency

elements

is due to

am identified,

be answered.

This resxlmhwas supportedbythefollowinggrants:MuscularDystmphy Association, American Heart Association #870992, and NIH Grant ROI 39669. WewculdliketothankDr.Angel was a fellow of the Arthritis Foun3ation. Fellicer for his fruitful discussions.

ST

1. Hers, H.G. (1963) Biochem. J. 86,11-16. 2. Pcanpe, J: C. (1932) Ned. Tijdschr. Gemrskd. 76,304-311. 3. Courteclussf, v., Royer F., Habib, R., Monnifer, C., and Denvos, J. (1965) Arch. Franc. Fediat. 22,1153-1164. 4. EM@, A. G., Gomz, M. R., Seybold, M. E., and La&x-t, E. H. (1973) Neurology 23,95-106. 5. M&le.r, M., and Di Mauro, S. (1977) Neurology 27,178-184. 6. H&s, H.G., vanHoof, F., and deBamy, T. (1989) In The Metabolic Basis of Inherited Disease (C.R. Striver, A.L. Beaudet, W. Sly, and D. Valle, Eds.). Vol.1, pp. 425-452. McXraw-Hill, New York. 7. Beratis, N.G., IaBadie, G.U., and Him&horn, K. (1978) J. Clin. Invest. 62,1264-1274. 8. Beratis, N.G., La&die, G.U., and Him&horn, K. (1983) Am. J. Hus.Genet. 35,21-33. 9. Brown, B. I., and Brown, D. H. (1965) Bicchim. Biophys. Acta. 110,124-133. 10. Hasilik, A., and Neufeld, E. F. J. Biol. Chem. (1980) 255,4937-4945. 11. Komfeld, S. (1986) J. Clin. Invest. 77,1-6. 12. LaBadie, G. U., Harris, H., Beratis, N. G., and Him&horn, K. (1985) Am. J. Mrm. Genet. (1985) 37,Al2 (abstract). 13. LaBadie, G.U. (1986) Ph.D. Thesis, City University of New York, Mt. Sinai Hospital. 14. Reuser, A. J. J., Kroos, M., Cude Elferink, R. P. J., and Tager, J. M. J. (1985) Biol. Chem. 260,8336-8341. 15. Reuser, A. J. J., Kroos, M., Williamson, R., Swallow, D., Tager, J. M., at-d Galjaard, H. (1987) J. Clin. Invest. 79,1689-1699. 16. !Qrtiniuk, F., M&&r, M., Pellicer, A., Tzall, S., IaBadie, G. U., Ellenbqen, A., and Hirschhom, R. (1986) Proc. NatI. Acad. Sci. USA 83,9641-9644. 17. Martiniuk, F., M&&r, M., Tzall, S., Meredith, G., and Him&horn, R. (1990) DNA and Cell Bio. 9‘85-94. 18. Martiniuk, F., Mehler, M., Tzall, S., Meredith, G., and Hir?XhhOm, R. (1990) Am. J. H\rm. mt. 47,73-78. 1514

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19. van der Ploeg, A.T. Hcefslot, L.H., Hoogeveen-Westeweld, M., Petersen, (1989) Am. J. Hum. Genet. 44,787-793. E.M., and Reuser, A.J.J. 20. Martiniuk, F., Bodkin, M., Tzall, S., and I3irs&hom, R. (1991) r%lA ti Cell Biolcgy, In press. 21. Tzall, s., Martiniuk, F., Adler, A., and Hire&horn, R. (1990) Nut. Acids Res. 18,1661. R. (1990) Nut. Acids Res. 22. Tzall, S., Martiniuk, F., and Hirwhhom, 18,193O. 23. Tzall, S., Martiniuk, F., Ozelius, L., Gusella, J., and Hire&horn, R. (1991) Nut. Acids Res. In press. 24. Tzall, S., Martiniuk, F., and Hirschhorn, R. (1991) Nut. Acids Res. In press. 25. Hcefsloot, L-H., Hcogeveen-Westerveld, M., Kmos, M., van Beeumen, J., Reuser, A.J.J., and Oostra, B.A. (1988) EM83 J. 7,1697-1704. 26. Sambrook, J., Fritsch, E.F, and Maniatis, T. (1989) Molecular Clonirq: A UbxatoryManual. ColdSpringHarborLabxatory Press, ColdSpring Harbor,NY. 27. Martiniuk, F., Bodkin, M., Tzall, S., at-d HiE&hOm, R. (1990) Am. J. M. Genet. 47,440-445. 28. Graham, F.L., and van der Eb, A.J. (1973) Virology 52,456-467. 29. Wigler, M., Pellicer, A., Silverstein, S., and &el, R. (1978) -11 14,725-731. 30. Martiniuk, F., and Hirschhom, R. (1981) Biochim. Bisphys. Acta. 658,248-261. 31. %X+kl.iUk, F., PelliCer, A., Mehhr, M. and Hi.rs&hom, R. (1986) SopMtic Cell and Mol. Gal&. 12,1-12. 32. Sanqer, F., Nicklen, S. and Coulson, A.R. Prcc. Natl. Acad. Sci., uSA (1977) 74,5463-5468.

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