Glycosylation of thyroglobulin and response to thyrotropin of human cultured thyroid cells

Vol.

104,

No.

February

4, 1982

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

26, 1982

Pages

1573-1578

GLYCOSYLATION OF THYROGLOBULIN AND RESPONSE TO THYROTROPIN OF HUMAN CULTURED THYROID CELLS.

A. Tanini,

R. Toccafondi,

Metabolic

Unit, Viale

Received

December

S. Aterini,

Clinica

Medica

Morgagni

17,

C.M. Rotella

85,

III,

and R. Zonefrati

University

Firenze,

I-50134

of Florence Italy

1981

SUMMARY: As native thyroglobulin reversibly inhibits TSH-induced cyclic AMP accumulation in cultured thyroid cells, we studied whether the glycosylation of this iodoprotein plays a role in this inhibition. Preincubation with human asialo thyroglobulin and asialo-agalacto thyroglobulin inhibited TSHstimulated CAMP accumulation after 60 min reaching the maximum after a 120 min. A dose-dependent reduction of CAMP production was found after a 120 min preincubation with graded amounts of asialo and asialo-agalacto thyroglobulin at doses respectively 10 and 200 times lower than native thyroglobulin. Such an inhibitory effect did not appear completely reversible: in fact 70 Ii!il! after their removal from incubation medium a response to TSH was found to be lower than control.

INTRODUCTION In a previous (Tg)

inhibits

thyroid Tg is ing

study

we have demonstrated

thyrotropin-induced

cells

(1).

Moreover

much more active

to bovine

plasma

determine

jrhether

interfere

with

to characterize

MATERIALS

than

this

Consiglio native

membrane.

the the

cyclic

effect

inhibition,

human native

AMP accumulation et al.

(2)

Tg in inhibiting

In this

preincubation

acute

that

respect

with

have also

asialo

shown

that

asialo

(TSH)

bind-

experiments

to

and asialo-agalacto

of TSH in human cultured if

in human cultured

thyrotropin we conducted

thyroglobulin

thyroid

Tg can cells,

and

present.

AND METHODS

The bovine TSH (bTSH, Batch no 53/11) was generously supplied by the National Institute for Biological Standards and Control, Holly Hill, London. Vibrio Cholerae neuraminidase and Escherichia Coli B-galactosidase were obtained from Behringwerke AG, Marburg, W. Germany. One Unit of V.Cholerae neuraminidase is defined as the ability to release 1 Fimol of N-acetyl-neu-

1573

0006-291X/82/041573-06$01.00/0 Cop.vrrghl 0 1982 by Academic Press, Inc. ,4 II rrghrs of reproducrion in an.v form reAen,ed.

Vol. 104, No. 4, 1982

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AND BIOPHYSICAL

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raminic acid from humanal-acid glycoprotein in one minute at 37°C. One Unit of E.Coli B-galactosidase is equivalent to 1 umol lactose hydrolyzed per minute at 25°C. Cyclic 3',5'-adenosine monophosphate (CAMP) and 3-isobutyl1-methylxanthine (IMX) were purchased from Sigma Chem. Co, St. Louis, MO.; 2,8-3H adenosine 3',5'-cyclic phosphate ammonium salt from the Radiochemical Centre, Amersham, England; Sephacryl S-300 from Pharmacia, Uppsala, Sweden; all other reagents of analytical grade from Merck, Darmstadt, W. Germany. Enzyme dispersed thyroid cells, obtained from normal thyroid tissue of patients undergoing neck surgery for non-thyroidal pathology, were cultured as previously described (3). The effect of bovine TSH was studied by incubating cultured cells in Krebs Ringer Bicarbonate (KRB) buffer, pH 7.4, containing 0.6 mM IMX, 10 mM glucose and 2.0 g/l human serum albumin (HSA). CAMP was measured in the freeze-dried 2,000 g supernatant of the ethanoltreated homogenate by radioreceptor assay (4). The DNA content was determined in the pellet by fluorometry (5). Asialo Tg was prepared by incubating native Tg (10 mg) purified and characterized according to the procedure of Salvatore et al. (6), with 300 mU of V.Cholerae neuraminidase for 2 hr at 37°C in 0.01 M Na acetate, pH 5.0, containing bovine serum albumin (2 g/l), being the final reaction volume 0.7 ml. Asialo-agalacto Tg was prepared by incubating asialo Tg (10 mg) with E.Coli B-galactosidase in 40 mM potassium phosphate, pH 7.2, containing 10 mM MgS04, The reaction was carried out at 37°C for 48 hr by adding 20 U of enzyme at the beginning of the reaction and another 20 U at 24 hr; 20 ~1 of toluene were added as preservative. The final volume was 2.8 ml. Both reactions were stopped by dilution, material was purified on Sephacryl S-300 and the fractions corresponding to 650,000 m.w. were collected. Thyroglobulin

concentration

'% -10.0). by A,gO(~,cm-

was measured

RESULTS Basal pmol/ug

CAMP accumulation DNA (mean+SD:

120 min incubation agalacto

Tg,

0.8620.23)

in

from

in human thyroid after

presence

0.01

in the

of 0.6 mM IMX.

same experimental

pmol/pg

DNA, respectively).

dium a CAMP increase Preincubation

from

CAMP accumulation

for

60 min,

asialo-agalacto

creased

after

120 min with

PM) produced

reached

graded

When cultured

amounts

with

a time

value)

of human asialo 1.574

during

was added

1287+217) human asialo

dependent

Tg (1.5

inhibitory

after

120 min and slightly

Tg (0.05

were

UN) and

effect

1. The inhibition asialo

to the me-

was observed.

(53% for

cells

a

CAMP accum-

value

thyroid

to 1.20

0.75+0.08-0.88t1.12

mu/ml)

as shown in Fig.

the maximal

Tg of control 4 hours.

TSH (2.5

0.66

Tg and asialomodify

(mean?SD:

30 m-in to 4 hours

bTSH stimulated after

Human asialo

1108 to 1532% (near&D:

Tg (0.5

from culture

significantly

conditions

asialo-agalacto

peared

not

When bovine

from

ranged

7 days of primary

to 4.0 uM, did

ulation

cells

on ao-

Tg and 24%

preincubated

defor

to 3.0 PM) a dose-de-

Vol. 104, No. 4, 1982

0

BIOCHEMICAL

AND BIOPHYSICAL

b6H ImUlml)

0

BASELINE

RESEARCH COMMUNICATIONS

BASELINE

blSH(mU/ml)

I.5 $4 HUHAN ASIALO Tg

12 8 4.4 8 z 2 0 s "r 12 ?I

0.5 NH HUHAN ASIALO AGALACTOlg

8

0 0

0

001

1

005 0.l MOLARITY(PM)

05

0

02

30 60 120 240 DURATIONOF PREINCUBATION lmin)

Figure 1. Effect of 120 min preincubation with human asialo Tg and asialo-agalacto Tg on CAMP accumulation induced by TSH in human cultured thyroid cells. About 1.5~10~ thyroid cells were preincubated in triplicate at 37°C in McCoy's 5a medium supplemented with 20% FCS, in presence of asialo Tg at 0.05-3.0 PM or asialo-agalacto Tg at 2.5 nM-0.5 uM. At the end of preincubation cells were stimulated at 37'C in KRB buffer, pH 7.4, containing glucose (2.0 g/l), HSA (2.0 g/l) and IMX (0.6 mM) with 2.5 mu/ml of bTSH. Results are expressed as pmol cAMP/Pg DNA; vertical bars represent deviation of mean. Figure 2. Effect of different preincubation times with human asialo Tg -~ (1.5 uM) and asialo-agalacto Tg (0.5 vM) on CAMP accumulation induced by bTSH in human cultured thyroid cells. About 1.5~10~ thyroid cells were preincubated in triplicate varying from 30-240 min at 37°C in McCoy's 5a medium supplemented with 20% FCS, in presence of asialo Tg or asialo-agalacto Tg. At the end of preincubation cells were stimulated with bTSH as in Figure 1.

pendent of

0.1

reduction UM asialo

amounts

order

permanent

to

study

reduction

120

2).

min

with

TSH

native

Tg

subsequently

ulated

with

bTSH

asialo

Tg and asialo-agalacto cells

to

at

bTSH

washed

three

not

Tg. return

effects thyroid

asialo

times

Tg

times (15

min

with

from

a dose

different

a dose-dependent

from a dose of 5.0 nM (Fig.

responsiveness, pM),

120

IIM) produced

inhibitory

(1.5

was found

for

r-84 to 0.05

these

different

did

preincubation

CAMP accumulation whether

of

The

CAMP accumulation

Tg (2.5

nM),

roid

(15

(Fig.

of bTSH induced

In

Tg

Tg

of asialo-agalacto

decrease

for

of bTSH stimulated

to

As

shown

to

normal

cells (1.5

with 120 in

could

uM)

fresh min)

Table 90 min

be

were or

I, after

the the

to

a

preincubated

asialo-agalacto

medium after

due

2).

and

then

removal

response removal

of of

of

stim-

thy-

asialo

vol.

104,

No.

BIOCHEMICAL

4, 1982

AND BIOPHYSICAL

TABLE Effect of bTSH on CAMP accumulation with native, asialo and asialo-agalacto

Time

in

RESEARCH COMMUNICATIONS

I

human cultured thyroid cells Tg, 90 min after Tg removal

(min) after Tg removal

CAMP (pmol/pg

Control

preincubated from medium.

Percent maximal

ONA)

of TSH response

0.62+0.05

bTSH

10.13t1.15

100

Native

Tg

0 90

5.12kO.45 10.73-tO.98

50 106

Asialo

Tg

0 90

4.3620.32 6.58tO.37

43 65

0 90

5.17to.14 7.8OtO.54

51 77

Asialo-agalacto

Tg

About 1.5~10~ cells were preincubated in triplicate for 120 min at 37°C in KRB buffer with native Tg (1.5 uM), asialo Tg (1.5 PM) or asialo-agalacto Tg (15 nM) Thyroglobulins were then removed by washing and thyroid cells were stimulated at 37°C in KRB buffer, pH 7.4, containing glucose (2.0 g/l), HSA (2.0 g/l) and X%X (0.6 mM) with 2.5 mu/ml of bovine TSH. Results are expressed as pmol cAMP/ug DNA (mean?SD).

and asialo-agalacto

Tg from

values,

respectively).

removal

a response

the

On the similar

incubation

medium

contrary,

at the

to control

(65 and 77% of control

same time

after

native

Tg

was found.

DISCUSSION Present

data

action

between

native

thyroglobulin,

fective

at high

extend

Tg

induced

for

doses

(1).

lower native alo

of

(0.1

of

at

cultured

PM and 5.0

Tg (i.e.

least

Tg

thyroid

cells.

1 hr

with

course

asialo-agalacto cells

at

doses

contrary

Tg appears

the not

1576

Tg are

inef-

the

effect

to that

of native

are

also

cells.

Tg and asialo-aga-

able

respectively

than

for

inhibitory

Tg

inter-

As observed

reduces

of

the

of thyroid

asialo

significantly

to be similar

On the

concerning

CAMP accumulation

nM, respectively)

1.0 PM).

Tg and asialo-agalacto

and

thyroid

cells

time

Tg appears asialo

the

thyroid

The

(1)

Tg and asialo-agalacto

in modifying

accumulation.

However,

response

asialo

of cultured

and asialo-agalacto

observations

and cultured

both

a period

CAMP

previous

thyroglobulin

The preincubation lacto

our

the minimal inhibitory

to be completely

to

thyrotropin of

thyroglobulin

inhibit 10 and

found effect

Tg

asialo

the 200

TSH

times

by us for of both

reversible,

asicon-

Vol. 104, No. 4, 1982 trary

to that

of cultured

BIOCHEMICAL

observed thyroid

lower

than

findings

modifications

molecules

cles

but remain (2)

plasma

bound

where

the

removal

after

response

of asialo

90 min it

when compared

stable

binding

residues

the

to native

Tg

is

still

Tg by rat

In conclusion

asialo

the

effect

cule

not

allowing

respect

Tg and its Besides

our

to carbohydrate

is

prolonged

results

who have described

to that

of adenylate

glycosylated

in the

for

bound

Tg.

to be more potent

cyclase-cA!iP

6 chain

with

of asialo-

of native

cultured

synthesized

the more

consistent

thyroglobulin.

to TSH of mammalian

compo-

cyclase-CAMP

uptake

Tg appear

native

by the newly

are

a higher

when compared

than

therefore

on adenylate

effect

galac-

to the Tg mol-

with

the TSH action

of TSH

Subsequently

chain

Tg and asialo-agalacto

be mediated

with

iodination. the

the folli-

from that

molecule,

of TSH action

completely

and interfering

undergo

different

of

thyroglo-

into

It is conceivable

TSH stimulation

could

secreted

in a locus

process

synthesized

(7).

hemilobes

refractoriness

following

this

thyroid

inhibitors

may explain

(8)

not

of carbohydrate in the

the newly are

of Consiglio

follicles

membranes.

Van den Hove and Couvreur

role

molecule

B chain

in inhibiting

to plasma

the

In fact

to the membranes

thyroglobulin

system

the observation

concerning

membranes.

into

is more active

and stable

extent

of thyroglobulin

tyrosine

nents,

(9-10)

even if

the

medium,

acid are added to complete

incomplete

agalacto

(7)

an incomplete

to be released

that

to a large

B chain

with

and sialic

ecule

In fact,

after

incubation

preparation

on the

bulin

tose

the

confirm

to thyroid

receptor

Tg.

to bTSH improves

Tg from

in acellular

binding

native

RESEARCH COMMUNICATIONS

controls.

These et al.

by us with

cells

and asialo-agalacto

AND BIOPHYSICAL

These

findings

thyroid

cells

system.

In fact

thyroglobulin to plasma

nolemembrane

TSH action.

ACKNOWLEDGMENT We are indebted to Drs. Gaetano Salvatore and Eduardo Consiglio, Naples, Italy, for their helpful discussions and suggestions in preparing thyroglobulins. The study was financially supported by the National Research Council (Progetto Finalizzato "Controllo della Crescita Neoplastica", CT 80.01467.

96).

REFERENCES 1.

Aterini, S., Rotella, R. (1980) FEBS Letters

C.M., 121:

Tanini, 44-46.

A.,

1577

Zonefrati,

R.

and Toccafondi,

Vol. 104, No. 4, 1982 2. 3. 4. 5. 6. 7. 8. 9. 10.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Consiglio, E., Salvatore, G., Rall, J.E. and Kohn, L.O. (1979) J. Biol. Chem. 254: 5065-5076. Toccafondi R., Aterini, S., Medici, M.A., Rotella, C.M., Tanini, A. and Zonefrati, R. (1980) Clin. exp. Immunol. 40: 532-539. Brown, B.L., Albano, J.D.M., Ekins, R.P., Sgherzi, A.M. and Tampion, W. J. 121: 561-562. (1971) Biochem. Kissane, J.M. and Robins, E. (1958) J. Biol. Chem. 233: 184-188. Salvatore, G., Salvatore, M., Cahnmann, H.J. and Robbins, J. (1964) J. Biol. Chem. 239: 3267-3274. Consiglio, E., Shifrin, S., Yavin, Z., Ambesi-Impiombato, F.S., Rall, J. E Salvatore, G. and Kohn, L.D. (1981) J. Biol. Chem. 256: 10592-10599. Y& den Hove, M.F. and Couvreur, M. (1981) Ann. Endocrinol. 42: 23A. Rapoport, B. and Adams, R.J. (1976) J. Biol. Chem. 251: 6653-6661. Takasu, M., Charrier, B., Mauchamp, J. and Lissitzky, S. (1978) Eur. J. Biochem. 90: 131-138.

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