Two plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein

Molecular and Biochemical Parasitology, 17 (1985) 61-77

61

Elsevier MBP 00584

T W O PLASMODIUM FALCIPARUM M E R O Z O I T E S U R F A C E P O L Y P E P T I D E S S H A R E E P I T O P E S W I T H A S I N G L E M r 185 000 PARASITE GLYCOPROTEIN

R A N D A L L F. H O W A R D 1, H A R O L D A. STANLEY 1, G A R Y H. CAMPBELL 2, SUSAN G. L A N G R E T H 3 and ROBERT T. REESE 1

~lmmunology Department, IMM14, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037, ZMalaria Branch, Centers for Disease Control Atlanta, GA 30333, and 3Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, U.S.A. Received 29 November 1984; revised version accepted 31 May 1985)

The malarial parasite Plasmodiumfalciparum synthesizes a major glycoprotein (gp) of M r 185 000 during its asexual blood cycle. Immunoprecipitation of [3SS]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of M r 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable a m o u n t s of radiolabeled saccharide. The smaller M polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp185. Our results suggest that gp185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite. Key words: Glycoproteins; Plasmodium falciparum; Merozoite surface; Malaria; Monoclonal antibodies

INTRODUCTION

Plasmodium falciparum synthesizes a high molecular weight polypeptide during the

Abbreviations: gp, glycoprotein; McAb, monoclonal antibody; Mr, relative molecular weight; p, polypeptide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; TCA, trichloroacetic acid.

0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

62 trophozoitc

{lVid ~cllJz~mt ~l;.t~c~, ()I l l l t r { t c i } ' t h l t K ; t l i c ,

dcx, c l t ) p m c n i

",c\ciai

molcctilai

weights r a n g i n g 1roi~l 1~5 000 t<) 200 ()(,ill ha',c bccn ascribed t~ v,h;lt i', probablT, lilt.' sanlc p r o t e i n based ,,n lt', lime <>1 synthesis, abilit~ I<, bc lal~clccl \~ith l a d i o l a b e l c d s u g a r s , arid a p p a r c i / !

localization

in the c l c c t r o p h o r c t i c

mobiliix

choice o f m o l c c u h i r

by indirect immunolluorcsccrk'c

j I - 4 I. D i l t c r c n c c ,

<,i lhi>, prl.)lCllt tilDi.)il~ isohttcs and lht.'i l chine-,. Lh<

tt c i g l / l i]larkctl prt~tc:in~, (sr~c, c l r i l l \ ,~ m \ i ~ S i l i L :illd laigl~ \ o r s \ I s lo~.t

percent polyl_lcr_vhtl'Itltlc gcl~ prob;~bl) ~lc:ct4inl i o l ~h~', '~ariabilll~ ] ':.0 i \~'~ x\ ili rclci !< this nlolcculc as gp IXS, ~ glyo)pl~,tcin h a \ m e :l l / , q 185(100 [3! [ ) t i r i n g the s c h i z o m s i a g c , g p l X 5 i> I h o u g h t i<)M-pl~>ccsscd i n h , , m a l l c r ! l o n g l y c o s y latcd nlotc.cttlc,; I2,7,x I lh,_' prc.'~,Cllt c ()illl/itinit.;.lthH1 (lcllloiqsiratt_'s t h a l t h c s c a l l d o t h e r polypeptidcs appctll b)bc S t l t l C l t l r a i I \ r c h i l u d t~> ~p185. In l;lc {ik" eptXS-rchitccl p o l y p e p t i d e s can hc d i \ idcd iilt<, al least tx~o gl'tiilp~,, tlllC COlltaii]ll'l~ wimt till(: p r o b a b l } the p r e v i o u s l 3 described nonglyt_os), kited p o l y p c p t i d c s t f,4.1)152-e4 ~ ()00 ;.ind a sccoi'id g r o u p t.'onlpo~,ed ,.)[ '4'l\c<)s\hltcd t'x)lbpcptidcs ,q / t 12(i-4(i()0~i t3iochcmical ;.il]d electit)r/ inicrcmcop> .{\idiots !{ii-ti1,.'I utdic-;_ll, ill:it ,. {)ilL'

from

e a c h o f ! iwsc l Z l ( ) u [ m

!}111\ t3c? c ~.llllt)( )[lclll "- OJ l hc CXl 1m c l l u h i r

Ill{Ill'Ix.

)~

"surface CO\it," ~l tilt ilc'c I]]t'lt)/*'il~ MATERIAIS

,\Nl)

Nit !ttf~l)1",

Cultivation q/para.~tl, ~

l h c M /a/~iparum isohitcs F V O (Vietnami. F ( B - I t ( ' o l o m bia), H o n d u r a s I / ( ' l ' ~ c . I n d o c h i n a 1. P a p u a - N e w G u i n e a 144. ;.liqd ~icrr;i I.c~11¢ weft. grown in h u m a n c r \ t h r o c v t c s in xitr~ with R P M I 164(I m e d i u m tlr,,inc Scientilic. Irvine, C A ) and O" humaN serum b 3 s l a n d a r d techniques [9]. Cult urcs were',3 nclaronized by c o n c e n t r a t i n g I r o p h o z o i t c - - o r schizont-stagc parasites with Physiogcl [10}. Proteins in the trophoz,)itc or s c h i l o n t stagm, v,'crc r a d i o l a b e l c d ~ i t h t ~ ' S i m c t h i o n i n c (40-2001a('i ml ~ \nlcrqaain (~oip. , \ r l i n g t o n ttcights, I L ) i n mcthionine-frcc medium: with [~H]ghtc,~-,ammc (c:l. 10Ill.i('[ ml ~, Nc\~ England Nuclear. Boston. M A ) m c o m p l e t e m e d i u m : ~ilh I ' H l i s o l c u c i n c (160 l.t('i ml . A m c r s h a m l m isolcticillc-frcc m e d i u m ; or with a m~xiurc of [ ' t l l a r g i n i n c , [~tt]lysiilc. [~Hlvalinc i N E N t . [ ~||]isolcut i n t . and [ ~tI]t:, ro~,im i \ m c r s h a m }. each at I00 ~.t('i I'11I 1, ill 1])Cdilll]] dcfi~.icii~ m thcs~ a m i n o acids [3I. Ccti~ wcrc washed in p h o s p h a t c - b u l f e r e d saline i PBS) and ~torcd at --70(',

\11 U:',{III]H[ d

i~l~ll ili~ilptqdtCd

l{Idh'~],~l[3Cl \~.~15 cicqq!lillilCv} d ~, Iolh,x,.>

M a t c r i a l w a s p r c c l p i t ; t ~ t x i i n I m l , , I , , > l ~ t S ' ; t r i c h l o r . a c c l i c a c i d ( l t \ i i , , i f i n i n lhv p r e c i p i t a t e wa', rct;uucd on \Vhatlvuln (iI',.'( iilicr~.. :lnd v, ashcd V,ilh ,~ ml "1'('\. followed bx 2 ml .':.t,.h,~t II(~ imd 05C; cthamq,

l'hc lihcts ,~.crc di!cd, and lh,:

radioactivity ol the :k:id-insolubh: malcrial coul-itcd alter the -iddition ~:I Stint-4 (Packard. [)ox~ncr<, i~i~,,c. II.i in :~ liquid scintillation t o u r e d I mnlcctcd hum
11][~ i/I;.iCl()lllt)icc.'ttit.':-,

Monoclonal an!d~udt<~ (tlcAD;

] m: m o n o c h m a ]

amibodie~ iMc'\b

4 - I 3 - 4 B . 4--f 5..

63 8G, 6-1-1C, 23-5-6, 23-36-7, and 31-24-1) were produced as previously described [11,12]. These antibodies were initially selected and antibody titers determined by indirect immunofluorescence using acetone-fixed parasitized erythrocytes and merozoites.

Immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitations were performed as previously described [3] with the following modifications. Buffer A contained 10 lag ml -~ leupeptin and 10 lag m1-1 pepstatin instead of 2% Trasylol and 40 mM NaF. Insoluble material was removed by centrifugation at 100 000 × g for 45 rain, and the soluble antigen incubated with an immunoadsorbent consisting of McAb-protein A-Sepharose for McAbs 23-5-6, 23-36-7, and 31-24-1 or McAb-rabbit anti-mouse antibody-protein A-Sepharose for McAbs 4-13-4B, 4-15-8G, and 6-1-1C. The rabbit anti-mouse antibodies were affinity-purified from the serum of rabbits injected with mouse IgG (Cappel Laboratories, Cochranville, PA) using a mouse IgG-CNBr Sepharose column. 50 tag of rabbit anti-mouse antibody were added per immunoprecipitation when used. Proteins were solubilized in SDS and mercaptoethanol and electrophoresed on 8% polyacrylamide gels [13] in parallel with 14C-labeled molecular weight markers (see figure legends). The gels were fixed, impregnated with 2,5-diphenyloxazole, dried, and fluorographed [ 14] using Kodak X-Omat film and Dupont Lightning-plus intensifying screen.

Immunolabelingfor electron microscopy. Cultures of the FCB- I or FVO isolate were enriched for schizont-infected erythrocytes on Percoll density gradients [15]. The schizonts were washed and recultured in RPMI 1640 with 10% human serum for 2 h to allow the release of merozoites. After this incubation the preparations were washed in RPMI 1640 without serum and fixed for 5 rain in 0.1% glutaraldehyde buffered to p H 7.4 with RPMI 1640. The lightly fixed cells were washed in PBS followed by 1 M glycine, and another PBS rinse before exposure to antibodies. All washes included centrifugation for 5 min at 5000 rpm in a Beckman Microfuge 12 with the horizontal rotor. The schizont/merozoite sample was approximately 5 Ill packed cell volume. These were incubated for 30 min at 25°C with 500 lal o f a 1:20 dilution of antibody-containing mouse ascites fluid (produced using either the mouse-mouse hybridoma or the parent myeloma cell line) and washed 3 times with PBS. The samples were then incubated for 30 min at 25°C with a 1:2 dilution (in PBS) of ferritin-conjugated goat IgG specific for mouse IgG heavy and light chains (Cappel Laboratories). The cells were then washed 3 times with PBS, fixed with 2% glutaraldehyde, and processed for electron microscopy as previously described [16].

Differential extraction of radiolabeledproteins. Ghosts were prepared from frozen (-70°C), radiolabeled parasitized erythrocytes [3], and dissolved in a buffered NaC1 solution containing 2% Triton X-114 [17]. The insoluble material was removed by

64 centrifugation (100000 X ,g, 15 rain, 4°C). Subsequent separations on a sucrose: cushion were performed as described [17]. Concentrated solutions of detergents. NaCI, and Tris-HC1 were added to the separated aqueous and detergent phases to equal our standard immtinoprecipitation conditions. Proteins were m m m n o p r e c i p i t a t e d sequentially with the indicated monoclonal antibodies, separated b} S D S - P A G E. and visualized by f]uorography. In sonication experiments, either water or 1 0 r a m E D ' I A , ptt 7.5, was added with 500 tag ml -~ leupeptm and 5 lag ml ~ pcpstatin to frozen radiolabcled parasitized erythrocytes. The suspension was sonicated in a cup horn (Heat Systems-Ultrasonics, Plainview, NY) for three 30-s periods (80-90 W). Samples were placed on ice for 5 rain between sonications, and 10 min after the final treatment. The soluble and particulalc fractions were separated by centrifugation ( 100000 )> <~,,,60 rain, 4 C I . The particulatv fraction was resonicated (5 s) in water, 10 mM E D T A , or0.15 M Na('l and recentrifuged. This particulate fraction was dissolved in detergent-containing Buffer A and either stored at -70°( ' or immediately processed for immunoprccipitation assa3 (scc' above). 1"he water soluble fractions from the centrifugation steps were diluted m hall with Buffer A for immunoprecipitation. Equivalent proportions of these fractions wet< divided a m o n g the i m m u n o a d s o r b c n t s for prccipitationanalvsis Proteins boundt~, the monoclonal antibodies were dissolved in SDS and separated by S I ) S - P A G t ! . Additional experiments were performed by sonicating and fractionaling unlabeled. uninfected h u m a n erythrocytes or EVO-infecied crythrocyics as de,,,cribed abo~e. The proteins were dissolxcd in SI)S mercaptocthanol buffer, separated by S D S - P A G t : . and stained with Coomassie blue. RfSULTS

Two categories oJanti-<~,,pl85 monoclonal antibodies.

Eluorescem-labeled antibodies which bind to surface antigens of extracellular merozoites, as ~cll as lo m e m b r a n e or surface antigens of intracellular merozoites prior to erythrocyte lysis. ~tlcn yield it rim-like indirect immtinofluorescence pattern. Several monoclonal antibodies giving such a picture were produced. Six of these (McAb 4- 13-4B, 4-15-8(i. {-,-1- 1(', 23-5-¢~. 23-36-7, and 31-24-1) b o u n d a [~SS]methionine-labeled parasite polypeptide ot <'l/ 185 000. M c A b s 23-5-6, 23-36-7, and 31-24-1 also immunoprecipitated polypeptides of M r 120 000, 90 000, 88 000, 46 000, and 40 000 (McAb 31-24-1, Fig. 1, lane 31. These ~i 11 be designated the group A polypeptides (p). The p185, p46, and p40 bands were' routinely the most prominent with these three McAbs. M c A b 4-t3-4B. 4-15-8G, and 6-1-1C specifically immunoprecipitated p 185 and additional major polypcptidcs o f 3,,1 152000, 106 000, and 83 000 ( M c A b 4-13-4B, lane 21. These will bc called the group 1~ polypeptides. Some less prominent polypeptides were also bound, but nc~t consistently. For simplicity, and unless explicitly stated otherwise, what is found true for one g r o u p m e m b e r is true for all the antibodies of the group. Thus, these six m o n o c h m a I antibodies recognize two different groups of polypeptides wiih p l85 being the ~nl\ apparent c o m m o n denominator.

65

.................~ 8 5 ~52

,=~ ~

-18-5

m m

n

1

2

3

m

4

5

6

Fig. 1. SDS-PAGE of proteins and glycoproteins bound by anti-gpl85 McAbs. Parasitized erythrocytes (FVO isolate), incubated with [3SS]methionine(lanes 1-3; 4% ring, 2% trophozoite, 79% schizont, 15% segmenter) or [3H]glucosamine(lanes 4-6; 8% ring, 10% trophozoite, 63% schizont, 19% segmenter at end of labelingperiod after overnight incubation ofphysiogel top), were dissolvedin BufferA. The proteins were precipitated with immunoadsorbents consisting of rabbit anti-mouse antibodies alone (lanes 1, 4), McAb 4-13-4B-rabbit anti-mouse (lanes 2, 5), and McAb 31-24-1 (lanes 3, 6) antibodies bound to protein A-Sepharose. The bound polypeptides were eluted and electropboresed on SDS gels along with the ~4C-labeled marker proteins myosin (200000), [3-galactosidase(116000), pbosphorylase b (97 400), serum albumin (69 000), gammaglobulin (53 000), and carbonic anbydrase (30 000) indicated by marks to the left of lanes 1 and 4. Mr (× 10-3) of the resolved proteins are shown on the right of the figure. The arrowhead indicates the dye front for lanes 4-6.

Glycosylated and nonglycosylated domains ofgpl85.

A polypeptide o f M r 185 000 has previously been suggested to be a glycoprotein [3]. W h e n the glycoproteins of the F V O isolate were radiolabeled with [3H]glucosamine a n d i m m u n o p r e c i p i t a t e d with the six McAbs, all of these reagents i m m u n o p r e c i p i t a t e d gp 185 showing that the M r 185 000 p r o t e i n is in fact gp 185. In addition, M c A b 31-24-1 again i m m u n o p r e c i p i t a t e d radiolabeled group A polypeptides of M r 120 000, 90 000, 88 000, 46 000, a n d 40 000 (Fig. 1, lane 6), a l t h o u g h gp 120, gp90, and gp88 are frequently quantitatively m i n o r glycoproteins and are difficult to see in the r e p r o d u c t i o n (see also Fig. 5, lane 8). A n a d d i t i o n a l b a n d of M r 48 000 a n d one or more polypeptides migrating with the dye front (M r < 30 000) were also frequently observed. In contrast, the group B-specific M c A b 4-13-4B i m m u n o p r e c i p i t a t e d only gp 185 from the [3H]glucosamine-labeled polypeptides (Fig. 1, lane 5). This was despite the fact that other i m m u n o p r e c i p i t a b l e glycoproteins were

66

present as demonstrated by precipitation with immune Aotus trivirgalus monke'+ serum [3]. Therefore, it appears that the g r o u p A polypeptides are in fact glycoprotems while - with the exception o f g p 185 - the group B polypeptides may not be glycosylated at all. To demonstrate that these McAbs bind the same gp185 molecule rather than two> antigenically distinct molecules, each with an apparent M of 185 000, an i m m u n o d e pletion experiment was performed. [3SS]Methionine-labeled antigen from the FVO isolate was first pre-adsorbcd with an irnmunoadsorbent consisting oi parent tnyelom+t culture fluid b o u n d to protein A-Sepharose (Fig. 2, lanes 1 and 5). The labeled antigen+ remaining in the supernatant were then adsorbed four times v+ith eithcr the g r o u p B-specific M c A b 4-13-4B (the first and fourth immunoprecipitates arc shown m lanes 2 and 3) or the group A-specific M c A b 23-5-6 (the first and fourth arc in lanes 6 and 71. Lastly, the supernatants were immunoprecipitated with the heterologous McAb (lanes

-gp185

200-

i w

11697-

-

p152

-gpj2O

~lm

~

-- pluQ

w

6953-

---'-- -

43-

:8g:t8

30-

1

2

3

4

5

6

7

8

Fig. 2. lmmunodepletion of gplS5 with McAbs. 13SS]Methioninc-labeled FV() pn~tein,, iI)~ ring, S~7 trophozoite, 71% schizont, 21 ~7~segmenter at end of labeling) were mmmnoprecipitatcd sequentially (lanes 1-4 and 5-8) with nonspecific myeloma supernatant (lanes 1 and 5), and with McAb 4-13-4B four times (the first and fourth imnlunoprecipitatcs tire m lanes 2 and 3) to deplete the sample ol gp185 followed by the heterologous McAb 23-5-6 (4) to detect remaining proteins, (~l with McAh 23-5-(~ tour limes (first and fourth, lanes 6 and 7)and then with McAb 4-13-4B (lane 8). Proteins were rcsobcd h,+ SI)S-PAGE along with ]4C-labeled marker proteins (left margin); lluorography was tot 10 days. Specilicaltv immunoprecipitat+ ed polypeptides are indicated.

67

4 and 8, respectively). Both antibodies removed nearly all of the gp185 during the adsorptions with homologous reagent (lanes 2, 3 and 6, 7) leaving practically no gp 185 to be adsorbed with heterologous antibody. While there was a large reduction in soluble gp 185, the lower M r polypeptides not adsorbed by the group B-specific McAb 4-13-4B were still bound by the group A-specific McAb 23-5-6 (lane 4). Likewise, the group B-specific antibody immunoprecipitated p152, p106, and p83 (lane 8) which were not removed by the group A-specific antibody. In fact, despite the prior removal of gp185, there was a nearly quantitative precipitation of the smaller polypeptides. Similarly, when [3H]glucosamine-labeled polypeptides were immunodepleted of gp185 with McAb 4-13-4B (Fig. 3, lanes 2-5), McAb 23-5-6 still immunoprecipitated gp46 and gp40 (Fig. 3, lane 6); gp 120, gp90, and gp88 were not detected in this antigen preparation. Conversely, after McAb 23-5-6 removed all the glycoproteins (lanes 8-11), McAb 4-13-4B failed to bind any radiolabeled proteins (lane 12). These experiments demonstrated that there is a single gp 185 molecule bearing epitopes recognized by both groups of McAbs. In addition, they showed that the smaller polypeptides were not merely coprecipitated with gp 185, but rather share epitopes with gp 185, and hence are structurally related to this glycoprotein. Unrelated antibodies (e.g., McAbs to

200 -

,,,,,, ~

.....

- , m ....,, - - .

-gp185

69-

301 1

2

3

4

5

6

7

8

9

10

11

12

Fig. 3. Immunodepletion of [3H]glucosamine-labeled gp 185. Radiolabeled FVO proteins (43% trophozoite, 57% schizont) were sequentially reacted with immunoadsorbents as described in the text and subjected to SDS-PAGE (fluorography for 20 days). Lane 1, rabbit anti-mouse alone; lanes 2-5, McAb 4-13-4B; and lane 6, McAb 23-5-6. Lane 7, rabbit anti-mouse alone; lanes 8-11, McAb 23-5-6; lane 12, McAb 4-13-4B. Coelectrophoresed marker proteins are indicated in the left margin.

68 unrelated parasite proteins or culture supernatants from the parent myeloma cell line~ failed to remove any of the group A or group B polypeptides. Gp 185 appears to constitute a relatively large portion of the [35S]methionine-labeled material of the trophozoite and schizont stages [ 1-3]. To estimate the amount of gp 185 synthesized by P. falciparum during the latter part of the intracellular growth cycle. parasitized cells were radiolabeled with [35S]methionine, [3H]isoleucine. or a mixture of five 3H-labeled amino acids (arginine, isoleucine, lysine, tyrosine, and valine). One aliquot was precipitated with TCA to assess total incorporated radioactivity while a second aliquot was immunoprecipitated with McAb 31-24- 1. In experiments m which gp185 constituted > 90c7;, of the ntaterial immunoprecipitated las judged by St)SP A G E and fluorography), we found that gp185 comprised at least 2.9c~{, 2.()c;~, attd 1.1% of the total acid-precipitable material labeled respectively with [3H]isoleucine, [35S]methionine, and the 3H-labeled amino acid mixture.

Electron microscopic localization of the antigens.

Indirect immunofluorcscence data suggested that both group A- and group B-specific McAbs bind antigens at the surface of the merozoite (see above). The results of others suggest that p83, one of the nonglycosylated antigens, is an externally exposed protein on the merozoite surface [7]. Therefore, we attempted to test this by suspension immunolabeling ofextracellular merozoites followed by electron microscopic analysis. In addition, we wanted to determine whether antibodies can detect any of the group A glycoproteins on the surface of the merozoite. Naturally released merozoites were lightly fixed and immunolabeled with the group A-specific McAbs 31-24-1 (Fig. 4B), 23-5-6, and 23-36-7 and the group B-specific McAbs 4-13-4B and 4-15-8G. Fig. 4 shows that the fine ultrastructural details of the merozoite, for example, the brushy surface coat, tile plasma membrane, and the pellicular complex (subsurface) membranes, were retained during the process. Four oi the five antibodies tested (McAb 31-24- 1, Fig. 4B and -Fable I) bound evenly about the, entire circumference of the merozoite surface. Controls were performed in parallel with either ascites fluid from mice injected with the parent myeloma (Fig. 4A) or a McAb to an internal protein [11,18]. Both controls were negative with only an occasional ferritin granule bound to the surface of the merozoite. The failure of McAb 23-5-6 to bind to the intact merozoite was not due to insufficient antibody since it had a fluorescence titer of 1:2560 while McAb 31-24-1 had a fluorescence titer of only 1:640 and showed good binding. Thus, with the exception of McAb 23-5-6, the antibodies that were tested bind specifically to the surface of the merozoite. Since gp 185 has not been found on the surface of the merozoite [7,19], these data suggest that as least two oI the lower M~ polypeptides (one group A and one group B polypeptide) are merozoHe surface antigens.

Epitope specificity oJ the McAbs.

The differing abilities of the group A- find group B-specific McAbs to bind proteins from certain isolates of P../alciparum and to bind t~,

69

Fig. 4. Electron micrographs showing localization of antigens to the surface of the merozoite. Isolated merozoites were lightly fixed and exposed to ascites fluid from mice injected with (A) the parent myeloma, or (B) the hybridoma producing McAb 31-24-1, followed by ferritin-conjugated goat anti-mouse IgG. Samples were prepared for electron microscopy after post-fixation (see Materials and Methods section). The magnification for both merozoites is identical; scale bar = 0.25 lam.

molecules in the surface o f the extracellular m e r o z o i t e were used to d e t e r m i n e the e p i t o p e specificity o f the i n d i v i d u a l M c A b s . The g r o u p A-specific a n t i b o d i e s could be d i v i d e d into two g r o u p s b a s e d on the ability of M c A b 23-5-6, but not 23-36-7 o r 31-24-1, to bind all of the isolates tested (Table I). Likewise, M c A b 23-5-6 was the only one o f the three a n t i b o d i e s which did not b i n d to the surface o f extracellular m e r o zoites even t h o u g h sufficient M c A b 23-5-6 was present to react with fixed parasite antigen by indirect i m m u n o f l u o r e s c e n c e (see above). Thus, M c A b 23-5-6 binds an e p i t o p e readily d i s t i n g u i s h a b l e f r o m the one (or p r o b a b l y two) different epitopes b o u n d by the o t h e r two g r o u p A-specific M c A b s . A similar analysis o f the g r o u p B-specific M c A b s indicates that M c A b 4-13-4B reacts with a different epitope (one lacking on the P a p u a - N e w G u i n e a isolate) t h a n d o M c A b s 4-15-8G a n d 6-1-1C, even t h o u g h all three a n t i b o d i e s b i n d to the m e r o z o i t e surface of, for example, the F V O a n d H o n d u r a s I isolates. Therefore, it is o b v i o u s that the g r o u p B-specific a n t i b o d i e s also recognize at least two different epitopes, one by M c A b 4-13-4B a n d at least one by the o t h e r two M c A b s . In light o f earlier d a t a [20], it is not surprising t h a t n o n e o f the three a n t i b o d i e s binds an e p i t o p e c o n s e r v e d on all the isolates tested.

7(I TABLE I Isolate diversit_,e ol gp185 cpitopes Anti-gp 185

,'4urtacc labeled Inerozoitc (EM) <

F'ixcd parasitized erythrocytes (IlF'Y'

Group specificity

McAb

FVO lton ~'

PNCi

A

23-5-6 23-36-7 31-24-1

* ~ *

>

4-13-4B

4-15-8(i 6-1-I(

Indo S1

I('B-I

1:\'O

NI) <~ ND NI)

+

~

N[)

~

i

N[)

~

~

NI)

,

NI)

" Trophozoite and schizont-stage parasites were prepared tot indirect immunoIluorescencc as described m Materials and Methods. b Parasite isolates are: Honduras I/CDC (Hon): Papua-New-Guinea 144 (PNG); Indochma 111ndol: Sierra Leone (SL). ' Naturally released mero:,oites were surface labeled for electron microscopy as described m Materials a ncl Methods. d ND = not done.

Selective extraction o[ the polypeptides.

The preceding ultrastructural data showed

t h a t o n e o r m o r e g r o u p A a n d g r o u p B m o l e c u l e s are e x t r a c e l l u l a r l y e x p o s e d o n the s u r f a c e o f the m e r o z o i t e .

H o w e v e r , there is p r e s e n t l y n o i n f o r m a t i o n

indicating

w h e t h e r these p o l y p e p t i d e s are c o m p o n e n t s o f the e x t r a c e l l u l a r m a t r i x o r arc integral membrane

p r o t e i n s . Since i n t e g r a l m e m b r a n e

p r o t e i n s r e q u i r e d e t e r g e n t l o r their

e x t r a c t i o n , we h a v e used the n o n i o n i c d e t e r g e n t F r i t o n X - I 1 4 to e x t r a c t p a r a s i t e p r o t e i n s f r o m a m i x t u r e o f t r o p h o z o i t e - a n d s c h i z o n t - s t a g e p a r a s i t i z e d cells. This p a r t i c u l a r d e t e r g e n t has the useful p r o p e r t y o f r e a d i l y p a r t i t i o n i n g p r o t e i n s into d e t e r g e n t - s o l u b l e ( h y d r o p h o b i c ) a n d w a t e r - s o l u b l e ( h y d r o p h i l i c ) p h a s e s at 3 0 ° C [ 17]. F o r e x a m p l e , the h u m a n

erythrocyte membrane

proteins acetvlcholinesterasc and

B a n d 3 are p a r t i t i o n e d into the d e t e r g e n t p h a s e while e r y t h r o c y t e g l y c e r a l d e h y d e - 3 p h o s p h a t e d e h y d r o g e n a s e a n d o t h e r s o l u b l e p r o t e i n s such as c a t a l a s e a n d m y o g l o b i n are l o c a l i z e d to the a q u e o u s p h a s e [17]. [35S]Methionine-labeled

p a r a s i t e p r o t e i n s w e r e s o l u b i l i z e d in T r i t o n X-114 a n d

s e p a r a t e d into a q u e o u s a n d d e t e r g e n t phases. A d s o r b e n t s c o n t a i n i n g no a n t i b o d y . M c A b 4-13-4B o r M c A b 31-24-1 w e r e t h e n used s e q u e n t i a l l y to p r o b e tot the p o l y p e p tides. F r o m this it was f o u n d t h a t a m a j o r p o r t i o n o f b o t h the g r o u p A a n d g r o u p B p o l y p e p t i d e s was in the a q u e o u s r a t h e r t h a n the d e t e r g e n t p h a s e s (Fig. 5, lanes I - 6 t . In this p a r t i c u l a r e x p e r i m e n t , the t w o b a n d s o f M

< 4 0 0 0 0 in lane 4 ,ire probablT,

c o n t a m i n a n t s f r o m the p r e v i o u s i m m u n o p r e c i p i t a t i o n with an u n r e l a t e d a n t i b o d y : the r e l a t e d n e s s o f the o t h e r m i n o r b a n d s to the g r o u p A a n d g r o u p B pol.,,pcptides is

71

695343-

0-

1

2

3

4

5

6

7

8

Fig. 5. Phase separation of parasite polypeptides after solubilization with Triton X-I14. Radiolabeled parasite proteins were partitioned into detergent (lanes 1, 3, 5, 7) and aqueous phases (lanes 2, 4, 6, 8) and immunoprecipitated as described in the text. (Left) [3sS]Methionine-labeledpolypeptides (6% ring, 25% trophozoite, 49% schizont, 19% segmenter, plus free merozoites at harvest) bound to protein A-Sepharose alone (lanes l, 2), McAb 4-13-4B (lanes 3, 4), McAb 31-24-1 (lanes 5, 6). (Right) [3H]Glucosamine-labeled glycoproteins (20% trophozoite, 70% schizont, 10% segmenter at harvest) bound to McAb 31-24-1(lanes 7, 8). [35S]Methionine-and [3H]glucosamine-labeledproteins were electrophoresed on separate SDS gels and fluorographed. The arrowheads indicate the tops and dye fronts of the two resolving gels. ~4C-labeled marker proteins are shown to the left of lanes 1and 7 and the Mr values (× 10-3) of the parasite proteins are given to the right of lanes 6 and 8. u n k n o w n . Similar f r a c t i o n a t i o n was observed with [3H]glucosamine-labeled glycoproteins following solubilization with T r i t o n X-114 a n d i m m u n o p r e c i p i t a t i o n with the g r o u p A-specific M c A b 31-24-1 (Fig. 5, lanes 7, 8). These data suggest that all of the g r o u p A a n d group B polypeptides c o n t a i n a significant p r o p o r t i o n of hydrophilic residues, m a k i n g it unlikely that these polypeptides are integral m e m b r a n e proteins. This i n t e r p r e t a t i o n was further tested to see whether gp185 a n d any o f the other g r o u p A or group B molecules were released into a water soluble, n o n s e d i m e n t a b l e form d u r i n g sonication of the parasites. [35S]Methionine-labeled parasitized erythrocytes were sonicated in a q u e o u s m e d i u m a n d centrifuged at 100000 X g for 60 min, c o n d i t i o n s which in other eucaryotes are likely to separate subcellular c o m p o n e n t s into soluble (i.e., cytosolic) a n d particulate fractions [21]. U n d e r these conditions, most of the gp185 was i m m u n o p r e c i p i t a t e d from the cytosolic fraction (Fig. 6, lanes 3-6). We observed some experiment-to-experiment variability in the p r o p o r t i o n of

72

A

-gp185

116 -

69-

~

5343-

1

2

3

4

5

6

:trin

200-

~

- gp185

695343-

3013 14

7

8

9

10

15

16

17

18

11 12

Fig. 6. Release ol soluble proteins by sonication. [3~S]Melhionine-labeled (lanes 1-0: physiogel lop pulse-l~t beled 15 rain at 29(~,~ ring, 31% trophozoite, 32!,~ schizom, 8~ segmenter),tmlabeled dane> 7~12L and [3H]glucosamine-labeled material (lanes 13-18; physiogel top labeled 5 h until 6(} rings, 32c} trophozolte, 51% schizont, 11c~ segmenter, and free merozoites) were sonicated and fractionated as described m Materials and Methods. (A) Radiolabeled material (lanes 1-6"1was solubilized in Buffer .-\ and the proteins resolved by SDS-PAGE after equi~alenl w)lumcs of lhc particulate fraction (lancs 1. ~. 51 and lhc soluble iracnoT~ (lanes 2, 4, 6"1 were immunoprecipitated with rabbit anti-mouse alone (lanes 1, 2), McAb 13 4B (lanes 3 . 4 i and McAb 23-5-6 (lanes 5. {~).(B) Uninfected erythrocytes (lanes 7-9) and FVO-inh.'cted (60{ f parasitemiai erythrocytes (lanes 10- 12) were also fractionated, An aliquot otthe cytosolic fraction after the first (lane> :

73

gp185 in the two fractions; in two cases (e.g., Fig. 6, lanes 1-6), the soluble fraction contained more than 50% of the total detectable gp185, and in two other experiments there were roughly equal amounts of gp185 in the soluble and particulate fractions. This variability is likely to result from differences in the amount of gp185 trapped within membrane vesicles formed during sonication. Sonication experiments were also performed on both unlabeled, uninfected erythrocytes and unlabeled FVO-infected erythrocytes (Fig. 6, lanes 7-12). Results with uninfected erythrocytes indicated that Band 3, the major Coomassie-stained integral membrane protein of the erythrocyte, as well as such cytoskeletal proteins as actin and most of the spectrin, were present in the particulate fraction (lane 9). Soluble proteins including carbonic anhydrase and hemoglobin were quantitatively separated into the cytosolic fraction (lanes 7, 8). Diminished, but still measurable amounts of the latter molecules were also present in the supernatant after the second sonication (lane 8). After Coomassie blue staining, the same qualitative results were observed with parasitized erythrocytes (Fig. 6, lanes 10-12). Band 3 was still quantitatively fractionated with the particulate material. While it was difficult to determine precisely where the erythrocyte actin was located, most of the spectrin was still in the particulate fraction (lane 12). These data indicate that not only is an integral membrane protein (Band 3) sedimented, but that cytoskeletal proteins known to interact with Band 3 are sedimented as well. When [3H]glucosamine-labeled parasitized cells were sonicated, fractionated by ultracentrifugation, and the proteins immunoprecipitated with a group B-specific McAb (Fig. 6, lanes 13-15) or a group A-specific antibody (lanes 16-18), sugar-labeled gp185 was found predominantly in the cytosolic fractions (lanes 13, 14, 16, 17). Thus, the localization of sugar-labeled gp185 was similar to that of [35S]methionine-labeled gp185. In contrast, gp46 and gp40 were observed predominantly in the particulate fraction (lane 18), indicating that these molecules fractionate independently of gp 185 during cell disruption. Using [35S]methionine-labeled parasites, thus far we have been unable to routinely detect p83 or the other group B polypeptides after sonication. In summary, more gp 185 is present in the cytosolic fraction than in the particulate fraction after sonication (Fig. 6). This indicates that a substantial proportion of this molecule is liberated by rupture of the infected erythrocyte and parasite membranes.

and 10) and second (lanes 8, 11) centrifugations and an equivalent portion of the particulate fraction (lanes 9, 12) were separated by SDS-PAGE along with unlabeled phosphorylase b, catalase (60000), ovalbumin, and bovine carbonic anhydrase, and stained with Coomassie blue. carb anh, human erythrocyte carbonic anhydrase; Hb, u and [3chains of hemoglobin. [3H]Glucosamine-labeled parasite proteins were immunoprecipitated with the group B-specific McAb 4-13-4B (lanes 13-15) or the group A-specific McAb 23-5-6(lanes 16-18) after fractionation of the parasites into the first (lanes 13, 16) and second (lanes 14, 17) cytosolic and the particulate fractions (lanes 15, 18). Material was solubilized in Buffer A prior to immunoprecipitation. The fluorograph (6 days exposure) after SDS-PAGE is shown. The molecular weights of the marker proteins are indicated to the left of each of the three panels.

74 The observation that gp 185, gp46, and gp40 fail to partition into the detcrgcnt phase after Triton X-114 extraction (Fig. 5) makes it unlikely that these glycoproteins arc transmembrane proteins. In contrast with the high molecular weight glycoprotein. gp46 and gp40 were sedimented principally with the particulate fraction after sonication, as were the cvtoskeletal proteins actin and spectrin and the integral membrane' protein Band 3 (Fig. (ii. Fhc interaction of these cvtoskeletal proteins with the Band ++ protein may be responsible for their sedimentation with the patticuiate !ractiol11221. Likewise, the sedimentation of gp46 and gp40 nlav result froln tileli +Itlteractioll wit i~ other proteins or lipids in the particulate fracti~m. DISCUSSION In the present communication x~e have shown that the 185000 polypeptide oi t ~ falciparum is a glycoprotem and that it comprises lCf or more otthe newly synthesized. acid-insoluble parasite protein. McAbs which bind gp185 identilv two di|Terent popu lations of molecules. Group A consists of the glycoproteins gp 185. gp 120, gp90, gp88, gp46, and gp40 while group B includes gp185, p152, p106, and p83 which, with the exception of gp185, cannot be detectably labeled with [~H]glucosamine. I h e polypeptides of both group A and group B are specifically immunopreclpitated by the McAbs after removal of gp 185, not merci?,' coprecipitated with this glycoprotein. Thus, gp 185 consists of at least two domains which are antigenically and biochemicallv distinct: on
75 Since at least two different epitopes are shared by gp185 and the other group A polypeptides (Table I), it seems unlikely that the group A polypeptides are encoded by separate genes which have diverged from the gp185 gene. Nevertheless, the present results do not eliminate two other possibilities: the smaller glycoprotein may be synthesized de novo by less c o m m o n mechanisms such as transcriptional processing [25], dead-end translation [26], or second-site initiation of translation [27]. Alternatively, the group A molecules may be related only as a result of the attachment of similar or identical oligosaccharide chains rather than shared amino acid sequences [28]. Additional experiments will be required to distinguish among the processing, de novo synthesis, and glycosylation possibilities. Using the group A-specific McAbs 23-36-7 and 31-24-1 and the group B-specific McAbs 4-13-4B and 4-15-8G, we showed directly by immunolabeling and electron microscopy that antigens from both group A and group B are present on the merozoite surface and exposed to extracellular antibodies (see also Fig. 1C and D, ref. 18). Data obtained by immunoprecipitation of radio-iodinated merozoite proteins and by immunodetection of unlabeled merozoite proteins on Western blots [23] suggested that p83 is on the surface of the merozoite whereas gp185, p152, and p106 are not [7,19]. Consequently, it is likely that, of the group B polypeptides, p83 is on the surface of the merozoite. It is likely that gp46 a n d / o r gp40 are also on the surface of the merozoite since gp46 and gp40 were present in the merozoite [3], and two polypeptides of similar size were labeled in trophozoites with monosaccharides as well as being radioiodinated in merozoite preparations [3,19,29,30]. In addition, gp46 and gp40 are two of the merozoite proteins present in the parasite following merozoite invasion [3]. With the addition of these two or three polypeptides to the previously identified p 140 [31], p 130 [32], p75 [31], p59 (p180) [31], and gp56 [18], there now appear to be at least seven polypeptides on the surface of the merozoite which are exposed to extracellular antibodies. Based on our detergent extraction and phase partitioning data, our working hypothesis is that the group A and group B polypeptides are not transmembrane molecules, having instead the characteristics typical of soluble proteins or peripheral membrane proteins. Following this line of reasoning and given our electron microscopic data which suggest that polypeptides from group A and group B are on the merozoite surface and are exposed to the extracellular milieu, some of these molecules are more likely to be components of the surface coat than of the plasma membrane itself. Since gp46 and gp40 were not strongly lipophilic as assayed with Triton, we infer from our sonication-release data that they can interact with other proteins or possibly certain lipids. Under the appropriate conditions, these may result in a sedimentable complex. Such interaction(s) may be necessary to maintain the matrix of the surface coat of the extracellular merozoite. Interactions between certain coat components and integral membrane molecules may also be responsible for maintaining the integrity of the surface coat. Another merozoite surface molecule, gp56, may serve the purpose of anchoring the surface coat proteins to the plasma membrane; one population of gp56

76

molecules appears tither to be modified (e.g., acylated) ill such a l-~lanncr that gp5O itself becomes tipophllic or to be tightly associated with another taydrophobic molecule [181. ACKNOW[.EDGEMENI',

The authors thank l)ianc l.vszczasz and Ina If rim for excellent technical assistance. Dr. A. Ziemiecki for suggesting the lriton X - l 1 4 e x p e r i m e n t s . I)rs, F" Ardeshir,,l Flint, and R. Ramasamy for critical reading, and ('. Bechtel-(iroat ior typing the manuscript. This ,aork ~ a s supported by the Agency for International De\elopment~ Contract N timber [) PE-0453-('-(10-1()17-00 and PASA Nu mber BST-0453-DZ-402200. D o n o r blood samples were obtained |rorrl the General Clinical Research Center Grant RR00833 from tile Division o l Research Resources. This is Publication Numbe~ 3652-IMM of the Research Institute of Scripps Clinic. REFEREN('I!S

1 2

Kilejian. A. 41 OhiO)Stage-specific proteins and glycoprotem~, ofPlasmodiumlal~iparum: ldcntilicauol~ of a n t i g e n s u n i q u e t,~ s c h i z o n t s a n d m e r o z o i t e s . Proc. Nail. A c a d Sci. IT S . A 77. 3 6 9 5 - 3 6 9 9 H o l d e r . A ~ , \ a n d I i c e m a n , R R t1'482i B i o s y n f l l c s i s a n d p r o c e s s i n g
ailtigci]

lcioglli/¢d

h', i i n m u l / c

SOltim and a m o t l o c l o n a ]

allti'r~ld',

I

Exp. M e d .

l Sf;

1528-153~4 3

tlo,a, ard. R I

4

( t9~44} Swnlhcsis o l ttlc'rozoilc" p r o l e m s {ind gl}~opti,tc'i~l'., dttrlllg tt>

{lllti it)e'~c~, 14.[

s c h i z o g o n 3 o l t~[zl,,mtJdit4m

ialciparum

Mol

BiochenL

ttle ervthrol_;tic stage', o l t t l e htllllitl~ illaiaFla parasite a n t i b o d i c > . M o l . t'~i~,.hc'll/. 5

PalaSit,,t

Plasmodhon/alcipa#umdctccW,.t b,~ illt/llOCl
-, 247-2f~5

Ncwbohl, ( I

",ch~xc~ Nit. Bo\lc. !).P;. M c B r i d e . . I S

K.N. I]984j

pc,..ii~lc

Parasitol

6

Pal-asito]. 10. 3 lk~- i tJ.

Hall. R.. McBride-. I . M o t g a l l . ( i . 1 air. ,X.. Z o l g . . i . W . . \Vallik,_'r. I). and ~>oail0..I i l~<3i ,\lltlgt.TP, <,I

\

iilolccuial

M c L e a n . , \ . *\i!.,,>~

ba!,i. Ior nlltll[i spccitic lllllllCllll(k

R I,~1 ,tnd Bt~,x;i*.

t, ltlidFId

~<1~,[ Bi,,clic'ni

I1, ~3 ? t,.~7

( 1984i *\ h i g h m o l e c u l a l \,,tight ~lil{i~t'll ii1 ]>/H~tIIfJ

S a u l . : \ . . Mylu'r. P . >,cholicld. 1 . ;tnd lkid:,t,l]. (

diumjalcipa#um rcc~,Mmzcd by i n h i b i t o i 3 n l o n o c l o n a l antibodies. Parasite [ m l / ! t i i l o l (,. t9-50 7

Flet'nlaI~. R.R. :ll/ti tt~*ldci, i\ ,% (lu)~<3) <<,ulface ctntigens ol m a l a r i a iliel~ZOltc's, -\ lligh il/cJtt_,v'tllai

~eight plu'c'tll n~ll - I)l OutsSSt:Cl II, a13 ,R.~,[)()0 n]ol ~,t [()l'l]l L'XpI'Usscd OIl lilt' .tlFlacc' t,l /~l(1.~#llOdllll#Z

/alciparum ll/t.logt)Hv-~..l 8

Hall, R . O s l a n d polymoTphisnl,

~,

l',Xp. Mc'd 15S, 1 6 4 7 - 1 6 5 t

il)dc. I I

Simmoi>,

I).I

ol Plasmodmm fill~ llselrul#? M o l . l'liod~em, l)arasitol 9 11,}

tl,g~,.', l & .

ai'ld Sc&lHc: I { ;

and i~iological >,igllilicallCc o f P191). ~l iI/a]oi -,illfLit\." an{igCll

Trager. \\:. ;llld .l,.l/>,cn.

I.B. ( 19761 t l u m a n

673-67g R e c s c , R I., I . a n g i c t h , S.( i. n n d

!I,}S-I'! t>rocc,,,.mg.

,i {he e l ; lhi
I1. ¢~I-S0.

i l l a l a r i a i parasites m tZtlll[lll/lt)t!, ctllillr~'

v%it~llcc' I ql~

[ l a g e r . W. ( 19791 I s o l a t i o n ot s t a g e s oi the m i m a i i pai!alsitu' t'l,l*l#l+~

dium,/'~Ueipatu.~ Il,qii ccilttliC ai~d lroli~ animal blood. B u l l Wt I O 57 C<,iiF,pi I I - t ~, 11

tto,aard, R l . . :-;taiHc,,

l lA

. ('alnpbcll. ( l . l ] .

ptillctalt: llLiorcscCllt.c paltu'li1 lit

12

('ampbcll. tilt.

M,IIc~. 1 .1t.. thldsOll. I ) ,

a l l l i b o d v ctlaiLictt. 'l'/,ectlittl] ot

and Rcu',.v. i~. 1. (19841 Pr~>ivin. ~..ponsiblc ~,* ,,

t'[a';lnodium lgUciparu#n. &m. I I'rop M c d l l>g. 3 ~,. 1055 1i)5 c) l-rallCO, l . I .

PiNct#lONHtl+lllfliCip~lrigltt

and :\ildrVslcik

P \ ' I . ( Iqb<4l .Mon<,cl,mal

{lilt i~cr> ~ m . . I I-r,,l~ Mc'd t I \ 7 I -, t051 - 1054

77 13 14 15 16 17 18

19

20 21 22 23 24

25 26 27 28 29

30 31 32

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