Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1219-1225

September 30, 1987

THE MEMBRANE-ANCHOR OF PARAMECIUM TEMPERATURE-SPECIFIC SURFACE ANTIGENS IS A GLYCOSYLINOSITOL PHOSPHOLIPID

Yvonne

Capdeville,

M. L u c i a

and C n r i s t i a n e

Centre Centre

Cardoso

de Gdn@tique

National 91190

Mol~culaire,

de la Recherche Gif/Yvette,

+Molteno

de A l m e i d a , +

Deregnaucourt

Scientifique,

France

Institute,

Cambridge,

England

Received August 19, 1987

SUMMARY: The temperature-specific G surface antigen of P a r a m e c i u m p r i m a u r e l i a s t r a i n 156 was b i o s y n t h e t i c a l l y l a b e l e d Oy[~H3myrist-~c ~ ~n its membrane-bound form, Out not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phosphotipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic i m m u n o d e t e r m i n a n t catted the crossreacting determinant. The P a r a m e c i u m enzyme, capable of converting its membranebound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal t i p a s e . From t h i s evidence we propose t h a t the Paramecium t e m p e r a t u r e - s p e c i f i c s u r f a c e a n t i g e n s are anchored i n the plasma membrane via a glycophosphotipid, and t h a t an endogenous phosphotipase C may be i n v o l v e d i n the a n t i g e n i c v a r i a t i o n process. ® 1987 Academic Press, Inc.

The s u r f a c e o f Paramecium a u r e i i a , m a i n l y by a

a ciliated

protozoan,

is c o a t e d

s i n g l e s e t o f m o l e c u l e s , t h e s u r f a c e a n t i g e n s (SAgs). These

high molecular weight proteins (250 - 300 kDa) Belong to a multigene family whose

expression

interatlelic

(2,

involves

mechanisms

3) exclusion,

and

of

their

mutual

intergenic

expression

is

(1) and

essentially

controlled by external factors, such as temperature (for a review, 4, 5).

A b b r e v i a t i o n s : SAg, surface a n t i g e n ; mSAg, membrane-bound surface a n t i g e n ; sSAg, s o l u b l e s u r f a c e a n t i g e n ; CRG, c r o s s r e a c t i n g g l y c o p r o t e i n ; VSG, variant surface glycoprotein; PIPLC, p h o s p h a t i d y l i n o s i t o l - s p e c i f i c phospholipase C; CRD, c r o s s r e a c t i n g d e t e r m i n a n t . + C o r r e s p o n d e n c e a d d r e s s : Y. C a p d e v i l l e , C e n t r e de G@n@tique M o l @ e u l a i r e , Centre N a t i o n a l de l a Recherche S c i e n t i f i q u e , 91190 G i f / Y v e t t e , France.

0006-291X/87 $1.50 1219

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 147, No. 3, 1987

Paramecium

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SAgs can be isolated either in a m e m b r a n e - b o u n d

form (mSAg)

which is amphipnilic or in a soluDle form (sSAg) which is hydrophilic (6). The membrane-bound

form

(mSAg),

found in celts stably expressing a given

SAg can be converted to the soluble form (sSAg) by incubating paramecia in ethanolic solution or in Triton X-IO0 (7). The conversion of mSAg to sSAg is

accompanied

by

immunodeterminant determinant)

release

of

a

particular

glycosidic

is located

in

the

COOH-terminal

glycopeptide

of

(for a review, 8). Together with sSAg molecules, there is

of a set

crossreacting

unmasking

crossreacting with the so-called CRD (for crossreacting

which

Trypanosoma VSGs

the

of 45-50

glycoproteins),

kDa

molecules,

which,

now

denoted

after reduetion~

CRGs

(for

crossreact with all

the sSAgs and also display the CRD (6, 7). This conversion is mediated by an

enzyme

which

phenantrolin

can

(7),

be in

inhibited the

phosphatidyiinositol-specific

same

by

Zn 2+

and

fashion

as

Cd 2+,

but

not

Trypanosoma

by

o-

brucei

phospholipase C (PIPLC) (9) which solubilizes

the VSGs, cleaving their lipid anchor (lO, ll). These features are similar to that found in surface proteins anchored in the plasma m e m b r a n e

via a phosphatidylinositol

(for a review,

12, 15).

Therefore, in order to demonstrate that Paramecium surface antigens display the same type of membrane anchor~ the strain 156 of Paramecium primaurelia, expressing the temperature-specific

?H/myristic acid,and t h e ,.

G SAg, was biosynthetically labeled by

capability

o f PIPLC f r o m T. b r u c e i

(14-16) or

.

from B a c i l l u s

cereus (17) f o r c o n v e r t i n g t h e mSAg i n t o

sSAg was checked.

MATERIALS AND METHODS Cellular fractions were prepared from strain 156 o f P_z_- p r i m a u r e l i a e x p r e s s i n g t h e s u r f a c e a n t i g e n G (156G SAg) and the i d e n t i f i c a t i o n of the expressed SAg was p e r f o r m e d as d e s c r i b e d p r e v i o u s l y (2). C e l l u l a r e t h a n o l i c extracts were o b t a i n e d by incubating cells (1 vol) in a salt ethanolic solution (4 vol) (0.45W NaC1, 15~ ethanol, 5 mM phosphate Duffer, pH 7) for 45 min at 4°C (18). The detailed preparations of ciliary membranes have been described in a previous paper (6). 156G SAg was purified by the method of Preer (18), followed By ultrafittration on Sephadex G-200 superfine (Pharmacia). Phcspholipase C from T. brucei (ILTat 1.25) was obtained by soluuilization of VSG-depleted membranes in n-beryl glycoside~ followed by chromatography on Affi-gel 501 (Bio-Rad) (19). Phospholipase C, type lit, from B_~.cereus, p-chloromercuripUenylsulfonic acid (pCMPSA), defatted BSA, leupeptin and p h e n y l m e t h y l s u l f o n y l f l u o r i e e (PMSF) were purchased from Sigma, 9,10-SH(n) myristic acid (55 Ci/mmol) and 9,10-~H(n) palmitic acid (50 Ci/mmol) from Amersham. Biosynthetic labeling with ~H]myristic acid: washed paramecia (2xlOS~ml) were incubated at 27°CJin lOml Dryl's mineral solution(20) with[3H] myristic acid-BSA complex or with ~H]palmitic acid-BSA complex (I0 mg BSA, 500 ~Ci) for 150 min. lO ml culture medium was then added and incubation continued for another 150 min. Cells were harvested and washed, then they were either lyzed in boiling 5~ SDS or submitted to ethanolic extraction in the presence or in the absence of 1 mM pCMPSA. After SDS-PAGE on a 5-15~ acrylamide gradient gel, performed as described previously (6), destained

1220

Vol. 147, No. 3, 1987

B I O C H E M I C AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

gels were treated with Amplify (Amersham) for 30 min, dried and exposed to Kodak X-Omat AR film at -70°C for I0 days. Lipase treatment: Phospholipase digestions of 156G mSAg were performed on fractions (ciliary membranes, 25 ~g; ethenolie cellular extracts, 40 pg; purified SAg, 20 ~g) prepared in the presence of imM pCMPSA (which was then removed by dialysis or by dilution in the case of ciliary membranes). The digestions were carried out for 50 min at 30°C in the following conditions: (a) in lO mM Tris-HC1, 150 mM NaC1 pH 7.4 (TBS) plus 0.5 mM dithiothreitol, 0.1% Triton X-114 and 0.01% sodium deoxycholate in the presence of (1 H1/ml) a f f i n i t y - p u r i f i e d phospholipase C of T. brueei; (b) in TBS plus 0.06% Triton X-114 in the presence of phospholipase C (lO units/ml) from B__~. cereus. The conversion of mSAg to sSAg was monitored by the immunological detection of the CRD exposure (7). Anti-CRD antibody was prepared by immunizing rabbits with VSG followed by affinity purification as describes in (lO). A n t i s e r u m 2555 raised against purified 156G sSAg was used in immunoblotting experiments as previously described (6). Protease inhibitors: 0.28mM PMSF, O.OlmM leupeptin and lmM EDTA were used during the course of extractions and sample preparations.

RESULTS Inhibition of endogenous conversion by pCMPSA:

Incubation of paramecia in

ethanolic solution led to the soluOilization of SAg along with CRGs and the appearance of anti-CRD binding epitopes in SAg and also in CRGs (Fig. l, lane I). However, when the same incubation was performed in the presence of 1 mM pCMPSA the anti-CRD binding was no longer detectable either on SAg or on CRGs (Fig. 1B, lane 2), and the CRG determinant, common to sSAgs, which

is specifically

recognized by antiserum 2355

(7)

was not r e v e a l e d ( Fig .

1A, lane 2). This i n d i c a t e s t h a t Paramecium has an endogenous enzyme, a b l e to disclose the CRD and another determinant common to sSAgs and CRGs, which ean be inhibited Oy the thiol reagent pCMPSA in a manner analogous to the trypanosomal PIPLC

(21).

Fatty acxlation o f mSAq:

Paramecia were biosynthetically labeled w i t h [ 3 ~

m y r i s t i e acid as described in M a t e r i a l s and Methods. The SAg appeared strongly labeled at 300 min while other polypeptides were already labeled at 150 min of incubation (Fig. 2B, lanes 1 and 2). When a sample of cells labeled for 500 min was extracted in ethanol under the usual eonditions, the label associated with SAg disappeared (lane 3). However,

when this

extraction was performed in the presence of 1 mM pCMPSA the SAg remained labeled ( l a n e 4), indicating t h a t ~ H I m y r i s t i c acid label was associated

w i t h the membrane-bound form of SAg. The o t h e r bands which remained l a b e l e d under both c o n d i t i o n s of e t h a n o l i c e x t r a c t i o n might correspond to p r o t e i n s directly

acylated

(22). Our a t t e m p t s to i n c o r p o r a t e 15HI p a l m i t i c acid unSer

the same c o n d i t i o n s d i d not r e s u l t i n l a b e l e d SAg. Treatment of m S A q b y f .

Orucei PIPLC: Ciliary membranes, ethanolic cellular

extracts and purified 156G SAg were prepared in the presence of pCMPSA 9 in order to inhibit endogenous conversion, excess inhibitor.

1221

and further dialysed to r e m o v e

Vol. 147, No. 3, 1 9 8 7

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

A A

B

1

1

1

2

3

B 4

1

2

3

4

SAg..~

SAg .l=,,-

c.G l

Q

®

Fig. i. Inhibition of conversion b ~ pCMPSA: 156G ethanolic cellular extracts prepared without (lane i) or with i mR pCMPSA (lane 2) were reduced and analyzed Dy SDS-PAGE and immunoOlotting. (A) immunoblot obtained with an anti-156G serum (AS 2355). (B) replica immunoDlot p~obed with anti-CRD. SAg: 156G SAg; CRG: crossreacting glycoproteins. Fig. 2. Fatty ~ o f mSAq: (A) and (B): Paramecia (2xlO 5 cells) were la-bel~ with ~H] myristic acid as described in Materials and Methods for 300 min. After 150 min of labeling, an aliquot of 5xlO 4 cell equivalents was h a r v e s t e d and l y s e d i n b o i l i n g 5W 5DS. At t h e end o f t h e l a b e l i n g experiment (500 min)~ the remaining cells were split into three aliquots of 5xlO4 cells. One was boiled in 5~ SD5 and the ether two were extracted with ethanol in the presence or in the absence of I mM pCMPSA. The four samples were submitted to SDS-PAGE: the Coomassie-mlue stained gel is shown in A and the corresponding fluorogramm in B. Lanes 1 and 2: whole cells lysed in SDS after 150 and 300 min of tsbeling~ respectively. Lanes 3 and 4: e t h a n e l i c e x t r a c t s o f c e l l s l a b e l e d f o r 300 min and p r e p a r e d i n the absence or presence o f 1 mM pCMPSA, r e s p e c t i v e l y . The h i g h m o l e c u l a r w e i g h t bands found i n e t h a n o l i c e x t r a c t s c o r r e s p o n d to SAg m o l e c u l e s , as SAg i s the o n l y high molecular weight protein extracted with ethanol (6). Note that a l e s s e r amount o f SAg m o l e c u l e s i s e x t r a c t e d w i t h e t h a n o l i n the presence o f pCHSA.

The lipase digestion could not De assessed by phase separation using Triton X-114

(23), as mSAg

Therefore~

molecules

were not recovered

this was assessed by checking

in the detergent

phase.

the Binding of purified anti-CRD

to SAg and to CRGs. The t r e a t m e n t by T. b r u c e i P I P L C (Fig. 3, lanes 4, 5, 7 and 9) led to the a p p e a r a n c e

of a n t i - C R D e p i t o p e s in SAg and also in C R G s

(Fig. 3B), and to the r e c o g n i t i o n

of CRGs Dy an a n t i - 1 5 6 G s e r u m (AS 2355)

(Fig. 3A). The B. c e r e u s P I P L C c o u l d also d i s c l o s e the CRD and lead to the CRG recognition by the antiserum

2355 (data net shown).

DISCUSSION

The r e s u l t s

fatty

described

acylated

in

in their

this

paper

demonstrate

membrane-bound

lost after conversion of mSAg to sSAg, s S A g can be p e r f o r m e d

that

Paramecium

form 9 since incorporated

SAgs

label

are

is

and that this conversion of mSAg to

by I. b r u c e i or B__~.c e r e u s PIPLC. S i n c e the T. b r u c e i

1222

Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2

3

-

4 +

_

5 +

6 -

7 q-

8 _

9 +

A SAg~

c.GI

Viq. 3. Treatment of mSAq b ~ T. Orueei PIPLC: Different fractions containing mSAg'were prepared and incubated with T. Drueei PIPLC, as indicated in Materials and Methods. Then, samples were reduced prior to analysis by 5DS-PAGE and immunoOlotting. (A) immunoDlot oOtained with an anti-156G serum (AS 2355); (B) replies immunoblot prooed with anti-CRD. Lane 1: ethsnolie cellular extract prepared in the absence of pCMPSA and used as control; lanes 2-9: different fractions (lanes 2-5: ciliary membranes, lanes 6 and 7: ethanolie cellular extract, lanes 8 and 9: purified 156G SAg) prepared in the presence of pCMPSA and ineuoated without (-) or with (+) T. Urueei PIPLC.

PIPLC has b e e n f o u n d substrate

(14-16)

to

selectively

we a r e

led

to conclude

a n c h o r e d in t h e p l a s m a m e m b r a n e secondly,

that in Paramecium

a phospholipase

C similar

recognize

features

that,

firstly,

by a g l y e o s y l i n o s i t o l

of

glycolipid

t h e SAgs a r e

phospholipid,

end

the endogenous conversion can be performed by

to that of T. b r u c e i ,

since

both enzymes

are

inhibited by the same reagents. Furthermore the new facts described in this paper point

to the possibility

post-translational

that P a r a m e c i u m

modification,

as that

1223

displays

found

the same

in T. b r u e e i

type

VSGs

of

(24).

Vol. 147, No. 3, 1987

B I O C H E M I C AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

Finally, it is tempting to assign a role to the phosphatidylinositol anchor of SAgs in combination with endogenous PIPLC in the antigenic variation process, as antigenic variation in P a r a m e c i u m

is triggered by external

factors and as SAg itself has been previously shown to be directly involved i n this phenomenon As for the CRGs, immunological

(25). found in ethanolie

means

end

only

cellular

following

extracts,

reduction,

detectable

their

binding

by to

a n t i b o d i e s a g a i n s t sSAgs or a g a i n s t CRD r e q u i r e s the a c t i o n o f endogenous or exogenous PIPLC to unmask the corresponding e p i t o p e s . Thus, the q u e s t i o n arises whether they derive from SAg by proteolytie events occurring before or a f t e r t h e a c t i o n of t h e PIPLC, o r whether they c o r r e s p o n d to a s e t of

other m e m b r a n e proteins that are also bound to the plasma m e m b r a n e by a glycolipid anchor. Results demonstrating

that they correspond to other

membrane proteins are reported elsewhere (26).

ACKNOWLEDGEMENTS We thank C. Bordier very much for fruitful discussions. This work was supported in part by the C.N.R.S. (grant 900264), and by the Ligue Nationale Fran£aise con,re le Cancer. C.D. was supported by a fellowship from l'Association pour la Recherche sur le Cancer. M.L.C. de Almeida received support from St John'S College, Cambridge.

REFERENCES I_~. Beale, G.H. (1952) Genetics 37, 62-74. 2 , Capdeville, Y. (1971) Hal. Gen. Gen. 112, 306-316. 3 . Capdeville, Y., Vierny, C. and Keller, A.M. (19783 Mol. Gen. Gen. 161, 23-29. 4__t.Beale , G.H. (1954) in: The genetics of P a r a m e c i u m aurelia (5all, G. ed) pp 77-123, University Press, Cambridge. 5 . Sonneborn, T.M. (1974) in: Handbook of genetics (King, R.C. ed) vol. 2, pp 469-594~ Plenum Press, New-York, London. 6 . Capdeville, Y., Deregnaucourt, C. and Keller, A.H. (1985) Experim. Cell Res. 161, 495-508. 7 . Capdeville, Y., Baltz, T., Deregnsucourt, C. and Keller, A.M. (19863 Experim. Cell Res. 167, 75-86. 8/_. Turner t M.3., Cardoso de Almeids, M.L., Gurnett, A.M., Raper, J. and Ward, a. (1989) Curr. Top. Microbial. Immunol. 117, 23-55. 9___~.Cardoso de Almeida, M.L., Allan, L.N. and Turner, N.J. (1984) a. Protoz. 31, 55-60. i0___t. _ Csrdoso de Almeida, M.L. and Turner, M.J. (1983) Nature 302, 349-352. Ii • Ferguson, M.A.J., Haldar, K. and Cross, G.A.M. (19853 5. Biol. Chem. 260, 4963-4968. 12. Low, H.G., Ferguson, M.A.J., Futerman, A.H. and 5ilman, I. (1986)TIBS ii, 212-215. !3. Cross, G.A.M. (19873 Cell 48, 179-181. !4 . BSlow, R. and Overs,h, P. (1986) 5. Biol. Chem. 2619 11918-11923. 15 . Hereld, D., Krakow, J.L., Bangs, 5.D., Hart, G.W. and Englund, P.T. (1986) 5. Biol. Chem. 261, 13813-13819.

1224

Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16 . Fox, 3.A., Duszenko, M., Ferguson, M.A.3., Low, M.G. and Cross, G.A.H. (1986) J. B i o l . Chem. 261, 15767-15771. 17 . Ikezawa, I . and Taguchi, R. (1981) in Meth. in Enzym. v o l . 71, pp. 751-741. 18. P r e e r , 3.R., J r . (1959) J. lmmunol. 83, 579-584. !9~ Ward, J., Cardoso de Almeida, M.L., Turner, M.3., Etges, R. and B o r d i e r , C.(1897) Mot. Bioohem. P a r a s i t o l . 23, 1-7. 20___~.D r y l , S. (1959) J. P r o t o z o o l . 6 ( s u p p l . ) , 25. 21. B51ow, R. and Overath, P. (1985) FEBS L e t t . 187, 105-110. 22__z. Olson, E.N., Towler, D.A. and G l a s e r , L. (1985) 3. B i o l . Chem. 260, 3784-5790. 23. B o r d i e r , C. (1981) 3. B i o l . Chem. 256, 1604-1607. 24. Bangs, i.D., Andrews, N.W., H a r t , G.W. and Englund, P.T. (1986) 3. C e l l Biol. I03, 255-263. 25. Capdeville, Y. (1979) J. Cell Physiol. 99, 383-393. 26. Deregnaueourt, C., Keller, A.M. and Capdeville, Y. (1987) submitted to Biochem. J.

1225