Vol.

176,

No.

May

15, 1991

BtOCHEMlCAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

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

Vol.

176,

lack

No.

3, 1991

BIOCHEMICAL

GM mRNA, while

and/or

amounts

(18,19).

been determined, junctions

many adult

(20).

AND

BIOPHYSICAL

onset patients

The organization

including

the

Tenrestriction

nu&er

RESEARCH

exhibit

mR?A of altered

of the stmctuml and sizes

of

COMMUNICATIONS

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

Vol.

176,

No.

BIOCHEMICAL

3, 1991

microgranspm-~ with the nemycin

AND

BIOPHYSICAL

and 40 mkrqram ofplamid, analog G-418 (Geneticin