Molecular and Biochemical Parasitology, 25 (1987) 165-174 165

Elsevier

MBP 00846

Localization of Plasmodium falciparum histidine-rich protein 1

in the erythrocyte skeleton under knobs
D i a n e W . T a y l o r 1, M a r c e l a P a r r a 1, G e o r g e B. C h a p m a n 1, M a r k E, S t e a r n s 2,
J o a n R e n e r 3, M a s a m i c h i A i k a w a 4, S h i g e h i k o U n i 4, S t e p h e n B. A l e y 5,
L i n d s e y J. P a n t o n 6 a n d R u s s e l l J. H o w a r d 6
1Department of Biology, Georgetown University, Washington, DC, U.S.A.; 2Pharmacology Department, Fox Chase Cancer
Center, Philadelphia, PA, U.S.A.; 3Hazelton Laboratories, Inc., Vienna, VA, U.S.A.; 41nstitute of Pathology, Case Western
Reserve University, Cleveland, OH, U.S.A.; 5Biomedical Research Institute, Rockville, MD, U.S.A.; 6Laboratory of Parasitic
Diseases, NIAID, NIH, Bethesda, MD, U.S.A.
(Received 16 March 1987; accepted 30 April 1987)

Plasmodium falciparum parasites that induce knobs in the host erythrocyte membrane (K + phenotype) synthesize a 90 kDa his-
tidine-rich protein (PfHRP-1), whereas knobless variants do not. A monoclonal antibody (mAb 89) to PfHRP-1, in combination
with cryo-thin section immunoelectron microscopy, localized the antigen in the parasitophorous vacuolar space and vesicles within
the erythrocyte cytosol. Additional immunoelectron microscopic studies showed that PfHRP-1 was also associated with submem-
branous electron-dense material under knobs and with microfilaments of the host erythrocyte skeletal network. Immunoftuores-
cence and immunoelectron microscopy of intact, non-fixed K + infected erythrocytes using mAb 89 and a rabbit antiserum raised
against purified PfHRP-1, failed to identify any surface exposed epitopes. These antibodies also failed to block cytoadherence of
infected erythrocytes to C32 melanoma cells or to affect macrophage phagocytosis of infected erythrocytes.

Key words: Plasmodium falciparum; Histidine-rich protein; Monoclonal anti-knob antibodies; Immunoelectron microscopy;
Erythrocyte skeleton

Introduction covered by the erythrocyte membrane [1,3,10-13].

The membranes of erythrocytes infected with
Plasmodium falciparum become altered morpho-
logically [1-3], antigenically [4-7] and function-
ally [8]. Some of these alterations are associated
with a parasite-induced structure called knobs [1,2,9-
11]. By transmission electron microscopy (EM),
knobs appear as protrusions composed of an
electron-dense inverted cup-shaped structure
Correspondence address: Diane W. Taylor, Dept. of Biology,
Georgetown University, Washington, DC 20057, U.S.A.
Abbreviations: EM, electron microscopy; HRP, histidine-rich
protein; IFA, indirect immunofluoreseent assay; K +, knobby
strains; K , knobless strains; mAb, monoclonal antibody; MC,
Malayan Camp; PBS, phosphate-buffered saline; PfHRP-1,
Plasmodium falciparum histidine-rich protein One; SDS-
PAGE, sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis; SL, Santa Lucia; FITC, fluorescein isothiocyanate.
Functionally, knobs are involved in mediating the
adherence of infected cells to capillary endothe-
lium [8,10,14,15].
Kilejian reported a correlation between the
presence of a protein rich in histidine (M r 80 000)
and the expression of knobs [16]. P. falciparum
produces at least three histidine-rich proteins
(HRP) [17-19]. Since the 'knob-associated' HRP
was described first, it has been designated PfHRP-
1 [19]. PfHRP-1 shows size diversity between iso-
lates of P. falciparum, and appears to be associ-
ated with the erythrocyte cytoskeleton [20].
The subcellular localization of PfHRP-1 re-
mains controversial. Several studies suggest that
it is a structural and/or functional component of
knobs [16,20-22]; however, one group has re-
ported the inability to identify PfHRP-1 in mem-
branes purified from knobby strain cells [23].

0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

166
Also, there is no consensus whether PfHRP-1 is
exposed on the cell surface or involved in me-
diating cytoadherence.
In this study, a monoclonal antibody (mAb 89)
specific for PfHRP-1 has been used to localize
PfHRP-1 and determine whether this protein is
involved in cytoadherence.
Materials and Methods
Parasites. Aotus trivirgatus monkeys were in-
fected with knobby strain (K +) or knobless strain
(K-) parasites of either the Malayan Camp (MC)
or the St. Lucia (SL) strains of P. falciparum [20].
Aliquots of cryopreserved blood containing ring-
stage parasites were thawed and cultured in vitro
[24]. Cloned K ÷ and K- parasites of the FCR-
3/IMA strain were obtained from T. Green (Uni-
versity of Missouri, Columbia, Missouri) and
maintained in continuous culture.
Production of mAb to PfHRP-1. A total of 9 fu-
sions using a variety of immunization schedules
and immunogens were required to produce a sin-
gle monoclonal antibody (mAb) to PfHRP-1. In
the successful fusion, immunization was as fol-
lows: a BALB/c mouse was immunized with ap-
proximately 107 K + SL-parasitized erythrocytes
emulsified in complete Freund's adjuvant, fol-
lowed by three boosts with 107 K + cells in incom-
plete adjuvant over a 4 month period, and a final
boost intraperitoneally with 107 cells 4 days prior
to hybridization with the P3-X63-NS/1-Ag.4.1
(NS/1) cell line [25]. Culture supernatants were
screened by a standard indirect radioimmunoas-
say using intact K ÷, K- and normal Aotus eryth-
rocytes as antigen. The hybrid cell line positive
for K ÷ but not K- infected cells was cloned twice
by limiting dilution and isotyped using an iso-
type-specific immunoassay [26].
Western blot analysis. Erythrocytes containing
late-trophozoites of P. falciparum were purified
on Percoll gradients to >t90% parasitemia [27].
Then, 108 infected or uninfected control cells were
solubilized with vortexing in 100 pA of sodium do-
decyl sulfate (SDS)-sample buffer and stored at
-90°C for <2 days until SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed [28].
Samples were incubated 10 min at 60°C before
loading. Electrophoresed proteins were blotted
onto nitrocellulose, and treated with a 1:50 dilu-
tion of serum, then probed with 125I-labeled pro-
tein A as described previously [17]. A parallel blot
was probed with a control mAb or normal mouse
serum. The distribution of a25I-radioactivity was
determined by autoradiography [29].
Production of rabbit anti-PfHRP-1 antiserum.
Triton X-100-insoluble material from Percoll-
purified, infected erythrocytes of K + MC strain
parasites was solubilized in SDS-sample buffer
and electrophoresed. The M r 92000 PfHRP-1
band was identified by Coomassie Blue staining,
excised, emulsified in Freund's complete adju-
vant, and injected into a rabbit to produce high
titer antiserum [30].
Indirect immunofluorescence studies. Hybridoma
culture supernatants, ascites fluid, and rabbit anti-
PfHRP-1 serum were examined by IFA analysis
using acetone-fixed smears of K ÷ and K- P. fal-
ciparum-infected erythrocytes according to the
procedure of Voller [31]. Surface fluorescence was
determined by incubating 50 txl of ascites diluted
in phosphate-buffered saline (PBS), pH 7.2, for
30 min with 25 p~l of a 10% suspension of infected
erythrocytes (>10% parasitemia). Cells were
subsequently treated with ftuorescein isothio-
cyanate (FITC)-labeled goat anti-mouse immu-
noglobulin antiserum. Wet mount preparations
were examined immediately using epifluores-
cence. To increase the sensitivity of the assay,
cells treated with mAb were sequentially incu-
bated in rabbit-anti-mouse immunoglobulin
serum, followed by FITC-labeled goat-anti-rabbit
immunoglobulin antiserum and examined as de-
scribed above. Preparations of erythrocyte cyto-
skeletons were prepared and processed for indi-
rect immunofluorescent assay (IFA) as previously
described [32].
Electron microscopic studies. The protocol for
cryo-ultrathin section immunoelectron micro-
scopy originally described by Tokuyasu [33] was
followed as outlined elsewhere [17]. In brief, P.
falciparum-infected erythrocytes were cultured in
vitro, aliquots were removed at 3, 14, 32 and 48

h, fixed in 0.25% glutaraldehyde/paraformal-
dehyde, frozen, sectioned approximately 450 nm
thick, and treated with mAb 89 or control sera,
followed by protein A-gold.
Intact, K + MC trophozoite-infected cells were
washed three times with PBS, and incubated 1 h
on ice with 50 txl of a 1:100 dilution of mAb as-
cites, normal mouse serum, hyperimmune serum
(from blood of mice used for fusion), or PBS.
After 3 washes with PBS, 50 txl of protein A-gold
(5 pxm diameter, prepared according to Muhl-
pfordt [34]), was added at 4°C for 1 h. Cells were
washed, fixed, embedded, stained and examined
by standard procedures [12].
K + and K purified, infected cells were also
treated with 0.1% (w/v) saponin in K-1 buffer [35]
(one volume cells to 2 volumes saponin) for 5 min,
centrifuged (18000 x g for 20 min) and washed
two times with K-1 buffer before incubation with
mAb 89 or control sera, followed by ferritin-con-
jugated rabbit-anti-mouse IgG (Cappell, Coch-
ranville, PA). Permeabilized cells were embed-
ded, stained and examined by standard
procedures [12].
P. falciparum-infected erythrocyte cytoskele-
tons were prepared for E M as previously de-
scribed [32]. In brief, infected erythrocytes were
applied to formvar-coated grids previously treated
with 0.1% poly-l-lysine, and extracted in situ with
0.1% Triton X-100 followed by 1% Triton X-100.
Extracted cytoskeletons were fixed with 0.1%
glutaraldehyde and then treated with 5% bovine
serum albumin, followed by a 1:200 dilution of
mAb or control sera for 30 min, and protein A-
gold [34]. Preparations were washed, fixed with
2.5% glutaraldehyde for 5 min, fixed with os-
mium tetroxide for 5 min, dehydrated in alcohol,
and subjected to critical point drying [36]. Prep-
arations were carbon coated and examined using
a J E O L S-100 electron microscope.
In vitro cytoadherence assay. MAb 89 was puri-
fied by protein A chromatography [37], dialyzed
against RPMI-1640 medium, and adjusted to 100
~g m1-1. IgG2a from normal mouse serum was
prepared identically. K + P. falciparum-infected
cells (MC, 15% parasitemia) were incubated with
100 txg m1-1 of m A b 89 or control IgG2a anti-
body, a 1:10 dilution of m A b 89 ascites or control
167
ascites, or 1:10 dilutions of rabbit anti-PfHRP-1
serum or control rabbit serum. Dilutions were
performed with RPMI-1640 medium. After 30
min at 37°C, the cells were added to formalin-
fixed C32 amelanotic melanoma cells for in vitro
assay of cytoadherence [38].
Macrophage phagocytosis. Aliquots of (100 Ixl) K +
infected erythrocytes were incubated 60 min on
ice with an equal volume (100 txl) of 1:50 dilu-
tions of normal mouse serum, mAb 89, control
ascites, or sera from African adults with high anti-
P. falciparum antibody titers. Cells were washed
and added to coverslips containing adherent mu-
rine peritoneal cells (obtained 2 h earlier from
normal BALB/c mice by peritoneal lavage) and
incubated for 1 h at room temperature. Cover-
slips were washed with PBS, briefly treated with
distilled water, dried, stained and examined. In
addition, antibody-treated infected-erythrocytes
were incubated in test tubes with normal or
thioglycollate-induced mouse peritoneal cells for
1 h at room temperature. Cells were washed and
smears prepared. The percentage of macro-
phages phagocytizing erythrocytes was deter-
mined by counting 500 macrophages.
Results
MAb 89 to PfHRP-1. Screening of approximately
2300 culture supernatants from 9 fusions identi-
fied one hybridoma cell line that secreted mAb to
PfHRP-1. This hybrid, designated mAb 89, was
cloned twice and determined to secrete an IgG2a
mAb.
PfHRP-1 has an M r of 92000 and 108000 in the
K + MC and SL strains of P. falciparum, respec-
tively, but is absent in K - parasites of these strains
[20]. MAb 89 recognized proteins of the appro-
priate M r in these K+-infected cells by Western
blot analysis (Fig. 1). MAb 89 did not bind to K -
or normal Aotus erythrocytes, nor did control
mAb or normal mouse serum react with K ÷
erythrocytes. Subsequent studies showed that
mAb 89 immune precipitated a protein of equiv-
alent M r from P. falciparum extracts metaboli-
cally labeled with [3H]histidine but not from ex-
tracts labeled with [3H]isoleucine (data not
shown).
168

< < with an African isolate (FCR-3). The K variants

t3,. O,. L) ¢J "~
of these strains and normal Aotus and human
erythrocytes were negative when treated with
~< < .,-, mAb 89.
L) td'~ ¢,.~ t..-
+ I + I t"- In synchronous cultures, PfHRP-1 could be de-
v v' 'v' v' :D
tected in young trophozoite-infected cells as bright
'patches' of fluorescence in the erythrocyte cyto-
sol and as diffuse staining in the vicinity of the
erythrocyte membrane (Fig. 2A). As the tropho-
200~
zoite matured to the stage of pigment accumula-
tion, only minute spots of mAb 89 fluorescence
were observed in the erythrocyte membrane (Fig.
108~ Q 2B). Infected erythrocytes remained positive
92~
throughout the remainder of the developmental
cycle (roughly 48 h), but mature schizonts and
66 ~ m
segmenters tended to be less bright. Cytoskeletal
preparations from K ÷ parasites were positive with
the predominant pattern being regularly-spaced
dots of fluorescence in the erythrocyte skeleton.
K cytoskeletons were totally negative (not
shown).
MONOCLONAL#89
Immunoelectron microscopic studies. Synchro-
Fig. 1. W e s t e r n blot analysis of m A b 89 with proteins from nized K + and K P. falciparum parasites were
K ~ and K P. falciparum-infected erythrocytes. M A b 89 re- examined by cryo-thin section immunoelectron
acted specifically with K + M C and SL parasite, but not with
microscopy using mAb 89 and relevant control
K- or normal cells. T h e lower band of Mr 66000 does not
represent a specific reaction since it was also present in the lane
ascites. PfHRP-1 could not be identified in K + and
containing erythrocytes. K - cells at 3 or 14 h of culture, or at 32 h in K-
cells (data not shown). However, the antigen was
Localization of PfHRP-1 by IFA analysis. By IFA readily identified at 32 h in K + parasitized cells
analysis, mAb 89 was found to bind to fixed (Fig. 3). Grains of gold were observed in the area
smears of K + erythrocytes, including Aotus of the parasitophorous vacuole apparently in as-
erythrocytes with parasite strains originally iso- sociation with both the parasite plasma mem-
lated from South East Asia (MC) and Central brane and vacuole membrane (Fig. 3A). PfHRP-
America (SL), and human erythrocytes infected 1 was also associated with membranous vesicles

Fig. 2. I F A results with P. falciparurn-infected erythrocytes of the K + phenotype. Photomicrographs show smears of (A) young,
i m m a t u r e K + trophozoite, (B) mature K - trophozoite, (C) cytoskeletons of K + trophozoite. Bar represents 5 ~m.
 169

Fig. 3. Immunoelectron microscopy on cryo-thin section of K ÷ P. falciparum-infected erythrocytes cultured in vitro 32 h and then
treated with m A b 89 and protein A-gold. (a) PfHRP-1 is not seen within the parasite (P) but is found external to the parasite
membrane (M) and in association with the parasitophorous vacuolar membrane (V). (b) PfI-IRP-1 is seen in membranous vesicles
and (c, d) in electron dense granules in the erythrocyte cytosol. (d, e) PfHRP-1 can be seen approaching the erythrocyte mem-
brane. Arrow identifies the erythrocyte surface (S). Bars represent 0.1 ~xm.
170
(Fig. 3 B , E ) and e l e c t r o n - d e n s e (Fig. 3 C , D ) gran-
ules in the e r y t h r o c y t e cytoplasm. P f H R P - 1 was
frequently o b s e r v e d near, or in opposition to, the
e r y t h r o c y t e m e m b r a n e (Fig. 3 D , E ) . H o w e v e r , in
this study, distinct k n o b structures were not seen
A L
k face of the erythrocyte m e m b r a n e and only a few
! I
B the host erythrocyte skeleton, cytoskeletal prep-
i~i!~~ iii~~ ~i~~i ~;;~~ ~~ ~~ ~:~ .... ~ ¢(!
Fig. 4. Localization of PfHRP-1 under knobs in saponin-
treated membrane preparations. (A) K + cells were treated with
mAb 89 and ferritin-labeled conjugate. Dense labeling with
ferritin can be seen on the parasitophorous vacuolar mem-
brane and electron-dense material under knobs (k). The par-
asite (P) was negative. (B) K + cells were treated with an ir-
relevant mAb plus ferritin. No labeling was seen (specificity
control). (C) K- parasites were treated with mAb 89 and were
found to lack knobs and to be negative for PfHRP-1. Bar rep-
resents 0.1 txm.
using this technique; thus, the binding of m A b 89
to knobs could not be assessed.
T h e o u t e r m e m b r a n e of infected erythrocytes
was partially solubilized by t r e a t m e n t with sa-
ponin, and the cells were then treated with m A b
89, followed by ferritin c o n j u g a t e d secondary an-
tibody. P f H R P - 1 was detected in high concentra-
• tions on the cytoplasmic face of the s u b m e m b r a n -
ous electron-dense material associated with knobs
(Fig. 4A). P f H R P - 1 was not present on the sur-
grains were seen on the cytoplasmic face of the
m e m b r a n e b e t w e e n knobs. K ÷ cells treated with
an irrelevant m A b (Fig. 4B) and K cells treated
with m A b 89 (Fig. 4C) were negative. Thus,
P f H R P - 1 was localized to the electron-dense ma-
terial u n d e r the erythrocyte m e m b r a n e at knobs•
T o localize the site of P f H R P - 1 insertion into
arations were treated with m A b 89 and protein
A - g o l d (Fig. 5). In one experiment, we were not
successful in extracting the cells completely and
individual microfilaments could not be discerned
(Fig. 5 A - C ) . In this experiment, p h o t o m i c r o -
graphs showed discrete clusters of gold (1(t-25
grains per cluster), 40-50 n m in d i a m e t e r on K ~
(Fig. 5A), but not on K - preparations (Fig. 5B).
H o w e v e r , control K - skeletons were heavily la-
beled w h e n treated with anti~erythrocyte serum
and protein A - g o l d (Fig. 5C). In routine experi-
ments, skeletons were completely extracted and
individual microfilaments clearly discernible (Fig.
5 D - E ) . In these preparations when K + prepara-
tions were treated with m A b 89, heavy labeling
was seen along microfilaments and at points of
intersection of skeletal filaments (Fig. 5E). In
control preparations, only low b a c k g r o u n d label-
ing was seen on microfilaments of K+ cells treated
with PBS (Fig. 5D), or normal m o u s e serum, but
heavy labeling was f o u n d on skeletons treated
with anti-erythrocyte serum (Fig. 5F). K cyto-
skeletons were routinely negative when treated
with m A b 89. By c o m p a r i s o n with o t h e r E M
studies using similar techniques to study h u m a n
erythrocyte cytoskeletons [39-41], the long fila-
ments measuring 3-10 n m x 45-70 n m in Fig.
5 D , E are p r e s u m e d to be c o m p o s e d of spectrin
and are labeled S in Fig. 5E. Nodular or junc-
tional areas (12-25 nm in diameter), where the
 171

Fig. 5. Whole mount immunoelectron microscopy of K ÷ and K- cytoskeletons. Electron micrographs A - C and D - F are from the
different experiments. (A) This panel demonstrates the binding of mAb 89 to K + cells that have been incompletely-extracted dur-
ing the solubilization process. Note discrete clumps of 11-17 gold grains (arrow). (B) A K- cell from the same experiment treated
with mAb 89. (C) A K - cell treated with serum from a mouse immunized with K ÷ P. falciparurn infected erythrocytes. Electron
micrographs A - C were taken at 25 000x. Electron micrographs D-F show K + cytoskeletons treated with (D) PBS, (E) mAb 89,
or (F) immune mouse serum, followed by protein A-gold. (E) Labeling along microfilaments of spectrin (S) and at nodular regions
(N) is clearly evident. Electron micrographs D-F were taken at 15 000x. Bar represents 50 nm.

filaments join the network, are probably the lo-
cation of actin + band 4.1 [39-41]. These areas
are marked N in Fig. 5E.
The epitope bound by mAb 89 is not expressed on
the surface of infected erythrocytes. To determine
if mAb 89 bound to an epitope exposed on the
surface of infected cells, four assays were con-
ducted. First, intact erythrocytes containing tro-
phozoites were incubated with m A b 89 or poly-
clonal rabbit anti-PfHRP-1 antiserum followed by
either a single or double-sandwich IFA. Aotus and
human erythrocytes infected with the 3 strains of
K + P. falciparum parasites were routinely nega-
tive. Acetone-fixed smears of the same cells were
strongly positive.
Second, K + and K - P . falciparum-infected
erythrocytes were treated with mAb 89 or control
sera and protein A-gold, and then prepared for
transmission electron microscopy. Grains of gold
were not observed on K ÷ cells treated with PBS
and protein A-gold. Only an occasional knob
(7/200) was observed to bind 1-3 grains of gold in
K ÷ cells treated with mAb 89.
Third, mAb 89 and rabbit anti-PfHRP-1 were
tested for the ability to block the binding of K ÷-
infected erythrocytes to melanoma cells, using the
in vitro cytoadherence assay of Udeinya et al.
[38]. Pretreatment of cells with mAb 89 (at 100
Ixg m1-1) or rabbit anti-PfHRP-1 serum at a 1:10
dilution did not affect cytoadherence (data not
shown).
172
Finally, mAb 89 was tested for the ability to
enhance immune phagocytosis by normal and ac-
tivated peritoneal macrophages. No enhance-
ment of Fc-mediated phagocytosis (i.e., number
of macrophages that phagocytized erythrocytes)
was observed compared to PBS and normal hu-
man sera controls. Sera from most Africans with
antimalarial antibodies significantly enhanced
phagocytosis (P<0.05).
Discussion
The protein identified by mAb 89 has all of the
characteristics of the P. falciparum PfHRP-1. It
is produced by K +, but not by K- parasites [16],
it first appears during the early trophozoite stage
of development [22], it is associated with the
erythrocyte membrane [20,22], it is not extracted
from infected cells by isotonic Triton X-100 [20]
and it shows molecular weight variations among
isolates [21].
P. falciparum parasites synthesize at least 2
other HRPs [17-19]; PfHRP-2 is 65-85 kDa
[17,19] and PfHRP-3 35 kDa [18,19]. Based on
the results from Western blotting, it is clear that
mAb 89 does not bind to these other P. falcipa-
rum HRPs. Thus, mAb 89 serves as a valuable
marker for the localization of PfHRP-1 within P.
falciparum-infected erythrocytes, and can be used
to verify the presence of this protein in new par-
asite isolates. Although the gene coding for
PfHRP-1 has recently been sequenced and
expression peptides used to purify antibodies from
human serum [9], to our knowledge this is the first
mAb reported to have specificity for a protein
with characteristics identical to those ascribed to
PfHRP-1.
Based on IFA and cryo-thin section EM re-
sults, there was little evidence for the presence of
PfHRP-1 within the parasite itself. This suggests
that the protein is either present in very low con-
centrations within the parasite due to rapid secre-
tion after its synthesis or it is modified following
export from the parasite so as to create the mAb
89 epitope.
PfHRP-1 was clearly present external to the
parasite in the parasitophorous vacuolar space
(Fig. 3A) and within organelles in the erytbro-
cyte cytoplasm (Fig. 3B-E). PfHRP-1 was found
within membranous vesicles, associated with the
membranes of vesicles, and around electron dense
granules (Fig. 3C,D). Because of heavy labeling
with protein A-gold, it is not clear if the dense
granules are also membrane bound. Previous
transmission EM studies of P. falciparum-in-
fected erythrocytes have described membrane-
bound vesicles within the erythrocyte cytosol
which are commonly referred to as Maurer's clefts
[1,3]. Recently, Aikawa et al. [12] demonstrated
that material with the same density as the mate-
rial found under knobs was present on some of
these membranous vesicles and clefts. They sug-
gested that the electron dense material moves
from the parasite into the parasitophorous vacu-
olar space where it becomes incorporated into
Maurer's clefts, moves through the cytosol, and
is finally inserted into the erythrocyte membrane
under knobs [12]. The current study shows that
PfHRP-1 is also present in Maurer's clefts. It is
tempting to speculate that the electron dense ma-
terial described by Aikawa et al. [12] is PfHRP-
1, but further studies are needed to verify the re-
lationship between the electron dense material
and PfHRP-1, and if they have the same pattern
of intracellular trafficking.
In this study, immunoelectron microscopy
studies have localized PfHRP-1 in the electron
dense material on the undersurface of knobs (Fig.
4A). A recent paper by Culvenor et al. (1987) re-
ported similar observations [9]. They affinity pur-
ified antibodies from immune human sera on a
column to which a k gtl0 expression peptide
(SD17) had been coupled. The purified antibod-
ies recognized a protein with the same biochem-
ical characteristics as PfHRP-1, and this protein
was found to be localized in knobs by post-
embedding immunoelectron microscopy. Thus
both studies, using different methodologies,
demonstrate the association of PfHRP-1 with
knobs.
IFA studies on Triton X-100 extracted cells
showed that PfHRP-1 was present in 'minute
spots' throughout the skeleton (Fig. 2C). To pin-
point the site of insertion of PfHRP-1 within the
skeleton, we used a whole-cell mounting tech-
nique which we recently developed for examining
changes in the architecture of the skeleton of in-
fected erythrocytes [32]. Knobs do not appear as

e l e c t r o n d e n s e s t r u c t u r e s in this t e c h n i q u e p r o b -
ably b e c a u s e l e a d a n d u r a n y l salts a r e n o t used.
In one e x p e r i m e n t , w h e r e we failed to extract part
o f t h e d e t e r g e n t - s o l u b l e m a t e r i a l f r o m t h e skele-
tons, a g g r e g a t e s o f g o l d r e a c h i n g 4 0 - 5 0 n m in di-
ameter were seen on K + but not on K- prepa-
r a t i o n s t r e a t e d with m A b 89 (Fig. 5 A , B ) . T h e
d i s t r i b u t i o n of the a g g r e g a t e s is c o n s i s t e n t with
k n o b s . I n p r e p a r a t i o n s w h e r e we w e r e successful
in r e v e a l i n g i n d i v i d u a l m i c r o f i l a m e n t s , specific la-
b e l i n g was s e e n a l o n g f i l a m e n t s a n d in high con-
c e n t r a t i o n o n s o m e , b u t n o t o t h e r , j u n c t i o n a l re-
gions (Fig. 5E). U n f o r t u n a t e l y , r e g i o n s l a b e l e d
with g o l d c o r r e s p o n d i n g in size to k n o b s (e.g.
75-100 n m ) in c o m p l e t e l y e x t r a c t e d p r e p a r a t i o n s
w e r e n o t s e e n , thus t h e p r e c i s e r e l a t i o n s h i p be-
tween knobs, PfHRP-1 and cytoskeletal compo-
n e n t s is u n c l e a r .
In this s t u d y , m A b to P f H R P - 1 d i d n o t b i n d to
t h e s u r f a c e o f n o n - f i x e d , intact i n f e c t e d cells in
IFA studies and, by immunoelectron microscopy,
t h e vast m a j o r i t y o f k n o b s w e r e n e g a t i v e for
P f H R P - 1 on t h e i r e x t e r n a l surface. In a d d i t i o n ,
m A b 89 d i d n o t b l o c k in v i t r o c y t o a d h e r e n c e o r
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