00ZZ-1767/85/1343-1946$02.

00/0
THEJOURNAL OP IMUUNOL~CY Voi. 134. No. 3. March 1985
Copyrlght Q 1985 by The American Assodatlon of Immunoioglsts Prtnted In U.S.A.

CHARACTEFUZATION WITHMONOCLONAL ANTIBODIES OF A SURFACE

ANTIGEN OF PLASMODIUM FALCIPARUM MEROZOITES'

PHILIPPE J. PIRSON AND MARGARET E. PERKINS

From theRockefeller University. 1230 York Avenue, New York, NY 10021

The merozoite, the extracellular form of the eryth- proteins of 155,000 and 130,000 m.w. on the merozoite
rocyte stage of the malarial parasite, invades the surface that bind to glycophorin (6).Immunocytochem-
erythrocyteanddevelopsintracellularly. Cloned istry demonstratedthat theglycophorin-binding proteins
hybridomacell linessecretingmonoclonalanti- are a component of the merozoite surface coat (7).
bodies directed against the merozoite surface were Several othermerozoite surface proteinsof P .falcipa-
selected by indirect immunofluorescent assay by rum have been identified (8-12). and it is possible that
using intact isolated merozoites. Monoclonal anti- some of these areinvolved in interaction withthe eryth-
bodies to a200,000 m.w. merozoite surface antigen rocyte. A merozoite antigen (m.w. 190.000 to 200,000)
were selected and were used to characterize this has been described in reports from several laboratories
protein and its role in erythrocyte invasion.Immu- (8. 9, 11, 12). In the present work, we used monoclonal
noelectron microscopy demonstrated that the anti- antibodies (McAb)*against the 200,000 m.w. antigen to
gen was located exclusively on the merozoite sur- additionally characterize the protein and its role in eryth-
face coat, distributed evenly over the entire surface.
The 200,000 m.w. protein incorporated [3H]glucosa- rocyte invasion.
mine, suggesting that it is a glycoprotein and could
be purified to homogeneity by using immuno-affin- MATERIALS AND METHODS
ity chromatography. Freshly isolated, invasive mer-Media and Reagents. RPMI 1640tissueculture medium was
ozoites retained the200,000 m.w. antigen, but the obtained from GIBCO. Grand Island, NY. Aprotinin, bovine serum
protein was rapidly cleaved to proteins of 90,000 albumin ( E A ) , deoxycholic acid, ethylenedlaminetetraacetic acid
and 50,000 m.w. when the merozoite was extracel- 401. (EDTA],gelatin, HEPES. and Tris. iodoacetamide, Nonidet P-40(NP-
phenylmethylsulfonylfluoride (PMSF), polyethylene glycol
lular. The 50,000 m.w. fragment was retained the 1500. protein A. pristane, andsome other chemicals were obtained
epitopebinding to monoclonalantibody 5Bl and from SigmaChemical Co., St. Louis, MO. Papain [Papaya latex) was
were labeled with [3H]glucosamine. Monoclonal anti- obtained from Worthington Biochemicals. Freehold, N J . Affi-gel 10,
bodies against the 200,000 m.w. antigen partially acrylamide. N.N'-methylene-bfs-acrylamide sodium dodecyl sulfate
(SDS] and all electrophoretic reagents were obtained from Bio-Rad
inhibited merozoite invasion into erythrocytes. Laboratories, Richmond,CA. Rabbit antisera specific for mouseIgG,
IgGl, IgG2a. IgG2b. IgG3, IgM. and fluorescein-isothiocyanate-con-
jugated rabbit anti-mouseI g G were fromCappel Laboratories, Coch-
The merozoite is the extracellular stageof the malarial ranvtlle, PA. ~-(~HHjClucosamine (IO to 30 Ci/mmolJ, ~ - [ ~ ~ S ] m e t h i o -
nine (>400Ci/mmol], ~-[~H]proline (15to 30 Ci/mmol], and Enhance
parasite that invades the erythrocyte. It is 1.5 &m in were obtained from New England Nuclear. hston, MA. The m.w.
diameter and is surrounded by a double bilayer mem- markers were purchased from BRL Inc.. Gaithersburg. MD.
brane, covered with a surface coat (1). The interaction Cultfvatlon a n d labellng of Plasmodlumfalciparum. The FCR-3
(Gambia] strain of P. falclparurn was cultued in vitro in human
between the merozoite and its host cell begins with the erythrocytes (13) in RPMI 1640-HEPES medium supplemented with
binding of the parasite tospecific receptors on the eryth- 10% human serum and20 mM glucose. Parasite development was
rocyte surface (2).In the caseof Plasmodiumfalciparurn, synchronized by repeated sequential treatmentwith gelatin(14) and
sorbitol (151. The cultures were treated with 5% sorbitol 3 hr after
the causative agentof a human malaria, the receptors on the appearanceof the firstrings.
the erythrocyte have been identified as glycophorin A Forbiosynthetic labeling. schizont-infected erythrocytes were
and B (3-5). Interest has centered on the identification concentrated to greater than 80% arasitemia with gelatin 38 hr
of merozoite surface antigens that are involved in the after the last sorbitol treatment. P
[ HJProline (100 rCi/ml) or [3H]
glucosamine (100 rCi/ml] were added, 4 hr after gelatin treatment,
interaction of the parasite with the erythrocyte during for 1 hr. [35S]Methionine(10 pCi/ml) was added. 4 hr after gelatln
invasion. In the host, such antigens could be the target treatment, for 2 hr. Typically, merozoite release began 2 hr after
of antibody-mediated immunityagainst theasexual labeling, and themedium was changed at thattime. Merozoites were
collected from the supernatant of cultures agitated for 30 min. 1 hr.
erythrocytic stageof malaria. Recently we have identified or longer. The procedure for collection of merozoites has been de-
scribed (3, 5).
Productfon of McAb. Female BALB/c mice (The JacksonLabora-
Received for publication Aprll23. 1984. tory. Bar Harbor, ME) were immunized with a membrane-enriched
Accepted for publication October23. 1984. fraction of isolated merozoites prepared as follows. Freshly isolated
The costs of publication of this article were defrayed in part by the (1 O ' O cells) were resuspended in 1 m10.25 M sucrose in
payment of page charges. Thls article must therefore be hereby marked merozoites
advertisement in accordance with 18 U.S.C. Section 1734 solely to lndi- phosphate-buffered saline (PBS)and were homogenized in a Potter-
cate thls fact. Elvehjem homogenizer with a tightly fitting Teflon pestle. The mer-
' This investigationreceived financial support from the United Nations ozoite suspension was layered directly on a 17-1111linear sucrose
Development Programme/World Bank/World Health Organization Specialgradient (10 to 60% sucrose in 50mM Tris-HC1. pH 7.2. with 1 mM
Programme for Research and Training in TropicalDiseases.National EDTA) and was centrifuged a t 27.000 x G for 2 hr at 4°C in the
Institutes of Health Grant Ab19585 to M.P. and National Science Foun- vertical rotor TV 8658. The fractions corresponding to 35 to 40%
dation Grant PCM-800713 toDr. C. de Duve. P. P. I s a postdoctoral fellow
of the Rockefeller University-InternationalInstitute of Cellular and Mo- * Abbreuiatlons used in this paper: IFA, indirect immunofluorescence
lecular Pathology Collaborative Programme. assay: McAb. monoclonal antibody.
1946
 Plasmodium fakiparum MEROZOITE SURFACE ANTIGEN 1947
sucrose contained parasite membranes. These were pooled. dialyzed, to theantibody were eluted with 0.3ml of a buffer containing 0.2N
and used as immunogen. Mice were injected i.p. with the membrane acetic acid, 0.15 M NaCI. and 20mM Tris-HC1. pH 2.0 (buffer B).
fraction (250 g protein) three times at 2-wk intervals. Four days SDS-polyacrylamidegelelectrophoresis. Labeled samples of
before fusion, the mice received a n i.v. injection of 100 g of the parasites were boiled for 2 to 5 rnin in sample buffer (0.1 M Tris-
membrane fraction. HCI.pH 6.8, 10% glycerol. 100 mM dithiothreitol, 2% SDS, and
Hybridoma cell lines were prepared by fusing spleen cells from 0.001%bromophenol blue). The samples were electrophoresed on a
immunized mice with the NS-1 myeloma cell line in the presenceof 5 to 15% polyacrylamide gel by using the buffers of Laemmli (22).
polyethylene glycol 1500 (16). Positive hybrids were screened for The gels were then treated with Enhance before drying for exposure
antibody secretion by a solid-phase ELISA (17) by using merozoite to X-omat AR 5 film (Kodak. Rochester, NY) (23).
extracts as antigen. Positive cellswere cloned twice and cloned Inuaston assay. Mature schizonts (42 to 44 post
hr invasion) were
hybrids secretingMcAb against themerozoite surface were selected diluted with erythrocytes to a final 2% hematocrit and 1 % parasit-
by immunofluorescence. emia. Aliquots (1 ml) of this suspension were placed in 24-well
Spent medium from in vitro-grown cloned hybrid cultures or microtiter plates and were incubated for 1 hr In a candle jar. The
ascites fluid from pristane-primed BALB/c mice bearing the hybrid- medium was thenreplaced with 200 pl of fresh medium containing
omas were used as the sourceof McAb. Ig isotypes of secreted McAb 50 pg (250 d m l ) or 100 pg (500 pdml) of purified antibody. The Ig
were determined by the dot-immunobinding assay on nitrocellulose fraction from ascties fluid was purified by ammonium sulphate
membranes (18). precipitation and Sephadexchromatography(24).Eachinvasion
Indirect immunofluorescence a s s a y 1FA. Thin smears of P.fa1- assay was done in dupiicate. Smears were made 12 hr later, and
ciparum merozoites were made onmicroscope slides andwere fixed ring stage parasites were counted per 10,000 cells. In a second type
in methanol. Supernatants from hybridoma cells were layered over of assay, antibodies were added to merozoites isolated as described
the smear for30 min a t 37°C in a humidified chamber. After three (3.5).
10 rnin washes in Trager's buffer (19). the slides were covered with
fluorescein-isothiocyanate-conjugated. affinity-purified rabbit anti- RESULTS
mouse Ig diluted 1/1 in PBS for 30 min a t 37°C. The slides were
washed multiple times In Trager's buffer and then with distilled
water, andwere mounted with 50%glycerol in PBS. The slides were Selection of McAb directed against merozoite sur-
examined by a Zeiss epifluorescence microscope. f a c e . Hybridoma cell lines secreting McAb against P.
Immunoeiectron microscopy. To 1 ml of merozoite suspension falciparum were first selected by ELISA assay by using
containing about loQmerozoites, 1 ml of 1% glutaraldehyde in 0.1 lysed schizonts as antigen. Positive wells were tested by
M cacodylate buffer. pH 7.4 and 0.25 M sucrose were added and
allowed to stand at0°C for 15 min. Thecell suspension waswashed IFA by using fixed, intact, nonacetone-treated isolated
twice with PBS containing 3%gelatin and 1% BSA. The cell sediment merozoites. Those reacting with the merozoite surface
was resuspendedin 1.5 ml of undiluted ascitic fluid, was incubated were cloned twice by limiting dilution. Cloned cell lines
for 1 hr at37'C on a rotator, and was washed three times with PBS-
1% BSA (suspensions were incubated for 10 minbetween centrifu- were again tested by IFA. Three of four parent cell lines
gations). The sediment was resuspended in 200 plof rabbit-anti- positive by ELISA secreted McAb reacting with the sur-
mouse IgG diluted once in PBS- 1 % BSA. was incubated 30 min a t face only. This indicates that the membrane fraction
23°C. and was washed four times as above. The sediment was
resuspended in 300 pl of 20 nm gold particles bound to protein A used to immunize mice contained little nonmembrane
(20),and was incubated on a rotator 30 min a t room temperature, material. Hybridoma line 5B1 gave the strongest immu-
washed three times with PBS (5 min a t 500 x G). resuspended in nofluorescence, and secreted McAbof the IgM isotype.
0.5%glutaraldehyde in 0.25M sucrose and0.1 M cacodylate buffer,
pH 7.4. for 15 min, and then processed
was fortransmission electron Subclones of this linewere used in all subsequentexper-
microscopy (21). To determine the extent of nonspecific binding, iments. The fluorescence pattern characteristic of this
merozoites wereincubated with rabbit anti-mouse IgG and thengold- McAb was a ring of staining corresponding to the perim-
protein A and then were processed for transmission electron mi- eter of the merozoite (Fig. la).Over 90%of the merozoites
croscopy. Mature and broken schizontswere added to the sampleof
merozoites for electronmicroscopy to observe the antibody reactivity were labeled with the McAb. Acetone treatment of mer-
with the erythrocyte surface and intracellular structures. ozoites did not result in additional (i.e., internal) fluores-
Antigen solubilfzatlon. For immunoprecipitation and immunoaf- cence. The resolution of the light microscope, however,
finity purification, biosynthetically labeled parasite sedimentswere
extracted for 1 h r a t4'C in 5 vol of extraction buffer containing 1% does not allow one to localize the antigen recognized by
P-40 and 0.1% deoxycholate in PBSI buffer (PBS, pH 8.0, 5 mM 581 more precisely. To achieve this, merozoites were
iodoacetamide. 2 mM PMSF, 5 mM EDTA, 2 mM Na&O5, and 17 treated with 5B1 McAb, rabbit anti-mouseIgC, and then
m U approtinin). The extract wascentrifuged (10,000x G, 10 mfn,
4°C) to remove the insoluble material. protein A-gold, and were examined by electron micros-
Immunoprecipitation. Aliquots of 100 gl of formalin-fixed Staph- copy. A s shown in Figure 1b, gold particles were located
ylococcus aureus (Cowan I strain) (Calbiochem-Behring Corp., La only on the external surfacecoat of the merozoites. The
Jolla. CA) in 10% solution (v/v) were washed twice with washing
buffer (WB) (PBS. pH 8.0. 0.1% SDS, 0.1% BSA. and 0.5%NP-40) in particles appeared to bind to the surface coat material;
1.5 ml Eppendorf microfuge tubes. The sedimentswere resuspended areas of the membrane that were exposed were not la-
in 200 pl of rabbit anti-mouse IgG (diluted four times in PBS), were beled. In the broken merozoite shown in Figure 1b, there
incubated 30 rnin a t room temperature, andwere washed four times was no internallabeling with gold nor was therelabel on
in WB. The sedimentswere then incubated with 1 ml of hybridoma
supernatant for 2 hr at room temperature with rotation, were cen- the surface of the erythrocyte or knobs. There was no
trifuged, and were washed three times. The final sediments was gold label in control samples incubatedwith rabbit anti-
incubated with 250 plof solubilized antigen (1 to 5 X IO5 cpm), mouse IgG. McAb 581 reacted alsowith the external
diluted with PBSI buffer. overnight a t 4°C. and thenwere extensively
washed as follows: twice in WB, twice in PBS-0.5 M NaCI-0.1% NP- surface of schizonts, which were released from the in-
40. once in PBS-1% NP-40, and once in PBS. Bound material was fected erythrocyte and were free from the erythrocyte
released by boiling the sediments in 100 pl of Laemmli sample buffer membrane and the parasitophorous vacuole membrane
(22)for 5 min.
Immunoaflinity chromatography. One milliliter of washed Affi- (Fig. IC).Gold particles were distributed evenly over the
gel 10 (Bio-Rad)was gently mixed for 5 hr with 1ml of McAb solution. entire surfaceof the schizont (Fig. IC).
The McAb was a n ammonium sulphateprecipitate of culture super- Characterization and purificationof the antigen rec-
natant and contained 100 wg protein/ml in buffer A (0.2 M N a HC03, ognized by McAb 5B1. Immunoprecipitation of [35S]me-
0.3 M NaCl, pH 8.0). Affi-gel beads were washed twice with buffer
A and were treated with 0.1 M ethanolamine for 1 hr. One milliliter thionine-labeled schizontsand merozoites with McAb
of biosynthetically labeled parasite material was added and then 581 is shown in Figure 2. From schizonts, the intrae-
mixed for 1 hr. The suspension was centrifuged and the supernatant rythrocytic stage, a single protein of 200,000 m.w. (Fig.
containing the nonbinding fraction was removed. The beads were
washed once with 20 mM Tris-HC1, pH 7.5, 0.5 M NaC1, and once 2, lanes b and c] was immunoprecipitated, but from
with 20 mM Tris-HC1, 0.15 M NaCl. The antigensspecifically bound merozoites two proteins of 200,000 and 50,000 m.w.
1948 Plasmodium
Jalciparum MEROZOITE SUKFACE ANTIGEN

a b c d e f a b c d e f g h i j k

200-

94-

43- '*
f

t
f-
t
68-

25- 43-

E'i</urc,2. Immunoprrripitatr withMrAb of [35S]methionine-labrlrd

:chizonts and mrrozoitrs. a. Total ["5S]methioninr-labrlrd schizonts: h.
inlnl~lnoprrripitatc withMrAh 5Hl .A8: c. immunoprrripitatr with McAb
5t3I .G I O : d . total [35S)mrthianinr-laheled merozoite. rollrrted 1athr from
sc~hizonts ina : c'. imrnunoprrripitate with McAb 5R1 .A8:J immunoprc-
cipitatr with MrAb 5H1 . C I O .
were immunoprecipitated.By using McAb coupled to Affi-
gcl beads. it was possibletopurify the 200.000 m.w.
protein from solubilized schizonts (Fig. 3, lane h). The
200.000 m.w. protein incorporated ["Hlglucosamine (Fig.
3 . lanes a and e) and ["Hjproline (Fig. 3 , lanes h and i).
When solubilized merozoites were added to McAb-Affi-
gel. the 50,000 m.w. protein and a number of lower m.w.
proteins were eluted with buffer B (pH 2.0) (Fig. 3. lane
k ) . Because the merozoites were collected from the same
25-
Figure 3. (3H]Clucosamine- and [3H]proline-labeled antigen binding to
MrAb 5 R 1 roupled to Affi-gel. Schizont-infected erythrocytes (42 hrpost
invasion) were labeled for1 hr with [3H]glucosamine(a tog. and k)or ["HI
proline ( h t o j ) . a , Total [3H]glucosamine-labeled schizonts addedto Affi-
gel-McAb: b. fraction not binding toAffi-gel-McAb: c. merozoites collected
at 2 h r from ~3H]glurosamine-labeled schizonts (rollertion began at 45 hr
post invasion) and added to McAb Affi-gel beads: d . merozoite fraction
not binding to McAb: e. schizont antigen eluted from McAb Affi-gel wtth
0.2 Nk acetic arid: g . (k)merozoite antlgen eluted from beads with 0.2 N
acetic acid (redured):-f.If and g ) merozoite antigen eluted and merozolte
extract incubated with Affi-gel McAb in the absence of protease inhibi-
tors: ( h )[3H]proline-labeled schizont antigen eluted fromMcAb 5R1-Affi-
gel with 0 . 2 N aceticarid: 1. [3H]proline-labeledschizontfraction not
binding to Affi-gel MrAb 5R1:j. total [3H]proline-labeled schizonts.
 Plasmodiumfalciparurn MEROZOITE SURFACE ANTIGEN 1949
pulse-labeled schizonts. allof the cleavage products must a b c d
be derived fromthe 200.000 m.w. protein. When protease
inhibitors were omitted from the solubilization buffer,
the 50,000m.w. protein was cleaved additionally (Fig. 3.
lanes g and h)yet still retained the [3H]glucosamine label. -200
The major ["H]glucosamine-labeled protein (m.w.60,000)
did not bind to the McAb-Affi gel column and appeared
unaltered in merozoites. The results shown in Figures 2
and 3 suggest that the 200,000 m.w. antigen is cleaved
to a protein of 50.000 m.w. and several others of lower
m.w. at the schizont stage. -67
Time course of proteolysis of 200,000 m.w. antigen. " 0
To examine the time course of proteolysis of the 200.000
m.w. protein, schizont andmerozoite samples were ana-
lyzed directly without immunoprecipitation by electro- c-43
phoresis. After collection, all samples were boiled imme-
diately in sample buffer and were applied to SDS-PAGE.
Schizonts were labeled for 1 h r with either ["Hjproline
(Fig. 4. lane a and Fig. 5, lane a) or [3H]glucosamine(Fig.
5, lane a) and were returned to culture until merozoite
release began,a t which timea schizont sample was taken
(Fig. 4. lane b. and Fig. 5, lane b). The majorprotein
labeled with proline was the200.000 m.w. antigen. Mer-
ozoites were collected from the cultures for30 min (Fig.
4, lane c) and 3 h r (Fig. 5, lane d).In merozoites collected
foronly 30 min, there was a smallreductioninthe Flgure 5. Pulse-chase ai [3H~glucosamine-labeled schizonts. a. schi-
labeled for 1 hr with [3H]glucosamine(100 pCi/ml): h. schizonts 2
200.000 m.w. protein, and proteins of 90,000and 50,000 zonts
hr after labeling. at time merozoite collection began: c. culture superna-
m.w. appeared (Fig. 4. lane c. indicated by arrows). Mer- tant from merozoites: d . merozoites collected from labeled schizonts over
ozoites incubated fora n additional 5 min showeda n even a 3 - h r period.
greater loss of the 200,000 m.w. protein (Fig. 4, lane e].
A protein of 130,000 m.w. wasfoundintheculture
supernatant afterremoval of the merozoites (Fig. 4. lanes
d and f). Washing of merozoites with PBS removed two

a b c d e f g h i j k I

-200

0 30 60 I20
TIME (MINI
Flgure 6. Effect of incubation and washing of merozoites on erythro-
cyte invasion. Isolated merozoites ( 1 0' cells) were incubated a t 37OC in
presence of culture medium for 0 to 120 min. After incubation. 50%of
the sample was added directly to erythrocytes (10' cells) and 50% was
washed with PBS and then added to erythrocytes. The percentage of
invasion represents the numberof rings per 10' erythrocytes. expressed
F i g u r e 4 . Time course of proteolysis of 200.000 m.w. antigen. Schi- as the percentageof the control infection (no incubation and no treatment)
zont-infected erythrocytes.42 h r post invasion. were labeled forI hr with that was 45 f 25 rings per IO' erythrocytes (mean f standard deviation.
100 pCi/ml of [3H]proline. Two hours after labeling. the media was re- n = 5):untreated merozoites, u, washed merozoites, -.
plared with fresh media and the cultures were returned to the candle jar
for 30 min. Merozoites released during this period were collected and proteins of 155,000 and 130.000 m.w. (Fig. 4. lanes g
divided into five aliquots for eachof the samples in c. e. g. I . a n d j. The
merozoites were removed from the media by centrifugation in a microfuge and h). The released proteins have been shown to be
and the supernatants were analyzed(d a n d f ) . a. Schizonts labeled for 1 merozoite surface proteins that bind to glycophorin (7).
hr: h. schizonts 2 hr later at the beginning of merozoite collection: c. In Fig. 4, lanes i to I), merozoites were incubated for 30
mrrozoites collected for 30 min: d . culture supernatant after removal of
merozoites: e . merozoites allowed to stand for 5 min a t roam temperature min in 0.2 ml of PBS in the presence and absence of
in RPMl 1640 and then centrifuged a second time:f. supernatant removed proteolytic inhibitors, Zn++(1 mM) and iodoacetamide (5
from merozoites in e : g . merozoites washed in PBS and centrifuged: h.
supernatant removed from washed merozoites: I . 0) a fourth and fifth
mM). In merozoitesincubatedwith the inhibitors, the
aliquot (representing only 50%the merozoites in c. e. and g ) were incu- 155.000 and 130.000 m.w. proteins remained with the
bated at 37°C for 30 min in PBS and Zn'+ ( 1 mM) and iodoacetamide (5 merozoite (Fig. 4, lane 1). and thecleavage of the 200,000
mM) u) or without inhibitors ( I ) . The supernatant was removedfrom
merozoites incubated without Zn++ and iodoacetamide (k)a n d with inhlb- antigen was partially inhibited.In the absenceof inhibi-
itors ( I ) . tors, the proteins werereleased into the buffer superna-
 1950 Phsmodium falcfparum MEROZOITE SURFACE ANTIGEN

tant (indicated by arrows in Fig. 4, lane k). The [3H] we characterized the 200,000 m.w. antigen additionally
glucosamine-labeled 200,000 m.w. proteinwas com- and assessed its role in merozoite invasion. Hybridoma
pletely lost in merozoites collected at 3 hr, and a labeled cell lines secreting McAb against the merozoite surface
band of 50,000 m.w. was present (Fig. 5, lane d). The were produced. The hybridoma line 5B1 secreted anti-
200,000 m.w. antigen was unaltered in the intracellular body against a 200,000 m.w. protein, and immunoelec-
schizont at the time of merozoite collection (Fig. 5. lane tron microscopy demonstrated that the antigen was lo-
b). Equal numbers of merozoites (approximately Os) 1 were cated exclusively on the merozoite surface coat and not
added to lanes c, e, and g in Figure 4. The large amount on the underlying cell membrane. The surface coat ma-
of protein in lanes d and j distorted lanes c, e, and g, terial wasloosely attached tothe membrane, and sheets
giving the appearance of unequal amount of 13H]proline of it appeared separatedfrom the surface.
label. The numberof merozoites in lanesi and j was 50% The protein also incorporated [3H]glucosamine,indicat-
of that in lanes c, e, and g, in Figure 4. ing that it was a glycoprotein; the [3H]glucosaminelabel
In another type of experiment, we examined the inva- was present in the 50.000 m.w. fragment, the epitope
siveness of merozoites incubated for various times and binding to the McAb. In previous experiments we found
in which increasingproteolysis of the 200,000m.w. pro- that isolated merozoite had lost the high m.w. glycopro-
tein occurred. The aged merozoites were considerably less teins synthesized in the schizont, and low m.w. break-
invasive than nonincubated merozoites; the rate of in- down products remained on the merozoite (9).We now
vasiveness of merozoites incubated for 120 min was only consider that thisobservation wasa consequence of pro-
23% of the control (Fig. 6).When merozoites were washed teolysis or excessive washing or merozoites before anal-
before being added to erythrocytes,the invasion rate was ysis by electrophoresis. As shown in the present work,
even lower, particularly after 60 min. Figure 6 presents washing of merozoites in buffer accelerates the break-
the resultsof five separate experiments. down of the 200,000 m.w. protein (Fig. 4, lane e). From
Inhibition of merozoite invasion by McAb 5B1 and electron microscopy studies it appeared that the loosely
rabbit antiserum. The effect ofMcAb 5Bl on invasion attached surface coat material was lost from merozoites
into erythrocytes was tested by adding the antibody to treated in the same manner. In addition, we found [3H]
both isolated merozoites and matureschizonts,and glucosamine-labeled protein at theschizont stage suscep-
counting the ring stages formed 12 or 14 hr later. The tible to pronase (9).This was probably a result of eryth-
results of these experiments are shown Table in I. McAb rocyte lysis caused by the high concentration of enzyme.
5B1 (250 pg/ml) reduced merozoite invasion by approxi- The high m.w. antigen (m.w. 195,000) identified by
mately 45% in four separate experiments. No inhibition Freeman and Holder (8)appears to be analogous to the
was observed in thepresence of antibodies from the 7H6 200,000 m.w. antigen identified here. Small differences
line, which did not bind to the merozoite surface. Com- in m.w. may be due to different strains used. Serologic
parable rates of inhibition were seen whether isolated cross-reactivity wasobserved between the 195,000m.w.
merozoites or mature schizontswere used. P.faZciparurn antigen, a 230,000 m.w. PZasmodfum yoe-
lii, and a 250,000 m.w. Plasmodium chabaudi antigen
DISCUSSION (25). A high m.w. (250,000)antigen of P . knowlesi,
The initial step in the complex process of invasion of shown by immunoelectron microscopy to be on the mer-
the malarial merozoite into erythrocytes is the attach- ozoite surface (26, 27),also belongs to the same classof
ment of the merozoite to specific receptors on the eryth- antigens.Freeman and Holder (8) showed thatthe
195,000 m.w. antigen present in the schizont wasproc-
rocyte surface. Plasmodium falciparum merozoites ap-
pear to recognize and attach to glycophorin A and B on essed or cleaved to proteins of 83,000 and 60,000 m.w.
on themerozoite surface (8,28). Theinfectivity of these
the erythrocyte surface (3-5). Merozoite surface proteins
that interact with theerythrocyte are of interest because isolated merozoites was not reported (28).We also found
antibodies to these components could block parasite in- proteins of 90,000 and 50,000 m.w. on the merozoite
vasion of its hostcell. that were immunoprecipitated by the McAb and thus
An important antigen of Plasmodium falciparum is must be derived from the 200,000 m.w. antigen. How-
the high m.w. protein, (190.000 to 200,000)identified by ever, we found that in freshly isolated merozoites, mero-
McAb in several laboratories(8.1 1). In the presentwork, zoites collected at 30 min, the 200,000m.w. protein was
retained with little proteolysis. We observed that after
TABLE 1 merozoites were isolated, the loss of the 200,000 m.w.
Inhibition of P. falcioarum merozoite fnuasion bu McAb protein was very rapid. Washing merozoites in buffer
Antibodya
Precipitates the Percent Inhibition of lnvaslonb appeared to increase proteolysis.
200.000 m.w.
(250 rg/ml) Antieen Expt. 1' Expt. 2' Expt. 3d Expt. 4' In the experimentshownin Figure 4, there was a
medium
Culture - 0 0 0 0
marked loss of the protein within a 5-min incubation,
7H6.A3 (IgC2a) - 5 6 8 - although in other experiments (not shown here) no loss
581 .A8 (IgM) 42 45 + 46 40 was observed until after1 hr of incubation. In merozoites
5Bl.B5 (IgM) + 23 31 45 -
5Bl.GlO (IgM) + 50- 43 41 collected at 3 hr, loss of the 200,000 m.w. protein was
McAb purified fromascites fluid a s described inMaterials and Meth- complete (Fig. 5).The ability of merozoites incubated for
ods. various times after isolation to invade erythrocytes was
bThe control rates of invasion were 1 9 . 23, 18. and 25 rings per IO3 tested. Because freshly isolated merozoites were more
erythrocytes for experiments 1 , 2, 3, and 4, respectively.
Invasion assay involved incubationof mature P.falciparum schizont- invasive than aged merozoites, it would appear that proc-
infected erythrocytes(0.7%parasetemia) with antibody andfresh eryth- essing or cleavage of the 200,000 m.w. protein to low
rocytes. Ring stages were counted 12 hr later. m.w. fragments is not a requirementfor the formation of
Isolated merozoites (10' cells) were Incubated with McAb before ad-
dition to erythrocytes (10'). and ring states were counted 4 hr later. viable, invasive merozoites. Conversely, the presence of
 Plasmodium fakiparum MEROZOITE
SURFACE ANTIGEN 1951
development. In Malarfa and the Red Cell. Pitman,
200.000m.w. protein does not confer infectivity, because rythrocytic London (Ciba Foundation Symposium 94).P. 45.
only 5 to 20% of the total number of merozoites isolated 2. Miller, L. H..F. M. McAuliffe. and S. J. Mason. 1977. Erythrocyte
during a 1-hrperiod were infective (TableI and Fig. 6).It receptors for malaria merozoites. Am. J . Trop. Med. Hyg. 26:204.
3. Perkins, M. 1981.The inhibitory effects of erythrocyte membrane
is more likely that theloss of infectivity is correlated with proteins on the in ultro invasion of the human malarial parasite
the shedding of the glycophorin-binding proteins, be- (Plasmodlumfalclparum) into its host cell. J . Cell. Blol. 90:563.
cause more than 50%of these proteins were found inthe 4. Pasvol, G.. J. S. Wainscoat. and D. J. Weatherall. 1982.Erythro-
cytes deficientin glycophorin resist invasionby the malarial parasite
supernatant after isolation of merozoites (7). The sensi- Plasmodlumfalciparum. Nature297~64.
tivity of the glycophorin-binding proteins to buffer wash- 5. Perkins, M. 1984.Binding of glycophorins to Plasmodiumfalclpa-
rum merozoites. Mol. Biochem. Parasitol. 10:67.
ing (6, 7) may explain the lower infectivity of washed 6. Perkins. M. E. 1984.Receptor mediated endocytosis of the malarial
merozoites shown in Figure 6. parasite by human erythrocytes. In The R e d Cell: SOrth Ann Arbor
The role of these surface proteins merozoite
in invasion Conference. G.J. Brewer. ed. A. R. Liss. New York. Pp. 361-376.
7. Perkins, M. E. 1984.Surface proteins of Plasmodium falclparum
is notunderstood. Aikawa and colleagues (29)have merozoites binding to the erythrocyte receptor glycophodn. J. Exp.
shown by electron microscopy that the surface coat of Med. 160: 788.
the merozoite forms the attachment site with the eryth- 8. Freeman, R. R., and A. A. Holder. 1982.Biosynthesisand processing
of a Plasmodium falciparum schizont antigen recognized by im-
rocyte surface, but is shed during the invasion process. mune serum andmonoclonal antibody. J . Exp. Med. 156:1528.
We have found that thegycophorin-binding proteinsare 9. Perkins. M. 1982.Surface proteins of schizont-infected erythrocytes
shed during invasion,and it is possible that the200,000 and merozoites of Plasmodiumfalciparum. Mol. Bfochem. Parasi-
tol. 5:55.
m.w. protein is also cleaved during this process. The 10. Perrin. L.H., and R. Dayal. 1982.Immunity to asexual erythrocytic
extreme lability of both these proteins may be related to stages of Plasmodiumfalciparum: role of defined antigens in hu-
moral response. Irnmunol. Rev. 61:245.
the fact that they have to be removed rapidly from the 11. Hall. R., J. McBride, G. Morgan, A. Tait, J. W. Zolg, D. Walliker,
merozoite during invasion. Shedding of the merozoite and J. Scaife. 1983.Antigens of the erythrocytic stages of the human
surface coat may affect theredistribution of glycophorin malaria parasite Plasrnodlumfalciparum detected by monoclonal
antibodies. Mol. Blochem. Parasitol. 7:247.
receptor molecules during invasion. That cleavage of the 12. Heidrich. H. G., W.Shrych. andJ. E. K.Mrema. 1983.Identification
200,000 m.w. protein could be linked to the release of of surface and internal antigensfrom spontaneously released Plas-
the glycophorin-binding proteins is suggested by the ob- modiumfalciparum merozoites by radio-iodination and metabolic
labeling. 2. Parasitenkd. 69~715.
servation that treatment of merozoites with Zn++ and 13. Trager. W.,and J. B. Jensen. 1976.Human malaria parasites in
iodoacetamide, inhibitors of sulhydralproteases, par- continuous culture. Science 193~673.
tially inhibit both the breakdown of the 200,000m.w. 14. Pasvol, G.. R. J. M. Wilson, M. E. Smalley, and J. Brown. 1978.
Separation of viable schizont-infected red blood cells of Plasmodium
protein and therelease of the 155.000 and 130.000m.w. falciparurn from human blood. Ann. Trop. Med. Parasitol. 7287.
proteins into the supernatant(Fig. 4). Because it was not 15. Lambros. C., and J. P. Vanderberg. 1979.Synchronisation of Plas-
rnodfum falclparum erythrocytic stages in culture. J . Parasltol
possible to achieve complete inhibition, we assume that 65418.
more than one enzyme is involved in the cleavage and 16. Kennett, R H.. K. A. Denis. A. S. Tung, and N. R. K l i i a n . 1978.
release process. The involvement of more than one en- Hybrid plasmacytoma production: fusions with adult spleen cells,
monoclonal spleen fragments. neonatalspleencells andhuman
zyme has also been proposed by others (27). spleen cells. Curr. Top. Microbiol. Immunol. 8:77.
McAb directed against the 200,000m.w. protein par- 17. Spencer,H. C., W. E.Collins, W.Chin, and J. C. Skinner. 1979.The
enzyme-linked immunoabsorbent assay (ELISA)for malaria. 1. The
tially blocked merozoite invasion. We have shown previ- use of in ultro-cultured Plasmodium falciparurnas antigen. Am. J.
ously that chymotrypsin treatment of merozoites selec- Trop. Med. Hyg. 28:927.
tively removes a 200,000 m.w. protein and a 130,000 18. Beyer, C. F. 1984. A *dot-immunobinding assay” on nitrocellulose
membrane for the determination of the immunoglobulin class of
m.w. protein from the surface, rendering the merozoite mouse monoclonal antibodies. J. Irnmunol. Methods 67:79.
incapable of erythrocyte invasion (9). Thus, it is possible 19. Trager, W. 1959.The enhanced folic and folinic acid contents of
that the200,000m.w. proteins are required in merozoite 20. Bendayan, erythrocytes infected with malaria parasites. Exp. Parasitol. 8~265.
M., J. Roth, A. Perrelet, and L.Orci. 1980.Quantitative
invasion and may interact with components of the eryth- immunochemical localization of pancreatic secretory proteins in sub-
rocyte surface. However, the 200,000m.w. protein does cellular compartments of the rat acinar cell. J. Histochem. Cyto-
not bind toglycophorin (6,7). It is possible that theMcAb 21. Langreth, chem. 28: 149.
S. G., J. B. Jensen, R. T. Reese, and W.Trager. 1978.
inhibits invasion by disrupting the association of the Fine structure of human malaria in ultro. J . Protozool. 25443.
200,000m.w. protein with the glycophorin-binding pro- 22. Laemmli. U.K. 1970.Cleavage of structural genesduring the assem-
bly of the head of bacteriophage T4.Nature 227:680.
teins. Additional experiments will be necessary to inves- 23. Laskey. R. A., and A. D. Mills. 1975.Quantitative film detection of
tigate the nature of organization of the 200,000 and ’H and I4C in polyacrylamide gels by fluorography. Eur. J. Biochem.
155.000m.w. proteins in the merozoite surface coat and 56:335.
24. Garvey, J. S.. N. E. Cremer, and D.H. Sussdorf. 1977.Methods in
their role in merozoite invasion. Immunology: a Laboratory Text for Instruction and Research. W.
A. Benjamin, Inc.. Reading, Massachusetts.
25. Holder, A. A.. R. R. Freeman, and C. I. Newbold. 1983.Serological
Acknowledgments. We would especially like to thank cross-reaction between high molecular weight proteins synthesized
Helen Shio for her expert assistance in theelectron mi- in blood schizonts of Plasmodium yoelli, Plasmodium chabaudl.
croscopic studies, Drs. Donald Harn (Harvard University and Plasmodiumfalciparum. Mol. Blochem. Parasitol. 9: 191.
26. Epstein. N., L. H. Miller, D. C. Kaushel, I. J. Udeinya, J. Rener, R.
Medical School) and Carl Beyer for their advice on the J. Howard, R. Asofsky, M. Aikawa, and R.E. Hess. 1981.Mono-
development of hybridoma cell lines, Dr. Miklbs Muller clonal antibodies against a specific surface determinantof malarial
for critical reading of the manuscript, and Drs. Peter (Plasmodlum knowlesi] merozoites block erythrocyte invasion. J.
Immunol. 127;212.
David and Louis Miller (National Institutes of Health) for 27. David, P. H.. T. J. HadIey, M. Aikawa. and L. H. Miller. 1984.
discussion of their results on P. knowlesi surface anti- Processing of a major parasite surfaceglycoprotein occurs during the
ultimate stages of differentiation in Plasmodium knowlesl malaria.
gens before publication. Mol. Biochem. Parasitol. 11:267.
28. Freeman. R. R., and A. A. Holder. 1983.Surface antigensof malar-
REFERENCES ala merozoites. J . Exp. Med. 158: 1647.
29. Aikawa. M.. L. H. Miller, J. Johnson, andJ. Rabbege. 1978.E M h -
1. Aikawa, M.. and L. H. Miller. 1983. Structural alteration of the rocyte entry by malaria parasites. A movingjunctionbetween e%h-
erythrocyte membraneduring malaria parasite invasion andintrae- rocyte and parasite. J. Cell. Blol. 77~72.