Treatment of glutathione synthetase deficient fibroblasts by inhibiting γ-glutamyl transpeptidase activity with serine and borate

Vol. 89, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 27, 1979

Pages 504-511

TREATMENT OF GLLJTATHIONE SYNTHETASE DEFICIENT INHIBITING Stephen

y-GLUTAMYL TRANSPEPTIDASE

P. Spielberg,

ACTIVITY

Jean DeB. Butler,*

Departments of Pharmacology and Pediatrics, Johns Baltimore,

FIBROBLASTS

BY

WITH SERINE AND BORATE

Kay MacDermot,

and Joseph

D. Schulman

and Experimental Therapeutics, Hopkins School of Medicine, Maryland 21205; and

Section

Received

on Human Biochemical and Developmental Genetics, NICHD, NIH, Bethesda, Maryland 20014

June 14, 1979 SUMMARY

Glutathione synthetase deficiency results in decreased cellular glutathione content and consequent overproduction of 5-oxoproline. Lserine in borate buffer inhibits y-glutamyl transpeptidase, the major Treatment of glutathione synthetase catabolic enzyme for glutathione. deficient fibroblasts with 4OmM serine and borate for 24 hours produced more than a 2-fold increase in cellular glutathione content. L-serine alone led to a smaller increase in glutathione level, and borate alone was without effect. On exposure to serine and borate, 5-oxoproline formation from L-glutamate was decreased to normal levels in glutathione presumably secondary to feedback synthetase deficient fibroblasts, inhibition of Y-glutamylcysteine synthetase by the increased intracellular Cellular free amino acid content was generally glutathione concentration. unaffected by such exposure although increases were observed in serine This model system suggests that y-glutamyl transpeptidase and phosphoserine. inhibition may be a rational approach to alleviating the effects of glutathione synthetase deficiency. Generalized 6.3.2.3.)

deficiency

results

consequent

anemia

(1,2)

with

conversion

of 5-oxoprolinase *To whom reprint and Developmental

intracellular

oxidant-mediated

with

(1,2).

cell

metabolic

it

to glutamate

@ 1979 by Academic Press, Inc. reproduction in any form reserved.

of

(1).

and

deficiency by hemolytic

function

occurs

(3).

secondary beyond

Inhibition

5to

the capacity of y-

Section on Human Biochemical requests should be addressed. 20014 Genetics, NICHD, NIH, Bethesda, Maryland

0006-291X/79/140504-08$01.00/0 Copyright All rights

manifested

to oxoproline,

(E-C.

content

Glutathione

leukocyte acidosis,

of y-glutamylcysteine to convert

damage,

polymorphonuclear

resultant

synthetase

glutathione

of 5-oxoproline

and defective

Oxoprolinuria, excess

in decreased

overproduction

is associated

of the enzyme glutathione

504

Vol.

89,

No.

BIOCHEMICAL

2, 1979

glutamylcysteine

synthetase

Ideal

(4). toward

therapy,

normal,

thus

by

would

decreasing

the

inhibition

of

reducing

oxoproline

production.

is

the

might

major be

particularly glutamyl

in

on

(8).

cultured

deficiency.

decrease

(7).

increase in

which

the

found in

conversion

cellular

glutathione

content

damage (5)

(6)

for

from

effect with

MATERIALS

L-serine, inhibitor

serine

site and

glutathione

y-

a of

borate

synthetase produces

content to

of

binding of

of

formation

serine-borate

acid

2.3.2.1.)

inhibition

a potent

glutathione C-glutamic

its

y-glutamyl

to

secondarily

content.

result

a patient exposure

14

and

increasing

(EC

and

is

the the

intracellular

while

transpeptidase

buffer,

examined

of

production

glutathione

competes

that

oxoproline

oxidant

may

COMMUNICATIONS

decrease

glutathione

borate

from

RESEARCH

synthetase

Inhibition

fibroblasts

We have

significant

of

We therefore

skin

of

cellular

presence

complex

enzyme

risk

for

increase

transpeptidase

serine-borate the

the

increase

y-Glutamyl

enzyme to

may

y-glutamylcysteine

catabolic

expected

BIOPHYSICAL

cystamine

however,

feedback

AND

and

an

a associated

5-oxoproline.

AND METHODS

Skin fibroblasts from a patient with glutathione synthetase deficiency and 5-oxoprolinuria (2) were maintained in Eagle's Minimal Essential Medium (N.I.H., #2 Eagle's Basal Medium, equilivant to Eagle's Minimum Essential Medium, Grand Island Biologicals Co., Cat. No. 320-1095, Grand Island, N.Y.) containing 10% fetal calf serum, fresh glutamine, and antibiotics. Studies we.ce performed on confluent cell cultures. L-serine was added to fresh culture medium in isotonic phosphate-buffered saline or sodium borate bufEer, pH 7.4, at concentrations indicated. For glutathione determinations, monolayers were rinsed three times with phosphate buffered saline and scraped into 0.02 N HCl. Glutathione was assayed by the Cell protein was determined according to Lowry, method of Tietze (9). -et (10). -al. Conversion to 5-oxoproline medium containing

14 of C-L-glutamic was examined after 40 mM serine-borate.

Acid 24

(U) hour

(New England pretreatment

Nuclear of cells

Corp.) with

The radioactive glutamic acid was repurified on a 0.4 x 6 cm. resin The column was column containing AG-50 (H+) X 8,100-200 mesh (Biorad). washed 3 times with 5 ml. of H 0 to remove any 5-oxoproline, and the The eluate was evaporated glutamate eluted with 2.5 ml o L? 3N NH40H. under nitrogen, resuspended in phosphate buffered saline, and 1OvCi Incubation was continued for added to the medium in each culture dish. 18 hours a't 37OC. For assay of extracellular 5-oxoproline, medium was proteins precipitated with l/10 volume of 50% trichloroacetic aspirated, and .the supernatant passed over a 0.4 x 12cm AG-50(H+) column. 5acid,

505

Vol.

89,

No.

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

oxoproline was eluted in 2 X 5 ml. water. Aliquots were mixed with Aquasol (New England Nuclear) and radioactivity determined in a scintillation counter. Intracellular 5-oxoproline was determined after washing the monolayers 3 times with phosphate buffered saline. Cells were scraped into water and l/10 volume of 50% trichloroacetic acid added. Proteins were moved by centrifugation and quantified by the method of Lowry, et al. (10). -re Tdentity of the labelled C-5-oxoproline was isolated and counted as above. product from the column was confirmed as oxoproline by high voltage paper electrophoresis in 0.05 N sodium acetate buffer, pH5.5. y-Clutamyl transpeptidase activity was assayed nitroanilide in the presence of glycylglycine (11). activity (2) and intracellular amino acid concentrations determined as previously described.

with y-glutamyl-pClutathione svnthetase (12) were

RESULTS L-serine inhibition was

more

inhibited was

than

markedly 95%

inhibited

fibroblast

y-glutamyl

enhanced by

by

borate

20 mM serine

INHIBITOR

transpeptidase buffer and

CONCENTRATION

(Fig. borate.

activity; 1).

Exposure

enzyme of

(mM)

Figure 1. Effect of serine-borate on fibroblast y-glutamyl Trypsinized fibroblasts were sonicated in (GGTP) activity. and y-glutamyl transpeptidase assayed at pH 9.0 (11) in the (OL-serine (O----O), serine borate a), or borate (x).

506

The

transpeptidase 0.1 M Tris presence of

Vol.

89,

No.

2, 1979

BIOCHEMICAL

Table Content Cell

I. of

AND

Effect of Glutathione

Line

BIOPHYSICAL

Serine-Borate Synthetase

on the Deficient

RESEARCH

Glutathione Fibroblasts"

Treatment

Normal

Fibroblast GSH (Pg/mg protein)

0

Glutathione

Synthetase

Deficient

0 40 mM serine 40 40

COMMUNICATIONS

6.9

+

0.7

(11)

1.9

+ 0.1

(4)

mM serine

4.4 2.4

+ 0.3 5 0.1

(6) (6)

mM borate

1.6

+- 0.2

(6)

and

borate

+ +

*Confluent fibroblasts were incubated in Eagle's MEM with 10% fetal calf serum with addition of the compounds indicated. Borate was added as sodium borate, cells were harvested and glutathione pH 7.4. After incubation for 24 hours, determined (9) as indicated in the text. + Different

than

untreated,

p
Student's

t-test.

13OJ / I00 -

90-

60-

&P 70P 5 0

GO-

5 c3 z-

5o

1

5

1

,

IO

40 L30RATEZ0hlM~

Figure

2.

on increase content.

Effect of borate concentration in glutathione synthetase Experimental conditions as

in deficient in Table

507

the I.

presence fibroblast

of

40 til glutathione

L-Serine (GSII)

Vol.

89, No.

2, 1979

glutathione medium

BIOCHEMICAL

synthetase at

37OC

intracellular

for

24 hours

GSH

content

increase,

and

borate

of

(up

to

borate

serine

(Fig.

6 through

was hour

measured pretreatment

of of

14

synthetase both

1). without

enhanced

the

COMMUNICATIONS

to

serine-borate

in

treatment C-L-glutamic

intracellularly serine-borate

with

4OmM

than

Serine

2-fold

alone

of

increased

cells and

in

glutathione

(4OmM)

by

was

to

normal

medium

(Fig.

decreased

40 mM during

3).

5-oxoproline

the

a smaller concentrations

(Fig.

compared

in

progressively

serine-borate to

to

Increasing

elevation

acid

increase

led

effect.

glutathione

deficient

with

RESEARCH

a more

was

Fibroblast

24 hours

glutathione

produced

alone

BIOPHYSICAL

fibroblasts

(Table

40 mM)

2).

Conversion

deficient

AND

increased when

5-oxoproline

4).

24

5-oxoproline

HOURS Figure 3. Time course of increase in glutathione glutathione synthetase deficient fibroblasts on borate. Cells were incubated for the indicated containing serine-borate and assayed as in Table

508

(GSH) exposure periods I.

in

content of to 4OmM serinewith medium

Vol.

89,

No.

2, 1979

BIOCHEMICAL

;H-S

GSH-S

Effect Etgure 4. C-5-oxoproline. untreated; Eta=

overproduction

Under mutant

these

DEFICIENT

and deficient

conditions

glutathione

fibroblasts

to

mutant

was amino

medium

with

RESEARCH

COMMUNICATIONS

DEFICIENT

synthetase

Intracellular exposure

BIOPHYSICAL

NORMAL 14

of serine-borate on conversion Experimental conditions are 40 mM serine-borate. GSH-S

in

glutathione

AND

normal

cells; cells

of C-glutamic acid detailed in the text.= = Glutathione Synthetase

5-oxoproline was

synthetase

production

normalized

in

activity

in

serine-borate. in

normal

or

unchanged. acid 4OmM

concentrations serine-borate

were except

509

unaltered for

increased

by

24

hour

L-serine

to =

Vol.

89,

(16

+ 3.6

to

No.

2.2

to

of

BIOCHEMICAL

67 & 9.1

+ 0.7

signs the

2, 1979

nanomoles/mg

nanomoles/mg

toxicity

AND

by

protein)

protein) light

BIOPHYSICAL

and

RESEARCH

phosphoserine

concentrations.

microscopy

(0.7

Cells

during

COMMUNICATIONS

did

serine-borate

+ 0.2

not

manifest

addition

to

medium. DISCUSSION The

result

clinical

both

signs

from

overproduction.

correction

of

synthetase

decreases

and

both

glutathione studies

content such

partially

function

defects

(14)

damage,

that

well

as

approach and

oxoproline

to

metabolic would

Inhibition

of

reported

to

is

(16)

toxic

rat the

are

unknown.

developed

which

might

and

the

a useful

tool

cellular

metabolism. The

by

for

renal

It be

be

possible

(13)

intracellular

catabolism

would

the

anemia

The

present

by

cell

production.

other

that

decreased

increases

The protected

inhibiting

glutathione advantage from

toxicity

use

glutathione

from

of oxidant

5-

is

of

such the

of

possible

two

major

here

of

metabolic

510

safe

(15).

inhibitors in

glutathione

in

tissue

culture

glutathione

and

the

been Borate

transpeptidase

useful

document

has

in viva -__

y-glutamyl

that

inhibitors roles

serine-borate

content

effects

reported the

by

therapeutically

exploring

of

shown

hemolytic

glutathione

cells

y-glutamylcysteine further

deficiency.

transpeptidase

long-term

investigations

serine-borate

that and

y-glutamyl

increase and

synthetase

at

diminished.

inhibition

deficiency,

is

acidosis

be

the

5-

aimed

of

have

with

5-oxoproline

therapy

be

should

studies

serine-borate

decreasing

but

associated

of

with

(4)

correct

inhibition

should

and

Inhibition

previous

glutathione

transpeptidase as

an

Our

deficiency

content

thus

formation

can

to

synthetase

abnormalities.

a-tocopherol

demonstrate

y-glutamyl

therapy

5-oxoproline

secondary

glutathione glutathione

Rational

synthesis.

leukocyte

of

intracellular

biochemical

glutathione

antioxidant

symptoms

decreased

oxoproline

reduce

and

could

synthetase may

provide

5-oxoproline

correction

consequences

be

in vitro -___ of

glutathione

in

Vol.

89,

No.

synthetase This -

2, 1979

deficiency

approach

inhibition

merit

AND

cellular a model

a "normal"

determined may

(low

represents of

geneticallv concept

BIOCHEMICAL

glutathione for

enzyme deficiency

consideration

BIOPHYSICAL

with

therapy

to

compensate

in

activity regard

and of

to

overproduction).

errors the

another certain

COMMUNICATIONS

oxoproline

inborn for

of

RESEARCH

of

metabolism

conscqucnccs enzyme. other

of

3

This

heritable

disorders. ACKNOWLEDGEMENTS This work was carried out in part in the Burroughs-Welcome Developmental Pharmacology Laboratory at The Johns Hopkins Hospital and was supported in part by N.I.H. general research support grant No. RR-5378 (SPS). We wish to thank Barbara Foley and Louise M. Schulden for expert Technical Assistance and Mrs. Penny Colbert and Ms. Dana Jarvis for typing the manuscript.

REFERENCES

1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16.

Idellner, V.P., Sekura, R., Meister, A., and Larsson, A., Proc. Natl. Acad. Sci. U.S.A., 71, 2505-2509 (1974). Spielberg, S.P., Kramer, L. I., Goodman, S.I., Butler, J., Tietze, F., Quinn, P., and Schulman, J.D., .J. Pediat., 91, 237-241 (1977). Spielberg, S.P., Boxer, L.A., Oliver, J.M., Allen, J.M., and Schulman, J.D., Brit. J. Haematol., in press. Griffith, O.W., Larsson, A., and Meister, A., Biochem. Biophys. Res. Comm., 919-925 (1977). 79, Richmond, P., and Meister, A., .J. Biol. Chem., 250, 1422-1426 (1975). Meister, A., Science, 180, 33-39 (1973). Revel, J.P., and Ball, E.C., J. Biol. Chem., 234, 577-582 (1959). Tate, S.S., and Meister, A., Proc. Natl. Acad. Sci. U.S.A., 2, 4806-4809 (1978) Tietze, F., Anal. Biochem.. 2, 502-522 (1969). Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J., J. Biol. Chem., 193, 265-275 (1951). Orlowski, ?l., and Meister, A., Biochim. Biophys. Acta, 2, 679-681 (1963). Schneider, J.A., Rosenbloom, F.M., Bradley, K.H. and Seegmiller, .J.E., Riochem. Biophys. Res. Comm., g, 527-53 (1967). Spielberg, S.P., Boxer, L.A., Corash, L.M., and Schulman, -J.D., Ann. Intern. Med., 90, 53-54 (1979). Boxer, L. A., Oliver, 3. M., Spielberg, S.P., Allen, J.M., and Schulman, 52, supp. 1, 145 (1973). J.D., Blood, C,riffith, O.W., Bridges, R.J., and hfeister, A., Proc. Natl. Acad. Sci. U.S.A., 5, 5405-5408 (1978). Valdes-Dapena, M.A., and Arey, J.B., 3. Pediat., &, 531-546 (1962).