Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ABNORMAL GANGLIOSIDE FIBROBLASTS

Gideon

ACCUMULATION IN CULTURED

FROM PATIENTS

Bach,

Maimon

WITH MUCOLIPIDOSIS

M. Cohen and Gertrude

IV

Kohn

Department of Human Genetics Hadassah-Hebrew University Medical Center P.O.B. 499, Jerusalem, Israel. Received

August

25,1975 SUMMARY

Extracts of cultured skin fibroblasts derived from patients with mucolipidosis IV showed a marked increase and altered distribution of GM3 and GD3 gangliosides. GD3 is elevated 1.5-2 times that of normal whereas GM3 is elevated No abnormalities were found in the neutral glycolipids. to a lesser extent. These two gangliosides apparently comprise most of the accumulated lipid-like material observed on ultrastructural analysis in this disease. Recently, cornea1

opacities,

abnormalities from

five

material

and,

glycoprotein

children

mental

revealed

to a lesser

extent,

of these

a mucolipidosis

(4).

accumulated

substances,

Abbreviations: N-acetyl Galactose

and motor

with

However,

assigned nor

the basic

granular

disorder,

(1,3).

Acid

derived

a lipid-like

indicative

of

storage

The simultaneous tissues fibroblasts

suggested (1)

and the name Mucolipidosis Neither defect

the nature

has as yet

GD3, NANA-cr(2->8)-NANA-u(2->3)-gal-8(1->4)-Glc-Ceramide GM3, NANA-a(2->3)-gal-8-(l->4)-Glc-Ceramide Neuraminic glc-Glucose

with

of these

in extraneurological

mucolipidoses

enzymatic

material

of cultured

no skeletal of biopsies

filled

origin

staining

analysis

to this

but

bodies

The lysosomal

enzymatic

congenital

microscopy

storage

of substances

described

retardation

amorphic

by histochemical

two types

bilateral

Electron

numerous

mucopolysaccharide.

any of the already was tentatively

manifesting

(1,2,3).

of tissues

was established

accumulation

NANA gal

progressive

and/or

organelles

excluded

old

have been described

a variety

(ML IV)

two year

been

of the identified.

IV

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMvtUNlCATlONS

Since all the ML IV patients are still limited

to cultured skin fibroblasts,

alive,

studies, by necessity,

and this paper identifies

were

GM3and GD3

gangliosides as the major accumulated substances in these cells. MATERIALSANDMETHODS. Cell Culture.-

Skin fibroblasts

obtained from three ML IV patients

normal controls were propagated in 100 mmplastic in an atmosphere of 95%air,

tissue culture

5% C02, using Ham's F-10 nutrient

and three

flasks

(Falcon),

mediumsupplemented

with 10% Fetal Calf Serum (GIBCOCo., Grand Island, N.Y. ).

Cultures with a

density of 5 x lo6 cells were used for biochemical studies. -Radioisotopes.-

[3H]-6-glucosamine (13.4 ci/mmol),

[3H]-l-galactose

were obtained from Amersham,Buckinghamshire, England. in these studies were of analytical

(13 Ci/mmol),

All other reagents used

grade.

Cultures were incubated with the appropriate

Radioactive labeling.-

48 hours prior to harvest.

isotope for

The labeling by [3H]-glucosamine or [3H]-galactose

was achieved in an F-10 glucose-free medium, to which 0.2 mg glucose/mlwas added with the radioactive Lipid extraction.-

precursor.

Cells were washed twice with 0.9% sodium chloride

protein determined by the Lowry technique (5).

Aliquots

containing

protein were homogenized in 19 volumes of chloroform-methanol 2:l Following centrifugation extracted

with chloroform-methanol 1:2 (v/v).

and partitioned

phase was extracted water (6). distilled

chloroform-methanol ratio

for two minutes.

supernatants were of 2:l with pure

The upper phase was collected

The dialysate (2:l)

was lyophilized,

while the lower

exhaustively

against

dissolved in 0.3 ml of

and then sonicated for two minutes in an ultrasonic

The lower phase of the extraction

under nitrogen,

(v/v>.

three more times with pure upper phase solvents containing

chloroform-methanol bath.

(6).

The resulting

The upper phases were combined and dialyzed water.

1 mg of

(10,000 g for 30 min), the insoluble residue was re-

combined,and adjusted to a final chloroform,

and total

mixture was evaporated to dryness

redissolved in 0.3 ml chloroform-methanol

(2:l)

and sonicated

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1. Accumulation of [3li]-glucosamine labeled compounds in fractionated fibroblasts, following chloroformmethanol extraction.

Fraction

ML IV fibroblasts cpm*

Normal fibroblasts cpm*

Total homogenate (freeze-thawed 3 x>

41000

33000

Upper phase

13000

5100

Lower phase

12000

12500

Insoluble residue **

14000

13000

* cpm represent total radioactivity in each fraction extracted from cells containing 1 mg protein. Conditions of labeling as described in Methods. **

The chloroform-methanol insoluble residue was suspended in 0.9% sodium chloride by vigorous shaking before counting.

Thin layer chromatography (TLC).precoated plates (Analtech).

This procedure was carried out on silica

The lower phase of the extraction

chromatographed in a 60:35:8 chloroform-methanol-water

solution was

mixture (v/v/v)

upper phase of the solution was developed in chloroform-methanol-2.5N (60:40:9).

Total lipids

were investigated

(7).

while the ammonia

by spraying the chromatography plates

with anthrone reagent which is sufficiently of lipid

Gel-G

sensitive

to detect at least lug

Gangliosides alone were detected by resorcinol

spray of TLC

plates (8). RESULTS. Fibroblasts

derived from ML IV patients

accumulation of [3H]-glucosamine-labeled

extraction

of [3H]-glucosamine-labeled solution

chloroform-methanol.

marked

compoundsin the upper phase following

incubation of the cells with the isotope. distribution

showeda consistently

Table 1 shows a representative compoundsin the various phases of the

from three ML IV affected

fibroblast

lines extracted

Similar results were obtained using [3H]-galactose

precursor. 1485

with as

BIOCHEMICAL

Vol. 66, No. 4, 1975

Table

Cell

Lipid-bound

2.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sialic acid in ML IV affected control fibroblasts.

Line

pg sialic acid/ mg protein

ML IV

4.95

ML IV

3.25

average

4.10

Control

2.04

Control

1.79

average

Since consists

1.91

the upper

mainly

determined.

phase

normal acid

More

via

that

than

twice

skin

N-acetyl Thin

layer

sides

in both

lipid

or quantitative

the detection in the lower theoretical Further

acid

was observed

that

phase,

it phase

evidence

2).

gangliosides

contain

N-acetyl

hexosamine

be labeled

a significant studied

abnormal

increase

of both

experiment phase

be

(GM3 and GD3) in (lo),

the

sialic

(7)

extracted

is

ganglioside

most

extremely

Although

of this distribution

no qualitative It

extracts.

should

sensitive GM3 is

to assume that the repeated

eliminated

from

GD3 and GM3 ganglio-

revealed

and ML IV cell

technique

1486

in two

(Fig.1).

solutions

by chromatography.

reasonable solvents

solutions,

control

spray

should

was

by [3H]-glucosamine

phase

in this

of the lower

It

fraction

medium.

of the upper

separated

for

(Table

two major

between

is

cells

of the

the anthrone

of lipids

upper

to control

nonetheless

content

differences out

sialic

in a low glucose

ML IV patients

The total

be pointed

as much lipid-bound

will

indicated

dialysis,

of this

chromatography

ML IV fibroblasts,

following acid

fibroblasts

mannosamine

mixture, sialic

neither

gangliosides

extraction

the lipid-bound

as compared

although

cultured of these

of this

of gangliosides,

of the ML IV cultures remembered

and normal

for

soluble

extraction

also by

ganglioside. in ML IV fibroblasts

BIOCHEMICAL

Vol. 66, No. 4, 1975

Figure

1.

Thin layer chromatography of upper phase (resorcinal spray): 1 - GM1 standard; 2 - GM3 standard; 3 and 4 - ML IV fibroblast extracts; 5 and 6 - control fibroblast extracts. Arrows indicate migration distance of GD3.

was obtained

by monitoring

labeledgangliosides located

and their

accumulation

vapor, radioactivity

control

whereas

fibroblast

layer

and strips,

one centimeter

quantitated.

This

of both

(Fig.2).

A 1.5-2

fold

values

was observed

in the

(approximately

lower

phase

extracts. 1487

[3H]-glucosamineThe gangliosides

wide, procedure

were also

GD3 and GM3 labeled

increase

in GM3 was much less

compounds

in

chromatography.

amounts

the increase

samine-labeled

thin

of radioactivity

of significant

Ml IV fibroblasts over

the pattern

following

by iodine

plate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

removed

were from

the

demonstrated

the

gangliosides

in

of GD3 in ML IV fibroblasts 40% of the total

pronounced. was similar

radioactivity)

The distribution in both

of gluco.

ML IV and normal

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

GD~ 6MJ

2400 -

-

-

1800 1200 s .-+ x Lk

A

5

600O*-,,r c 2400-

e

1800-

E

, \, .

/" \, .H. 1 I I I 1 I 1 1 , 1 h.-. 2 4 6 8 10 12 14 centimeter from origin

0

2.

Thin layer a - Normal

DISCUSSION. European

Within

symptomotology. abnormal indicative

Electron bodies

lipidoses were

very

quantities

minute

numbers

allowed

limited

of cells.

when compared

experiments

performed

gangliosides significantly

normal

control

in MI IV cells, approximately

(the

it

using

that

these

control

organs

the levels in normal

compounds close (This

exhibit

skin

1488

clinical of

individuals, analysis

disorder

nor

the

as one of the

been called

Mucolipidosis

fibroblasts

from which

because

somewhat

more variability or brain.

of GDS, and to a lesser cultured

skin

in such The extent

pattern

GMS,

(10)

when compared

gangliosides,

GD3 contains

only small

fibroblasts)

Furthermore, a different

IV.

of the relatively

such as liver

to 20% of total is because

of Eastern

identical

enzymatic

of this

in ML IV fibroblasts.

GDS comprising 10% in

neither

expect

gangliosides:

the accumulation

has tentatively

gangliosides

elevated

the almost

can be extracted

to those

all

of the affected

classification

one might

indicate

major

cells,

tissues Since

of material Therefore,

children,

with

to the use of cultured

experiments

are

in several

or mucolipidoses,

Our studies

Israeli

has demonstrated

substances.

manifestations

known

five

have presented microscopy

of lipid-like

clinical

two years

origin,

cytoplasmic

,_._ 16 18

chromatography of [3H]-glucosamine labeled control; b - ML IV fibroblast extracts.

the past

(Ashkenazi)

b

4

'z;p

Figure

,

to

of distribution compared

two labeled

sialic

to acids).

BIOCHEMICAL

Vol. 66, No. 4,1975

On the lipid

other

hand,

bands

following

differences

This

bands

in ML IV cells

also

observed

were

noted

than

lamellar

ive

of water

soluble

the accumulated

definitive

information

and both possibility aspect

water

exists currently

both

that under

and brain

this

cells

more,

microscopy

lysosomal

defect

synthetic

pathway

clinical

whereas (13).

manifestations

structure

granular

storage

of these

bodies

was strongly

mucopolysaccharides.

suggest-

Attempts

have not as yet

to yielded

nature.

accumulate

in ML IV affected

the enzyme ganglioside

syndrome

bodies

two gangliosides.

fewer

macromolecules,

fibroblast

storage

amorphic

fibroblasts,

sialidase

may be due to a sialidase

(GM3 sphingolipodystrophy) of the affected

patients.

GM3 ganglioside

due to the deficiency electron

of these

Although

chemical

quantitative

(ll),

the

deficiency,

an

investigation.

GM3 and GD3 accumulate,

disorder

soluble

for

In a new gangliosidosis, in liver

and/or

nor

the lamellar

granular

12 different

ML IV and control

deposits,

their

as to their

as substrates

that

in ML IV cells.

GD3 and GM3 gangliosides serve

no qualitative

partially

lamellar

glycoproteins

identify

suggests

organelles,

at least

when comparing

at least

to the concentric

were

Since

strongly

revealed

However,

observed

consist

organelles

solutions

spray.

were

observation

In addition

phase

anthrone

in these

extracts. observed

TLC of lower

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Unlike

accumulates

of N-acetyl-galactosamine

has indicated

that

two disorders

and therefore

GM3 accumulates

ML IV, mainly

in which in the former

transferase.

ML IV can be categorized

GM3 s p hingolipodystrophy These

(12),

is also

can easily

due to an impaired

present

completely

Furtheras a biodifferent

been distinguished.

ACKNOWLEDGEMENTS. The technical assistance of Miss Marcia Zeigler and Mr. Robert Dworkin is deeply appreciated. This investigation was supported in part by Joint Research Fund of Hadassah Hospital and the Hebrew University. REFERENCES. 1. 2. 3.

Berman, E.R., Livni, N., Shapira, E., Merin, S., and Levij, I.S. (1974) J. Pediatrics 84, 519-526. Russell, A., Yatsiv, S., Livni, N., and Kohn, G. J. Pediatrics (submitted for publication). Merin, S., Livni, N., Berman, E.R., and Yatsiv, S. (1975) Invest. Ophthal. l4, 437-445.

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Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

4.

RESEARCH COMMUNICATIONS

McKusick, V.A. (1972) Heritable disorders of connective tissue. 4th ed. pp. 641-665 Academic Press, NewYork. 5. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, J. (1951) J. Biol. Chem.193, 265-275. 6. Folch, J., Lees, M., and Sloane-Stanley, F. (1957) J. Biol. Chem. 226, 497-509. 7. Trevelyn, W.E., and Harrison, J.S. (1952) Biochem. J. so, 298-303. 81 Svennerholm, L. (1957) Biochem. Biophys. Acta 2, 604-611. 9. Warren, L. (1959) J. Biol. Chem.234, 1971-1975. 10. Dawson, G., Matalon, R., and Dorfman, A. (1972) J. Biol. Chem.247, 5944-5950. 11. Ohman, R., Rosenberg, A., and Svennerholm, L. (1970) Biochemistry 2, 3774-3782. 12. Max, S.R., Maclaren, N.K., Brady, R.O., Bradley, R.M., Rennels, M.B., Tanaka, J., Garcia, J.H., and Cornblath, M. (1974) New Eng. J. Med. 291, 929-931. 13. Fishman, P.H., Max, S.R., Tallman, J.F., Brady, R.O., Maclaren, N.K., and Cornblath, M. (1975) Science 187, 68-70.

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