BIOCHEMICAL

Vol. 69, No. 2, 1976

INTERCELLULAR ENZYME

ASSAYS

IN

Arnold Madeleine

Received

EXCHANGE SINGLE

Reuser, van

Dept.

AND BIOPHYSICAL

OF

HUMAN

LYSOSOMAL

ENZYMES:

FIBROBLASTS

Dicky Halley, Elly der Kamp, Maarten

of Cell Biology Erasmus University

December 29,

RESEARCH COMMUNICATIONS

AFTER

de Wit, Mulder

and Genetics, Rotterdam,

CO-CULTIVATION

Andre Hoogevcen, and Hans Galjaard

The

Medical Faculty, Netherlands

1975 SUMMARY

Intercellular exchange of N-acetyl-P-D-glucosaminidase (EC 3.2.1.30) P -galactosidase (EC 3.2.1.23) and acid a-glucosidase (EC 3.2.1.20) was studied after cocultivation of normal and enzyme deficient human fibroblasts in confluent cultures. Enzyme activities were measured in single cells using microchemical procedures. After co-cultivation of normal control fibroblasts and those from a patient with Sandhoff’s disease an increase of activity of N-acetylp -D-glucosaminidase was found in Sandhoff cells, together with a decrease of activity in normal control cells. After co-cultivation of normal fibroblasts and those from patients with glyno indication was found for intercellular cogenosis I I and GMl-gangliosidosis, transfer of acid a-glucosidase and p -galactosidase respectively. The significance of the results is discussed in respect of the hypothesis of Hickman and Neufeld about secretion and uptake of lysosomal enzymes. INTRODUCTlON Cultures study

of

of

human

lysosomal

stration

of

studies

(l-7).

therapy

in

A more

fundamental

raised

in

fibroblasts storage

enzyme

lysosomal

been

to

diseases.

interest

in

uptake

acid

hydrolases

increased

levels

cells

from

patients,

together

(8, and

9).

these

Hickman

and

subsequently

are

that

Neufeld taken

Copyright 0 I976 by Academic Press, Inc. All rights of reproduction in any form reserved.

in enzyme

and

a useful the

model

metabolic

culture

was

secretion

of

lysosomal

patients

with

I-cell

of

activity

were

found

in

via

decreased that

specific

lysosomal recognition

311

levels

the

of

in

in

the

admini-

several

be

a feasible

enzymes

was

disease.

For

several

medium

of

cultured

intracellular

enzymes sites

for by

reported might

from

with

system defect

replacement

cells

suggested up

be of

fibroblasts

suggested

cultured

to

Correction

storage

with

proven

diseases.

preparations

It has

studies

have

activities

have

to

be

order

to

reach

secreted their

Vol.

69, No.

2, 1976

lysosomal To

general of

deficient

whereas of

(further

donor to

studied

acceptor tion

the

enzyme

avoided tact

l-cell

exchange

and

was

destination.

investigate

cellular

BIOCHEMICAL

with

be

cells. normal

of

acid

hydrolases

In this for cells.

this

activities

RESEARCH

be

of

the

result

in

we

mixed

confluent

administration

of

transfer

of

were

exchange

of

with measured

in

single

the

inter-

cultures

of

normal

preparations

optimal

by

close

was con-

p -D-glucosaminidase and

deficiencies

uptake.

studied

enzyme

N-acetyl-

/j-galactosidase

patients were

enzyme

COMMUNICATIONS

a defective

have

way

The

from

BIGPHYSICAL

hypothesis

as hexosaminidase),

fibroblasts Enzyme

would

validity

conditions

acceptor

referred

using

disease

cells.

the and

three

AND

for cells

acid

a-glucosidase

these

enzymes

after

co-cultiva-

as

cells. METHODS

Cell culture and isolation: Cells were cultured in Ham’s FlO medium supplemented with 15% fetal calf serum and antibiotics. In addition to fibroblasts from control subjects fibroblasts were cultured from patients with Sandhoff’s disease (deficient in both lysosomal forms of hexosaminidase), GMI-gangliosidosis type 1 (deficient in lysosomal P-galactosidase) and glycogenosis II (deficient in lysosomal &-g/ucosidase). Preceeding co-cultivation cells from different strains were labeled with either latex or carbon particles. Suspensions of latex particles were obtained from Servo (Dow latex, 0.79 p) and were added at a concentration of 0.02 ml to 2 x 106 cells in 10 ml culture medium. Suspensions of carbon particles were obtained from George T Gurr (Indian Ink, cat no 51400) and were added at a concentration of 0.1 ml. The medium was removed after one day culturing, the cells were washed three times with 0.9% NaCl and trypsinized. Equal numbers of normal and enzyme deficient cells were mixed and confluent cultures were initiated. Growth in confluency was allowed for one to ten days. The cultures were trypsinized after this period of co-cultivation and reseeded in low density in petri dishes with a thin plastic foil (mylar) bottom, to enable single cell analysis. After cultivation for 20 hrs. differently labeled cells were identified with phase contrast microscopy and localized using a micro grid. Cultures were then freeze dried, whereupon preselected cells could be isolated by microdissection as described by Galjaard et al. (11). Enz me ass s: 4-methylumbelliferyl-2-acetamido-2-deoxy-P -D-glucopyranoside, --4-methylum ellrferyl-fi -D-galactopyranoside and 4-methylumbelliferyla-Dglucopyranoside were used as substrates for the determination of hexosaminidase, P -galactosidase and cr-glucosidase activity respectively. Detailed conditions for the assay of p-galactosidase are described by Galiaard et al (12). A method for the single cell analysis of cr-glucosidase is described by Reuser et al (13). To assay hexosaminidase activity single fibroblasts were incubated in 0.1 pl phosphate (20 mM)-citrate (10 mM) buffer heat inactivated, bovine serum one hour at 370C and stopped

pH 4.5 containing 5 mM substrate and 0.02X, albumin. The reaction was allowed to proceed for by the addition of 1 pl, 0.5 M carbonate buffer

312

Vol. 69, No. 2, 1976

pH 10.7. tosidase. ferone

BIOCHEMICAL

Fluorescence was Enzyme activities (MU).

Empty

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

measured in glass capilleries as described were calculated using a standard curve

pieces

of

plastic

foil

were

dissected

to

for p-galacmethylumbelli-

of

serve

as blanks.

RESULTS Normal latex

control

or

carbon

influence

for

for

ten

After

various

the

same

days

and

assayed 1A

from

control

was

present in

ten

moles 1.7

in

normal

Fig.

1B. MU

x

cells the

lo-l2

per

hr

distributions

GM normal of

mean

human

were

were

of lacking

confluent

cultured

separa-

co-cultivated

density

in

localized,

petri

dishes.

freeze

Hexosaminidase

not

numbers disease

were

low

did

in

cells

the

dried

activity

was

in

fibroblasts

Low

residual

activity

moles

MU

0.06 was

The

same

x

4.6

10 x

noticed

cells

was

of

-12

moles

in

the

increased

control

effect

-12

10

were

activity

activity

separately.

activity

Sandhoff

mean

hr.

hexosaminidase

changes

was

found

MU

was after

hr)

per

where-

hr.

After

distributions to

cells

per

0.7

x

as shown 10

-12

decreased three

days

to of

co-

prominent.

exchange

1 -gang1

the

cells

cultured

activity

of

per

lines

in

Methods.

equal

Sandhoff’s

cell

either

material

co-cultivated

reseeded the

of

when (mean

activity

less

enzyme

in

marked

MU

experiments

with

patient

the

both

hours

distribution

whereas

moles

20

and

with

types.

cells

mean

In similar

vated

both

co-cultivation

although

with

and

fibroblasts

cultivation

patient

for

frequency

mixed

then

marker

with

described,

and

labeled

experiments

a patient

experiment

culture

of

Sandhoff

The

from

a control

as described

subject

of

the

cultivation

single

shows

days

In

selectively

in

Fig.

in

period.

As

were that

several

were

periods.

as a confluent

isolated

In

hexosaminidase

time

a subsequent

and

cells showed

measured.

subject

of

deficient

experiments

activities

forms

for

enzyme

Control

a control

lysosomal

tely

as

enzyme

from

cultures

and

particles.

the

fibroblasts both

fibroblasts

of

iosidosis,

lacking

fibroblasts activity

/3-galactosidase

in for

both

was

lysosomal equal types

313

studied.

Fibroblasts

p-galactosidase,

were

co-culti-

The

frequency

numbers of

cells

for

eight

when

days. cultured

from

separately

a

are

Vol.

69, No.

BIOCHEMICAL

2, 1976

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Frequency I

Frequency

& 64-

0 0

Fig.

5

t t 1.5 25

I 15

I 45

t I 5.5 65 7.5 N-ocetyl-O-D-

1. Frequency distributions of hexosaminidase derived from a control subject and from A. Separate cultivation B. Co-cultivation for 10 days in confluency control patient

Frequency

activity a patient

A

14-

14-

12-

12-

lo-

IO-

8-

a-

6-

6-

4-

4-

2-

2r

I

I

I

I

I I

OJ

00123456 O-goloctosfdase

Fig.

fibroblasts disease.

Frequency I 46

r-’

o-

in single human with Sandhoff’s

activity

2.

(~10~‘~

Frequency distributions of P -galactosidase blasts derived from a control subject and sidosis. A, Separate cultivation B. Co-cultivation for 8 days in confluency control patient

314

00123456 moles MU/hr)

activity from

in

a patient

single with

human

fibro-

GM1

ganglio-

Vol.

69, No.

given

in

case

/3-galactosidase

The

mean

per

hr.

2, 1976

Fig.

2A.

activity

from

used

as acceptor

done

in

frequency

normal

cells

and

enzyme

II.

way

as

described

The

cultured

FWC

was

of

no

be

mean

activity

this

demonstrated

3.9

x

and

1.38

acid

enzyme. were

Fig. in

normal

10

fibrobiasts

we my

from x

lo-l3 of

of

normal

and

38

4.7

the

patient.

moles

MU

P -ga-

were

experiments

were

for and

seven

deficient

days. cells

respectively.

from

was

In neither

a-glucosidase The

fibroblasts

after

28.

co-cultivated

3A

cells

COMMUNICATIONS

Fig.

exchange

activity in

in

in

lacking

Cells

shown

-14

-13

shown

intercellular

of

of

10

II

above.

are

could

and

x

cr-glucosidase

cultures

alone

that

RESEARCH

are

demonstrated 1.36

exchange

study

activity

be

glycogenosis

to

BIOPHYSICAL

co-cultivation

with

cells

mixed

AND

indicates

distributions

separate

when

could

a patient

a similar

glycogenosis hr.

activity

This

Fibroblasts

No

after

respectively. occurred.

in

Distributions

of

lactosidase

The

BIOCHEMICAL

x

the 10

patient

-14

moles

with MU

per

co-cultivation.

Frequency

45

46 ]r :E

j

B

ILIZ-

12-

IO8-

B-

6-

6-

4-

4-

2O0

Fig.

3.

Frequency broblasts, sis II. A. Separate B. Co-cultivation -

0

2

4 acid

6 8 IO I2 a-glucos~dase

distributions derived from cultivation for patient

14 oclwity

of acid a control

7 days

in -

0 I, (xlOe”moles

cu-glucosidase subject and

confluency control

315

U’LI-

2 MUlhr)

4

6

8

IO

12

14

activity in single from a patient with

human figlycogeno-

Vol.

69, No.

No

transfer

2, 1976

of

BIOCHEMICAL

acid

AND

ct-glucosidase

BIOPHYSICAL

activity

from

RESEARCH

normal

to

COMMUNICATIONS

deficient

cells

was

found.

DISCUSSION Selective rial.

The

OS on and of

and former

the Neufeld

onother.

from

clinically

the

result

man

and

that

the

Neufeld theory for

We

studied

by

co-cultivation

res

thereby in

clear

cells

fits

in

by

for

the

loss

No

transfer

of

with

to

approach far

a loss of of

of

Hickman

in

in in

This

compored and

normal

or

to

recognition

sites

in on

evidence

has

recognition

sites

the

316

and

be

Hick-

Strong

been

(is).

a-glucosidase

in

confluent

the

of

cultu-

administration

to enzyme the

of

deficient increase

co-cultivation,

compensation

is accomseems

these

could enzymes

In this

l-cell

of indicate

cells

activity for

to

to occur

cells.

hexosaminidase.

hexosaminidose

hypothesis

Interestingly

No

a-glucosidase that

fibroblasts

authors

normal

fibroblasts.

indicate

one

activity.

as o result

normal

from

The

cells

hypothesis.

from

packaging

demonstrated

the

as well

Hickman

for

was

avoiding

from

cells,

of

cultured

and

deficient

intracellular

mate-

surface

enzyme

in

3).

specific

situation,

Neufeld

activity

might

vivo

activity

Sandhoff

2,

I-cells

enzyme

the

and

of

P -galactosidase

and

cell

is’ essential

enzymes.

hexosaminidase,

hexosaminidase

P-galactosidase

affecting

via

the

intracellular

is different

hexosaminidase

control

activity

with

lysosomal

exceeding

activity

studies

the

transfer

(1,

extracellular

hypothesis

defect

enzymes

all

of

normal

the

P-galactosidase

a mutation

of

the

mucopolysaccharidosis

on to

of

of on

uptake

metabolic

lysosomal

apply

of

of

experiments.

lysosomes

not

exchange

hexosaminidase

our

bosed

uptake

transfer

panied

in

is mainly

to

possibility

with

of

According

the

uptake sites

selective

the

potients

quantities

(14).

of

exchange

trying

enzymes The

the

implies

for

recognition

by

correction

might

presented have

This

different free

up

followed

Mutual

of

distinguished

specific

is taken

secretion

enzymes.

to

are

requires

thot

(10)

lysosomol

pinocytosis

process

molecule

cell

in

bulk

disease

enzyme

be the

packaging

the

simultaneous

case can

molecules.

demonstrated

not

be

explained

into lock by

Vol.

69, No.

2, 1976

A different

BIOCHEMICAL

explanation

GMl-gangliotidosis sites

on

the

cell

respectively. patient

No with

defect

in

the

results

glycogenosis

surface,

preventing

cross-reacting

to uptake

in

II are uptake

the

(16)

be

that

caused of

may

cells

mutation

BIGPHYSICAL

could

which

liver

a structural of

might

material II

material points

our

and

glycogenosis

cross-reacting however,

for

AND

RESEARCH

the by

enzyme

a defect

of

a patient

in

the

enzyme

this

the

and

demonstrated

support

deficiencies in

p-galactosidase be

COMMUNICATIONS

in

recognition cr-glucosidase

liver

hypothesis.

with

in

cells

Detection

of of

GMl-gangliosidosis

molecule

rather

a

(17) than

to

a

P-galactosidase.

ACKNOWLEDGMENT This study was supported by the Research (FUNGO) with financial Scientific Research (Z.W.O.).

Netherlands aid from

Foundation for the Netherlands

Fundamental Organization

Medical for Pure

REFERENCES 1. Kresse, H., and Neufeld, E.F. (1972) J. Biol. Chem, 247, 2 164-2170. 2. Figura, K. von, and Kresse, H. (1972) Biochem. Biophys. Res. Comm, 48, 262-269. 3. Bach, G., Friedman, R., Weissmann, B., and Neufeld, E.F. (1972) Proc. Nat. Acad. Sci, 69, 2048-2051. 4. Porter, M.T., Fluharty, A.L., and Kihara, H. (1971) Science, 172, 1263-1264. 5. Dawson, G., Matalon, R., and Li, Y. (1973) Pediat. Res, 7, 684-690. 6. Cantz, M., and Kresse, H. (1974) Eur. J. Biochem, 47, 581-590. 7. Enzyme Therapy in Lysosomal Storage Diseases, Edited by Tager, T.M., Hooghwinkel, G. J.M., Daems, W.Th. North-HOI land Publishing Company, Amsterdam, 1974. 8. Lightbody, J., Wiesmann, U., Hadorn, B., and Herschkowitz, N. (1971) Lancet, i, 451. 9. Wiesmann, U., Vastella, F., and Herschkowitz, N. (1971) N. Eng. J. Med, 285, 1090-1091. 10. Hickman, S., and Neufeld, E.F. (1972) Biochem. Biophys. Res. Comm, 49, 992-999. 11. Gal jaard, H., Hoogstraten, J.J. van, Josselin de Jong, J.E. de, and Mulder, M.P. (1974) Histochem. J. 6, 409-429. 12. Galjaard, H., Hoogeveen, A., Keyzer, W., Wit-Verbeek, E. de, and Vlek-Noot, C. (1974) Histochem. J. 6, 491-509.

317

Vol.

69, No.

13.

Reuser, A.J.J., Jongkind, them, in press. Jacques, B.J.: Lysosomes Dingle, J.T., and Fells,

14.

15. 16. 17.

2, 1976

BIOCHEMICAL

J.F., in H.,

AND

and

BIOPHYSICAL

Galjaard,

Biology and pp 395-520,

Amsterdam. Hickman, S., Shapiro, L.J., and Neufeld, Comm, 57, 55-61. Barsy, T. de, Jacquemin, P., Devos, P., 31, 156-165. Meisler, M., and Rattarzi, M.C. (1974)

318

H.

RESEARCH

(1976)

Pathology, North-Holland E.F.

vol

(1974)

and

Hers,

Am.

J.

J.

Histochem.

11, (1969) Publishing Biochem.

H.G. Hum.

COMMUNICATIONS

Edited by Company, Biophys.

(1972) Gen,

Cyto-

26,

Eur.

J.

683-691.

Ret. Biochem,