EUKARYOTIC CELL, Nov. 2010, p. 1661–1668
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 9, No. 11
The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel
Targets on the Surface of Entamoeba histolytica Trophozoites?†
Andrea Carpentieri,1‡ Daniel M. Ratner,1‡§ Sudip K. Ghosh,1¶ Sulagna Banerjee,1? G. Guy Bushkin,1
Jike Cui,1# Michael Lubrano,1†† Martin Steffen,2Catherine E. Costello,3Barry O’Keefe,4
Phillips W. Robbins,1and John Samuelson1*
Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 021181;
Department of Pathology and Laboratory Medicine, Boston University Medical School, Boston, Massachusetts 021182;
Mass Spectrometry Resource, Department of Biochemistry, Boston University Medical Center, Boston,
Massachusetts 021183; and Molecular Targets Development Program, Center for
Cancer Research, NCI-Frederick, Frederick, Maryland 217024
Received 6 July 2010/Accepted 6 September 2010
Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked
glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). Here, we show that glycoproteins with
unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiret-
roviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the
Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized
by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica
trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the
plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass
spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc
lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously
hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In
summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E.
histolytica that are rapidly replenished from large intracellular pools.
Entamoeba histolytica causes amebic dysentery and liver ab-
scess in the developing world (10, 20, 29). We are interested in
E. histolytica glycoproteins containing Asn-linked glycans (N-
glycans) for numerous reasons. E. histolytica makes an N-gly-
can precursor that contains 7 sugars (Man5GlcNAc2-PP-doli-
chol) rather than 14 sugars (Glc3Man9GlcNAc2-PP-dolichol)
made by most animals, plants, and fungi (21, 31, 44). E. histo-
lytica N-glycans are used for quality control of glycoprotein
folding in the endoplasmic reticulum (ER) lumen, and there is
positive selection for sites of N-linked glycosylation in secreted
and membrane proteins of E. histolytica (5, 11, 53).
Unprocessed Man5GlcNAc2, by far the most abundant E.
histolytica N-glycan, is present on the plasma membrane and
vesicular membranes (31). The antiretroviral lectin cyanovi-
rin-N, which is specific for ?-1,2-linked mannose present on
unprocessed N-glycans, binds E. histolytica N-glycans and
forms aggregates or caps on the surface of E. histolytica tro-
phozoites (1, 25, 31, 44, 45). E. histolytica glycoproteins are also
capped by the plant lectin concanavalin A (ConA), which has
a broader carbohydrate specificity (mannose and glucose) than
cyanovirin-N (3, 16, 18, 19). Heavy subunits of the Gal/GalNAc
lectin, the most important E. histolytica vaccine candidate, have
7 to 10 potential sites for N-linked glycosylation (32, 39, 43).
Inhibition of N-glycan synthesis results in Gal/GalNAc lectins
that are unable to bind to sugars on host epithelial cells.
Carbohydrates appear to be an important target on the sur-
face of E. histolytica as anti-proteophosphoglycan (PPG)
monoclonal antibodies bind to O-phosphodiester-linked gly-
cans and protect animal models from amebic infection (6, 33,
35, 40, 48). Lectin affinity columns are a powerful method for
enriching unique parasite glycoproteins that may be identified
by mass spectrometry (MS) of tryptic fragments (17, 55). For
example, we recently used the plant lectin wheat germ agglu-
tinin to dramatically enrich glycoproteins with short N-glycans
of Giardia (42).
The goal of the present studies was to explore further the
interaction of the antiretroviral lectin cyanovirin-N with E.
histolytica trophozoites in vitro. Questions asked included the
following: Are E. histolytica glycoproteins with N-glycans re-
plenished on the plasma membrane after capping with cyano-
virin-N? What is the effect of cyanovirin-N capping on other
amebic virulence factors and/or vaccine candidates (e.g., the
* Corresponding author. Mailing address: Department of Molecular
and Cell Biology, Boston University Goldman School of Dental Med-
icine, 72 East Concord Street, Evans 425, Boston, MA 02118. Phone:
(617) 414-1054. Fax: (617) 414-1041. E-mail: firstname.lastname@example.org.
‡ A.C. and D.M.R. contributed equally to this work.
§ Present address: Department of Bioengineering, University of
Washington, Seattle, WA 98195.
¶ Present address: Department of Biotechnology, Indian Institute of
Technology, Kharagpur, West Bengal, India.
? Present address: Department of Surgery, University of Minnesota,
Minneapolis, MN 55455.
# Present address: Center for Biomedical Informatics, Harvard
Medical School, Boston, MA 02115.
†† Present address: Albert Einstein College of Medicine, Bronx, NY
† Supplemental material for this article may be found at http://ec
?Published ahead of print on 17 September 2010.
Gal/GalNAc lectin and PPG)? Is capping by cyanovirin-N me-
diated by actin, as described for capping by the Gal/GalNAc
lectin and ConA? What is the effect of the cyanovirin-N on
amebic phagocytosis of mucin-coated beads, a surrogate assay
for virulence? Which trophozoite glycoproteins are potential
targets of cyanovirin-N (identified by mass spectrometry of
lectin-enriched E. histolytica proteins)? Are any of them po-
tential vaccine candidates?
MATERIALS AND METHODS
Fluorescence microscopy. Logarithmic-phase trophozoites of the genome
project HM1 strain of E. histolytica were chilled to release adherent organisms,
concentrated by low-speed centrifugation, and washed in chilled phosphate-
buffered saline (PBS) (29). For surface labeling, trophozoites were incubated for
30 min at 4°C in cyanovirin-N labeled with either Alexa Fluor 488 (green) or
Alexa Fluor 585 (red) (1, 31). Cyanovirin-N-labeled trophozoites were washed
three times in PBS and then fixed for 10 min at 4°C in 2% paraformaldehyde in
100 mM phosphate, pH 7.4. For capping experiments, trophozoites labeled with
cyanovirin-N were warmed to 37°C for 15 min prior to fixation.
To determine whether N-glycans are replenished on the surface of trophozo-
ites capped with cyanovirin-N, we treated capped and fixed organisms with PBS
containing 2% bovine serum albumin (BSA) to quench free aldehydes and then
labeled them with cyanovirin-N conjugated to a different Alexa Fluor dye. To
demonstrate actin fibrils, we permeabilized capped and fixed organisms with
0.1% Triton X-100 and then stained them with 0.1 mg/ml phalloidin conjugated
to Alexa Fluor 480 for 1 h at 4°C (18). To determine whether cyanovirin-N
cocaps other E. histolytica antigens, we incubated capped and fixed E. histolytica
with an Alexa Fluor-labeled mouse monoclonal antibody to the Gal/GalNAc
lectin (a generous gift of William Petri) (32, 39). Alternatively, capped and fixed
E. histolytica organisms were incubated with an Alexa Fluor-labeled mouse
monoclonal antibody to the E. histolytica PPG (a generous gift of Michael
Duche ˆne) (33).
For internal labeling with cyanovirin-N, we fixed E. histolytica trophozoites for
10 min at 4°C, and Triton X-100 was added to a final concentration of 0.1% for
1 min. Cells were gently pelleted by centrifugation, washed with PBS–2% BSA,
and then incubated with cyanovirin-N, as described above. Similar methods were
performed for labeling the surface and interior of E. histolytica with anti-Gal/
GalNAc antibodies and for determining whether Gal/GalNAc lectins are replen-
ished on the parasite surface after capping.
The nuclei of E. histolytica cells labeled with cyanovirin-N or the anti-Gal/
GalNAc antibody were stained with 0.1 ?g/ml 4?,6?-diamidino-2-phenylindole
(DAPI), SlowFade antifade solution (Invitrogen) was added, and organisms were
visualized with a DeltaVision deconvoluting microscope (Applied Precision,
Issaquah, WA) with channels for each fluorochrome. Images were taken at a
primary magnification of ?100 and deconvolved using Applied Precision’s
Phagocytosis of mucin-coated spheres. Assays for E. histolytica phagocytosis of
mucin-coated spheres were preformed, as described previously (18). Briefly, E.
histolytica trophozoites (105/ml) were incubated with microspheres (107/ml) in
culture medium for 15 min at 37°C and then fixed in 2% paraformaldehyde.
Phagocytosed beads within 100 cells in each group were counted with a fluores-
cence microscope. The results were plotted using a modified box and whiskers
plot to illustrate the significant shift in phagocytosed beads between the cyano-
virin-N-treated and untreated trophozoites. In addition, an analysis of variance
(ANOVA) was used to evaluate the statistical significance of the differences in
phagocytosis and calculate the P value.
Concanavalin A affinity chromatography of Entamoeba proteins. Logarithmic-
phase E. histolytica trophozoites were harvested on ice, washed in PBS, and
sonicated in an ice-water slurry containing 0.1% Triton X-100 and EDTA-free
Complete protease inhibitor cocktail (Roche). Insoluble material was removed
by centrifugation (at ?12,000 ? g). Soluble proteins were applied to a ConA-
Sepharose column (EY Laboratories, Inc.) (17, 55), and the column was subse-
quently rinsed with PBS. To avoid collecting proteins that were nonspecifically
bound to the ConA resin, we selectively eluted E. histolytica glycoproteins with 50
mM ?-methyl mannoside rather than with SDS. Proteins eluted from the ConA
column were run on SDS-PAGE gels containing a 4 to 20% gradient of acryl-
amide (Bio-Rad). In a parallel lane were E. histolytica proteins that were treated
twice with 1,000 units of peptide:N-glycanase F (PNGaseF; New England Bio-
labs) for 9 h at 37°C in NEB G7 phosphate buffer. E. histolytica proteins were
transferred to nitrocellulose membranes by electroporation, incubated with
horseradish peroxidase (HRP)-conjugated cyanovirin-N, and developed with
ECL chemiluminescent substrate (Pierce).
Mass spectrometry. Mass spectrometry of E. histolytica proteins was per-
formed using two different methods, as two different mass spectrometers were
used. For the linear trap quadrupole (LTQ) ProteomeX ion trap mass spectrom-
eter (Thermo Finnigan) present at the Boston University Proteomics Core Fa-
cility, E. histolytica peptides were prepared and analyzed using methods that were
essentially the same as those used to identify peptides from the E. histolytica cyst
wall (54) or from lectin affinity preparations of Giardia glycoproteins (42, 56). In
addition, some samples were run on a similar Thermo Finnigan mass spectrom-
eter at the Cancer Center at the Massachusetts Institute of Technology (MIT).
Mass spectra were compared to tryptic digests of E. histolytica proteins predicted
from whole-genome sequencing using SEQUEST, GPM (The Global Proteome
Machine Organization [www.thegpm.org]) open source software, or Mascot soft-
ware (13, 22, 23).
Two-dimensional protein gels were simulated from mass spectrometry data
using GPM, where the position of each protein was determined by its predicted
pI and mass, not including posttranslational modifications, and the size of the
spot was proportional to the number of observed ions corresponding to that
protein. These two-dimensional gels highlighted relative abundances of secreted
and plasma membrane proteins (defined by either an N-terminal ER-targeting
sequence or a transmembrane helix [TMH]) (24, 36) versus nucleocytoplasmic
proteins (defined by the absence of these features). The Excel files in the
supplemental material each show the merged results of four mass spectrometric
experiments using Mascot software. Proteins previously identified as hypotheti-
cal because they showed no homology to other eukaryotic proteins were assigned
simple names based upon their topology (e.g., unique nucleocytosolic protein,
unique secreted protein, unique type 1 membrane protein, unique glycosylphos-
phatidylinositol [GPI]-anchored protein, etc.). GPI anchors were predicted using
the algorithms of Eisenhaber et al. (15). Where there seemed a good match in
the nonredundant (NR) database as demonstrated by a high score with BLASTP
(2), we renamed the E. histolytica protein (e.g., “cysteine proteinase” or “disul-
fide isomerase” rather than “conserved hypothetical protein”).
To identify occupied N-glycan sites, we used a two-dimensional chromatogra-
phy approach. A peptide mixture from the tryptic digestion of ConA-enriched E.
histolytica glycoproteins was treated with PNGaseF to remove N-glycans and to
convert Asn to Asp. PNGaseF-treated peptides and an untreated control were
separated using strong cationic exchange (SCX) chromatography prior to Nano-
flow reversed-phase high-performance liquid chromatography (HPLC)-coupled
tandem mass spectrometry (MS/MS). SCX chromatography was performed on a
Beckman Coulter ProteomeLab PF2D using a PolySulfoethyl A column. The
buffers used were the following; buffer A, 7 mM KH2PO4, pH 2.65, 30% aceto-
nitrile (ACN; vol/vol); buffer B, 7 mM KH2PO4, 350 mM KCl, pH 2.65, 30%
ACN (vol/vol); buffer C, 50 mM K2HPO4, 500 mM NaCl, pH 7.5. Peptides were
separated using a linear gradient from 0% to 70% of buffer B in 30 min, from
70% to 100% of buffer B in 10 min, and then 100% of buffer B for 6 min. The
flow rate used was 0.5 ml/min. Thirteen 2-min fractions were collected. Each
fraction was dried to eliminate acetonitrile before LC-MS/MS.
LC-MS/MS was performed using a nanoAcquity ultra-performance liquid
chromatography (UPLC) capillary system (Waters Corp., Milford, MA), coupled
to an LTQ-Orbitrap hybrid mass spectrometer (ThermoFisher Scientific, San
Jose, CA) equipped with a TriVersa NanoMate ion source (Advion, Ithaca, NY).
Sample concentration and desalting were performed online using a nanoAcquity
UPLC trapping column (180 ?m by 20 mm; packed with 5-?m, 100-Å-pore-size
Symmetry C18material; Waters Corp.) at a flow rate of 15 ?l/min for 1 min.
Separation was accomplished on a nanoAcquity UPLC capillary column (100 ?m
by 100 mm; packed with 1.7-?m,130-Å-pore-size bridged ethyl hybrid [BEH] C18
material; Waters Corp.). A linear gradient of A and B buffers (buffer A, 3%
ACN–0.1% formic acid [FA]; buffer B, 97% ACN–0.1% FA) from 7% to 45%
buffer B over 124 min was used at a flow rate of 0.5 ?l/min to elute peptides into
the mass spectrometer. Columns were washed and reequilibrated between LC-
MS/MS experiments. Electrospray ionization was carried out at 1.7 kV using the
NanoMate, with the LTQ heated capillary set to 150°C.
Mass spectra were acquired in the Orbitrap in the positive-ion mode over the
range of m/z 300 to 2,000 at a resolution of 60,000. Mass accuracy after internal
calibration was within 4 ppm. Simultaneously, tandem MS spectra were acquired
using the LTQ for the five most abundant, multiply charged species in the mass
spectrum with signal intensities of ?8,000 noise levels. MS/MS collision energies
were set at 35%, using helium as the collision gas, and MS/MS spectra were
acquired over a range of m/z values dependent on the precursor ion. Dynamic
exclusion was set such that MS/MS for each species was acquired a maximum of
twice. All spectra were recorded in profile mode for further processing and
1662 CARPENTIERI ET AL.EUKARYOT. CELL
Xcalibur software was used for MS and MS/MS data analysis, while peptide
and protein assignments were conducted using Mascot to search against the E.
histolytica database employing an error window of 6 ppm on the precursor ions
and 0.6 Da on the fragment ions. Table 1 shows occupied N-glycan sites where
the predicted Asn was converted to Asp by PNGaseF treatment, resulting in a
shift in mass of ?1 Da.
MS data. Mass spectrometric data have been deposited in AmoebaDB (4).
E. histolytica glycoproteins are capped by the antiretroviral
lectin cyanovirin-N and then replenished from large intracel-
lular pools. Cyanovirin-N, which labels ?-1,2-linked mannose
residues in unprocessed N-glycans, evenly stains the surface of
E. histolytica trophozoites either kept at 4°C to prevent capping
or fixed prior to labeling (see Fig. S1 in the supplemental
material). Glycoproteins containing N-glycans are capped on
the surface of E. histolytica trophozoites when cyanovirin-N-
labeled trophozoites are warmed to 37°C (Fig. 1A and C, large
arrows). Subsequent labeling of fixed parasites with cyanovi-
rin-N conjugated to a different Alexa Fluor dye shows that
many glycoproteins containing N-glycans are replenished on
the surface of E. histolytica trophozoites away from the cap
(Fig. 1B and C, small arrows). Similarly, Gal/GalNAc lectins
that are capped by a monoclonal antibody are replenished on
the surface of E. histolytica away from the cap (Fig. 1D to F;
see also Fig. S1 in the supplemental material).
The source of the new glycoproteins with N-glycans on the
E. histolytica surface after capping is likely the large intracel-
lular pool of glycoproteins containing N-glycans. These glyco-
proteins are clearly visible on cyanovirin-N labeling of fixed
and permeabilized E. histolytica trophozoites (Fig. 1G). Cya-
novirin-N binds in a reticular or membrane pattern to glyco-
proteins of permeabilized E. histolytica. In contrast, the anti-
Gal/GalNAc lectin monoclonal antibody labels the membranes
and the contents of numerous secretory vesicles throughout
the E. histolytica trophozoite (Fig. 1H). A model for the re-
plenishment of capped E. histolytica surface glycoproteins from
large intracellular pools is shown in Fig. 1I and discussed
Cyanovirin-N caps the Gal/GalNAc lectin and glycoproteins
recognized by an anti-proteophosphoglycan antibody. Consis-
tent with the presence of 7 to 10 N-glycan sites on each heavy
subunit of the Gal/GalNAc lectin, cyanovirin-N cocaps the
Gal/GalNAc lectin (Fig. 2A to C). The presence of anti-Gal/
GalNAc lectin antibody labeling in areas away from the cap
(Fig. 2B and C) is consistent with spontaneous replenishment
of the Gal/GalNAc lectin from the large intracellular pools
concurrent with the capping event. Cyanovirin-N also cocaps
glycoproteins recognized by the anti-PPG antibodies (Fig. 2D
to F). There is binding of the anti-PPG in areas away from the
cap (Fig. 2E), consistent with replenishment of the PPG from
large intracellular pools (data not shown).
Cyanovirin-N inhibits phagocytosis by Entamoeba trophozo-
ites of mucin-coated beads. Filamentous actin, which is labeled
by the fungal toxin phalloidin, is important for amebic motility,
capping, and phagocytosis. Actin filaments accumulate in the
region of the cyanovirin-N induced cap (Fig. 2G to I), as has
been shown for caps by the plant lectin ConA and by the
monoclonal antibody to the Gal/GalNAc lectin.
Cyanovirin-N inhibits phagocytosis of mucin-coated beads
by E. histolytica trophozoites, a surrogate assay for amebic
virulence (Fig. 3A to C). While untreated E. histolytica tropho-
zoites phagocytose 43 ? 22 (mean ? standard deviation [SD])
TABLE 1. E. histolytica glycoproteins with occupied N-glycan sites as shown by PNGaseF treatment and mass spectrometry
Accession no. Protein name or description
Gal/GalNAc lectin heavy subunit
Gal/GalNAc lectin heavy subunit
Gal/GalNAc lectin intermediate subunit
Gal/GalNAc lectin intermediate subunit
Unique GPI-anchored glycoprotein
Secreted glycoprotein similar to Igl1
Unique secreted protein with PKEDQ repeats
Unique type 1 membrane glycoprotein
Unique basic secreted glycoprotein
Unique ER glycoprotein
Unique secreted glycoprotein
Unique secreted glycoprotein
Unique secreted glycoprotein
Unique type 1 transmembrane glycoprotein
Asp-rich type 1 membrane glycoprotein
Unique type 1 membrane glycoprotein
Unique type 1 membrane glycoprotein
Unique type 1 membrane glycoprotein
Unique type 2 membrane glycoprotein
Unique type 1 membrane glycoprotein
Unique GPI-anchored glycoprotein
aBoldface indicates where the predicted Asn was converted to Asp by PNGaseF treatment.
VOL. 9, 2010 ENTAMOEBA GLYCOPROTEINS TARGETED BY CYANOVIRIN-N1663
mucin-coated beads, cyanovirin-N-treated trophozoites phago-
cytose 9 ? 11 (mean ? SD) beads (P ? ? 0.005). The inhibition
of phagocytosis by cyanovirin-N is comparable to that caused
by overexpression of a dominant negative p21racmutant that
interferes with localization of actin filaments during phagocy-
E. histolytica membrane and secreted proteins are dramat-
ically enriched by affinity chromatography with ConA. Lectin
affinity chromatography was performed with ConA-Sepharose
because glycoproteins can be eluted with excess ?-methyl man-
noside. In contrast, glycoproteins bound to cyanovirin-N–
Sepharose may only be eluted with SDS that introduces non-
specifically bound contaminants. Western blotting showed that
cyanovirin-N conjugated to horseradish peroxidase binds to E.
histolytica glycoproteins that were enriched by ConA affinity
chromatography (Fig. 4A). In contrast, cyanovirin-N no longer
binds to Entamoeba glycoproteins treated with PNGaseF to
remove N-glycans (Fig. 4A). These results confirm that cyano-
virin-N is binding only to E. histolytica N-glycans.
In the absence of ConA affinity chromatography, the vast
majority (87%) of 302 E. histolytica proteins identified by mass
spectrometry of tryptic peptides are nucleocytosolic (Fig. 4B
and C, labeled blue). For example, when proteins are listed by
their Mascot score, there are 41 nucleocytosolic proteins be-
fore the first secreted protein, a cysteine proteinase (see Excel
file S1 in the supplemental material). While they are not the
focus of the present study, nucleocytosolic proteins (many of
which have greater than 50% peptide coverage) include en-
FIG. 1. Capped cyanovirin-N-binding glycoproteins and Gal/GalNAc lectins are rapidly replenished on the E. histolytica surface from large
intracellular pools. (A) Cyanovirin-N forms a tight green cap (large arrow) on the surface of trophozoites warmed for 15 min prior to fixation.
(B) The same organism was fixed and then labeled with red cyanovirin-N. Cyanovirin-N binding sites that are replenished on the E. histolytica
surface are marked with small arrows. (D) Similar results were obtained with a monoclonal antibody to the Gal/GalNAc lectin, which forms a tight
cap on trophozoites prior to fixation (green). (E) After fixation, the Gal/GalNAc lectin that is replenished on the parasite surface is shown with
a red anti-Gal/GalNAc antibody. Merged panels are as indicated. (G) Cyanovirin-N (red) binds to a large interior pool of glycoproteins in a fixed
and permeabilized ameba. (H) Similar results were obtained with antibodies to the Gal/GalNAc lectin. In panels G and H nuclei are stained with
DAPI (blue). Bar, 5 ?m. (I) A model for the redistribution of glycoproteins binding cyanovirin-N or anti-GalNAc antibodies from large
intracellular pools during capping. See Fig. S1 in the supplemental material for images of cyanovirin-N and anti-Gal/GalNAc antibody labeling of
uncapped parasites. Ab, antibody.
1664CARPENTIERI ET AL.EUKARYOT. CELL
zymes involved in fermentation, glycolysis, and protein synthe-
sis as well as chaperones and cytoskeletal proteins.
Following ConA affinity enrichment, the majority of E. his-
tolytica proteins identified (52%) were membrane or secreted,
as shown by the presence of N-terminal signals and/or trans-
membrane helices (Fig. 4B and C, labeled red). For example,
25 of the 30 proteins with the highest Mascot scores are se-
creted or membrane proteins rather than nucleocytosolic pro-
teins (see Excel file S2 in the supplemental material). These
glycoproteins (many of which have greater than 50% peptide
coverage) include well-characterized virulence factors such as
all three subunits of the Gal/GalNAc adherence lectin, as well
as lysosomal proteases and phosphatases (see Excel file S2) (7,
10, 29, 32, 39). ER chaperones, protein disulfide isomerases,
peptidyl-prolyl cis-trans isomerases, and calreticulin are all
abundant. Of particular interest for discovery of potential vac-
cine candidates are 27 unique type 1 membrane proteins and
six unique GPI-anchored proteins (see the FASTA file in the
supplemental material). In the absence of information with
regard to the location of any of these unique proteins, in the
Excel files in the supplemental material the proteins with
TMHs were arbitrarily assigned to the plasma membrane while
proteins with an N-terminal signal peptide and no TMHs were
assigned to the lysosome.
E. histolytica glycoproteins with occupied N-glycan sites in-
clude numerous proteins implicated in amebic pathogenesis.
ConA-enriched glycoproteins were treated with PNGaseF, and
peptides in which the predicted Asn was converted to Asp were
identified by a shift in mass of ?0.984 Da (Table 1). These
modified peptides (32 total), which represent occupied N-gly-
can sites, are absent from E. histolytica proteins that have not
been treated with PNGaseF. Glycoproteins (26 total) with oc-
cupied N-glycan sites include numerous well-characterized vir-
ulence factors and/or vaccine candidates (heavy and interme-
diate subunits of the Gal/GalNAc lectin, serine and cysteine
peptidases, and a receptor kinase) (Table 1) (8, 10, 32). Other
FIG. 2. Cyanovirin-N cocaps and partially depletes the Gal/GalNAc lectin and PPG. E. histolytica trophozoites were capped with cyanovirin-N
(red), fixed, and then labeled with monoclonal antibodies to the Gal/GalNAc lectin (green) or to PPG (green). In each case, cyanovirin-N is present
in a relatively tight cap (single large arrow), while the Gal/GalNAc lectin and PPG are each present in the cap and along the surface of the protist
(series of small arrows). A cyanovirin-N-induced cap on the surface of an E. histolytica trophozoite (G) colocalizes with phalloidin (H) that binds
filamentous actin. Merged panels are as indicated. Bar, 5 ?m. Ab, antibody.
VOL. 9, 2010ENTAMOEBA GLYCOPROTEINS TARGETED BY CYANOVIRIN-N1665
glycoproteins with occupied N-glycan sites include 17 unique
proteins that are secreted, membrane associated, or GPI an-
chored. Because some of these unique E. histolytica proteins
with occupied N-glycan sites are both short and abundant (e.g.,
EHI_077530 is 206 amino acids long with 56% peptide cover-
age and EHI_161040 is 180 amino acids long with 42% peptide
coverage), it is likely that they would make good vaccine can-
didates. A list of unique E. histolytica glycoproteins is shown in
the FASTA file in the supplemental material.
Capping is more complex than previously supposed. While
actin-mediated capping of amebic proteins has been described
(3, 16, 18, 19, 50), this is the first demonstration, to our knowl-
edge, of replenishment of surface antigens from large intracel-
lular pools. This process is shown in the model in Fig. 1I, where
the precap surface antigens are shown in green, and the precap
internal pool of antigens is shown in red. During capping of the
green antigens by cyanovirin-N or antibodies to the Gal/GalNAc
lectin, the red antigens move from internal pools to cover the
Because replacement occurs so quickly, the effects of cya-
novirin-N on amebic phagocytosis in vitro (shown here) and of
antibodies to the Gal/GalNAc lectin (32, 39, 43) and to PPG
(6, 33, 35, 40, 48) on amebic virulence in vivo are likely not
simply based upon clearing the relevant proteins from the
parasite surface. Instead, the effects of cyanovirin-N and of
antibodies to the Gal/GalNAc lectin or to PPG are likely also
mediated by perturbation of the cytoskeleton during capping
(3, 16, 18, 19, 50) and/or by signals transduced by various
receptors (Gal/GalNAc lectin and/or receptor kinases) (8).
Conversely, it does not appear that E. histolytica trophozoites
escape anti-Gal/GalNAc lectin or anti-PPG antibodies by cap-
ping and removing antigens from their surfaces as both the
Gal/GalNAc and PPG are rapidly replenished from large in-
While there was no surprise that the E. histolytica Gal/GalNAc
lectin has occupied N-glycan sites (32), it was not possible in
advance to predict that glycoproteins recognized by anti-PPG
antibodies are also capped by cyanovirin-N (6, 33, 35, 40, 48).
The latter result suggests that some E. histolytica glycoproteins
contain both N-glycans and O-phosphodiester-linked glycans.
E. histolytica glycoproteins include well-characterized viru-
lence factors, as well as numerous unique proteins that may be
novel vaccine candidates. ConA affinity chromatography en-
abled the identification of ?100 E. histolytica secreted and
membrane proteins by mass spectrometry. The gel-free mass
spectrometric methods used here are easier than cutting pro-
teins from two-dimensional protein gels and result in relatively
fewer cytosolic proteins identified than methods in which
membranes or lysosomes are isolated (12, 26, 37, 51, 52, 55,
56). However, these other mass spectrometric studies reveal
differences between virulent and avirulent strains of Entam-
oeba and demonstrate accessory proteins (e.g., Rabs) involved
in vesicle sorting, endocytosis, and protein secretion.
Gal/GalNAc lectins are among the most abundant E. histo-
lytica glycoproteins identified here, consistent with their prior
identification by monoclonal antibodies and their importance
in amebic pathogenesis (32, 39, 43). Dozens of unique and
abundant E. histolytica glycoproteins identified here by mass
spectrometry include new vaccine candidates (type 1 mem-
brane proteins and GPI-anchored proteins) and/or new pro-
teins involved in pathogenesis (secreted proteins). Of course,
vaccine candidates and proteins involved in pathogenesis may
be overlapping (e.g., the Gal/GalNAc lectin) (39). Recombi-
nant versions of these unique E. histolytica glycoproteins, many
of which are relatively small and not too Cys rich, might be
used to vaccinate animal models and so add to the relatively
short list of amebic vaccine candidates (Gal/GalNAc lectins,
serine-rich E. histolytica protein [SREHP], and the 29-kDa
protein) (9, 32, 39, 46, 47). Knockdown or knockout methods
might be used to test the roles of these proteins in amebic
virulence (27, 34).
These results suggest the possibility that E. histolytica N-
glycans may be a new target for antiamebic reagents. Unproc-
essed Man5GlcNAc2, by far the most abundant E. histolytica
N-glycan, is recognized by the antiretroviral lectin cyanovi-
rin-N that has been overexpressed in Lactobacillus (1, 25, 28,
31, 41, 46). Cyanovirin-N-expressing lactobacilli (“yogurt-
plus”) might be introduced into the gastrointestinal tract,
where the bacteria may have an antiamebic effect. Other bac-
terial lectins that target high-mannose N-glycans of HIV (e.g.,
griffithsin and banana lectin [BanLec]) may have even greater
efficacy than cyanovirin-N versus E. histolytica (38, 49). Con-
versely, it may be possible to vaccinate against amebic infection
FIG. 3. Cyanovirin-N
spheres by E. histolytica trophozoites. Fluorescence micrograph of a
control E. histolytica trophozoite (A) that phagocytoses many mucin-
coated spheres (red). In contrast, a representative cyanovirin-N-
treated E. histolytica trophozoite (B) phagocytoses many fewer red
spheres. Bar, 5 ?m. (C) Phagocytosis of mucin-coated beads, as illus-
trated by a modified box and whiskers plot, in which the means are
marked with a heavy horizontal line and the medians are marked by a
light horizontal line. The lower and upper medians are the edges of
each gray box, while the vertical bar shows the minimum and maximum
values and the 10th and 90th percentiles (crosses).
1666CARPENTIERI ET AL.EUKARYOT. CELL
with high-mannose N-glycans present on Saccharomyces mu-
tants (14, 30).
This work was supported by NIH grants AI44070 (to J.S.), GM31318
(to P.W.R.), and RR10888 (to C.E.C.). Support for D.M.R. was pro-
vided by the Training Program in Host Pathogen Interactions (T32
We thank Richard Cook of MIT for some mass spectrometry data.
1. Adams, E. W., D. M. Ratner, H. R. Bokesch, J. B. McMahon, B. R. O’Keefe,
and P. H. Seeberger. 2004. Oligosaccharide and glycoprotein microarrays as
tools in HIV glycobiology; glycan-dependent gp120/protein interactions.
Chem. Biol. 11:875–881.
2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,
and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25:3389–3402.
3. Arhets, P., P. Gounon, P. Sansonetti, and N. Guillen. 1995. Myosin II is
involved in capping and uroid formation in the human pathogen Entamoeba
histolytica. Infect. Immun. 63:4358–4367.
4. Aurrecoechea, C., J. Brestelli, B. P. Brunk, S. Fischer, B. Gajria, X. Gao, et
al. 2010. EuPathDB: a portal to eukaryotic pathogen databases. Nucleic
Acids Res. 38:D415–419.
5. Banerjee, S., P. Vishwanath, J. Cui, D. J. Kelleher, R. Gilmore, P. W.
Robbins, and J. Samuelson. 2007. Evolution of quality control of protein-
folding in the ER lumen. Proc. Natl. Acad. Sci. U. S. A. 104:11676–11681.
6. Bhattacharya, A., R. Arya, C. G. Clark, and J. P. Ackers. 2000. Absence of
lipophosphoglycan-like glycoconjugates in Entamoeba dispar. Parasitology
7. Bruchhaus, I., B. J. Loftus, N. Hall, and E. Tannich. 2003. The intestinal
protozoan parasite Entamoeba histolytica contains 20 cysteine protease
genes, of which only a small subset is expressed during in vitro cultivation.
Eukaryot. Cell 2:501–509.
8. Buss, S. N., S. Hamano, A. Vidrich, C. Evans, Y. Zhang, O. R. Crasta, B. W.
Sobral, C. A. Gilchrist, and W. A. Petri, Jr. 2010. Members of the Entamoeba
histolytica transmembrane kinase family play non-redundant roles in growth
and phagocytosis. Int. J. Parasitol. 40:833–843.
9. Chaudhry, O. A, and W. A. Petri, Jr. 2005. Vaccine prospects for amebiasis.
Expert Rev. Vaccines 4:657–668.
10. Clark, C. G., U. C. Alsmark, M. Tazreiter, Y. Saito-Nakano, V. Ali, S.
Marion, C. Weber, C. Mukherjee, et al. 2007. Structure and content of the
Entamoeba histolytica genome. Adv. Parasitol. 65:51–190.
11. Cui, J., T. Smith, P. W. Robbins, and J. Samuelson. 2009. Darwinian selec-
tion for sites of Asn-linked glycosylation in phylogenetically disparate eu-
karyotes and viruses. Proc. Natl. Acad. Sci. U. S. A. 106:13421–13426.
12. Davis, P. H., M. Chen, X. Zhang, C. G. Clark, R. R. Townsend, and S. L.
Stanley, Jr. 2009. Proteomic comparison of Entamoeba histolytica and En-
tamoeba dispar and the role of E. histolytica alcohol dehydrogenase 3 in
virulence. PLoS Negl. Trop. Dis. 3:e415.
13. Duncan, D. T., R. Craig, and A. J. Link. 2005. Parallel tandem: a program for
parallel processing of tandem mass spectra using PVM or MPI and X!Tan-
dem. J. Proteome Res. 4:1842–1847.
14. Dunlop, D. C. C. Bonomelli, F. Mansab, S. Vasiljevic, K. J. Doores, M. R.
Wormald, A. S. Palma, T. Feizi, D. J. Harvey, R. A. Dwek, M. Crispin, and
C. N. Scanlan. 2010. Polysaccharide mimicry of the epitope of the broadly
neutralizing anti-HIV antibody, 2G12, induces enhanced antibody responses
to self oligomannose glycans. Glycobiology 20:812–823.
15. Eisenhaber, F., B. Eisenhaber, W. Kubina, S. Maurer-Stroh, G. Neuberger,
G. Schneider, and M. Wildpaner. 2003. Prediction of lipid posttranslational
modifications and localization signals from protein sequences: big-Pi, NMT
and PTS1. Nucleic Acids Res. 31:3631–3634.
FIG. 4. E. histolytica secreted and membrane proteins are markedly enriched by a ConA affinity column. (A) Cyanovirin-N binding to a Western
blot of ConA-enriched E. histolytica proteins is removed by prior treatment with PNGaseF. Computer-derived, two-dimensional (2D) protein gels
show mass spectrometry data from a representative experiment where unfractionated E. histolytica trophozoite proteins (B) and trophozoite
proteins after ConA affinity (C) were identified. The size of each spot is proportional to the peptide coverage of the protein. Secreted proteins,
which are markedly enriched after ConA, are shown in red. Nucleocytosolic proteins, which are abundant in unfractionated proteins, are shown
VOL. 9, 2010 ENTAMOEBA GLYCOPROTEINS TARGETED BY CYANOVIRIN-N1667
16. Espinosa-Cantellano, M., and A. Martinez-Palomo. 1994. Entamoeba histo-
lytica: mechanism of surface receptor capping. Exp. Parasitol. 79:424–435.
17. Ghosh, D., O. Krokhin, M. Antonovici, W. Ens, K. G. Standing, R. C. Beavis,
and J. A. Wilkins. 2004. Lectin affinity as an approach to the proteomic
analysis of membrane glycoproteins. J. Proteome Res. 3:841–850.
18. Ghosh, S. K., and J. Samuelson. 1997. Involvement of p21racA, phosphoino-
sitide 3-kinase, and vacuolar ATPase in phagocytosis of bacteria and eryth-
rocytes by Entamoeba histolytica: suggestive evidence for coincidental evo-
lution of amebic invasiveness. Infect. Immun. 65:4243–4249.
19. Guillen, N. 1996. Role of signaling and cytoskeletal rearrangements in the
pathogenesis of Entamoeba histolytica. Trends Microbiol. 4:191–197.
20. Haque, R., C. D. Huston, M. Hughes, E. Houpt, and W. A. Petri, Jr. 2003.
Amebiasis. N. Engl. J. Med. 348:1565–1573.
21. Helenius, A., and M. Aebi. 2004. Roles of N-linked glycans in the endoplas-
mic reticulum. Annu. Rev. Biochem. 73:1019–1049.
22. Higdon, R., N. Kolker, A. Picone, G. van Belle, and E. Kolker. 2004. LIP
index for peptide classification using MS/MS and SEQUEST search via
logistic regression. OMICS 8:357–369.
23. Koenig, T., B. H. Menze, M. Kirchner, et al. 2008. Robust prediction of the
MASCOT score for an improved quality assessment in mass spectrometric
proteomics. J. Proteome Res. 7:3708–3717.
24. Krogh, A., B. Larsson, G. von Heijne, and E. L. Sonnhammer. 2001. Pre-
dicting transmembrane protein topology with a hidden Markov model: ap-
plication to complete genomes. J. Mol. Biol. 305:567–580.
25. Kwong, P. D., M. L. Doyle, D. J. Casper, C. Cicala, S. A. Leavitt, S. Majeed,
T. D. Steenbeke, M. Venturi, et al. 2002. HIV-1 evades antibody-mediated
neutralization through conformational masking of receptor-binding sites.
26. Leitsch, D., I. B. Wilson, K. Paschinger, and M. Duche ˆne. 2006. Comparison
of the proteome profiles of Entamoeba histolytica and its close but non-
pathogenic relative Entamoeba dispar. Wien. Klin. Wochenschr. 118:37–41.
27. Linford, A. S., H. Moreno, K. R. Good, H. Zhang, U. Singh, and W. A. Petri,
Jr. 2009. Short hairpin RNA-mediated knockdown of protein expression in
Entamoeba histolytica. BMC Microbiol. 9:38.
28. Liu, X., L. A. Lagenaur, D. A. Simpson, K. P. Essenmacher, C. L. Frazier-
Parker, Y. Liu, D. Tsai, S. S. Rao, D. H. Hamer, T. P. Parks, P. P. Lee, and
Q. Xu. 2006. Engineered vaginal lactobacillus strain for mucosal delivery of
the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrob.
Agents Chemother. 50:3250–3259.
29. Loftus, B., I. Anderson, R. Davies, U. C. Alsmark, J. Samuelson, P. Amedeo,
P. Roncaglia, M. Berriman, et al. 2005. The genome of the protist parasite
Entamoeba histolytica. Nature 433:865–868.
30. Luallen, R. J., C. Agrawal-Gamse, H. Fu, D. F. Smith, R. W. Doms, and Y.
Geng. 2010. Antibodies against Man?1,2-Man?1,2-Man oligosaccharide
structures recognize envelope glycoproteins from HIV-1 and SIV strains.
31. Magnelli, P., J. F. Cipollo, D. M. Ratner, J. Cui, D. Kelleher, R. Gilmore,
C. E. Costello, P. W. Robbins, and J. Samuelson. 2008. Unique Asn-linked
oligosaccharides of the human pathogen Entamoeba histolytica. J. Biol.
32. Mann, B. J., B. E. Torian, T. S. Vedvick, and W. A. Petri, Jr. 1991. Sequence
of a cysteine-rich galactose-specific lectin of Entamoeba histolytica. Proc.
Natl. Acad. Sci. U. S. A. 88:3248–3252.
33. Marinets, A., T. Zhang, N. Guillen, P. Gounon, B. Bohle, U. Vollmann, O.
Scheiner, G. Wiedermann, S. L. Stanley, and M. Duche ˆne. 1997. Protection
against invasive amebiasis by a single monoclonal antibody directed against
a lipophosphoglycan antigen localized on the surface of Entamoeba histo-
lytica. J. Exp. Med. 186:1557–1565.
34. Mirelman, D., M. Anbar, and R. Bracha. 2008. Epigenetic transcriptional
gene silencing in Entamoeba histolytica. IUBMB Life 60:598–604.
35. Moody-Haupt, S., J. H. Patterson, D. Mirelman, and M. J. McConville. 2000.
The major surface antigens of Entamoeba histolytica trophozoites are GPI-
anchored proteophosphoglycans. J. Mol. Biol. 297:409–420.
36. Nielsen, H., S. Brunak, and G. von Heijne. 1999. Machine learning ap-
proaches for the prediction of signal peptides and other protein sorting
signals. Protein Eng. 12:3–9.
37. Okada, M., C. D. Huston, M. Oue, B. J. Mann, W. A. Petri, Jr., K. Kita, and
T. Nozaki. 2006. Kinetics and strain variation of phagosome proteins of
Entamoeba histolytica by proteomic analysis. Mol. Biochem. Parasitol. 145:
38. O’Keefe, B. R., F. Vojdani, V. Buffa, R. J. Shattock, D. C. Montefiori, J.
Bakke, J. Mirsalis, A. L. d’Andrea, S. D. Hume, B. Bratcher, C. J. Saucedo,
J. B. McMahon, G. P. Pogue, and K. E. Palmer. 2009. Scaleable manufacture
of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as
a topical microbicide component. Proc. Natl. Acad. Sci. U. S. A. 106:6099–
39. Petri, W. A., Jr., R. Haque, and B. J. Mann. 2002. The bittersweet interface
of parasite and host: lectin-carbohydrate interactions during human invasion
by the parasite Entamoeba histolytica. Annu. Rev. Microbiol. 56:39–64.
40. Prasad, R., M. Tola, M. P. Sharma, and A. Bhattacharya. 1992. Recognition
of Entamoeba histolytica lipophosphoglycan by a strain-specific monoclonal
antibody and human immune sera. Mol. Biochem. Parasitol. 56:279–287.
41. Pusch, O., D. Boden, S. Hannify, F. Lee, L. D. Tucker, M. R. Boyd, J. M.
Wells, and B. Ramratnam. 2005. Bioengineering lactic acid bacteria to se-
crete the HIV-1 virucide cyanovirin. J. Acquir. Immune Defic. Syndr. 40:
42. Ratner, D. M., J. Cui, M. Steffen, L. L. Moore, P. W. Robbins, and J.
Samuelson. 2008. Changes in the N-glycome (glycoproteins with Asn-linked
glycans) of Giardia lamblia with differentiation from trophozoites to cysts.
Eukaryot. Cell 7:1930–1940.
43. Ravdin, J. I., J. E. John, L. I. Johnston, D. J. Innes, and R. L. Guerrant.
1985. Adherence of Entamoeba histolytica trophozoites to rat and human
colonic mucosa. Infect. Immun. 48:293–297.
44. Samuelson, J., S. Banerjee, P. Magnelli, J. Cui, D. J. Kelleher, R. Gilmore,
and P. W. Robbins. 2005. The diversity of protist and fungal dolichol-linked
precursors to Asn-linked glycans likely results from secondary loss of sets of
glycosyltransferases. Proc. Natl. Acad. Sci. U. S. A. 102:1548–1553.
45. Scanlan, C. N., R. Pantophlet, M. R. Wormald, E. Ollmann Saphire, R.
Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek, P. M. Rudd, and D. R.
Burton. 2002. The broadly neutralizing anti-human immunodeficiency virus
type 1 antibody 2G12 recognizes a cluster of ?132 mannose residues on the
outer face of gp120. J. Virol. 76:7306–7321.
46. Stanley, S. L, Jr. 2006. Vaccines for amoebiasis: barriers and opportunities.
47. Stanley, S. L., Jr., A. Becker, C. Kunz-Jenkins, L. Foster, and E. Li. 1990.
Cloning and expression of a membrane antigen of Entamoeba histolytica
possessing multiple tandem repeats. Proc. Natl. Acad. Sci. U. S. A. 87:4976–
48. Stanley, S. L., Jr., H. Huizenga, and E. Li. 1992. Isolation and partial
characterization of a surface glycoconjugate of Entamoeba histolytica. Mol.
Biochem. Parasitol. 50:127–138.
49. Swanson, M. D., H. C. Winter, I. J. Goldstein, and D. M. Markovitz. 2010.
A lectin isolated from bananas is a potent inhibitor of HIV replication.
J. Biol. Chem. 285:8646–8655.
50. Tavares, P., P. Sansonetti, and N. Guille ´n. 2000. Cell polarization and
adhesion in a motile pathogenic protozoan: role and fate of the Entamoeba
histolytica Gal/GalNAc lectin. Microbes Infect. 2:643–649.
51. Teixeira, J. E., and C. D. Huston. 2008. Participation of the serine-rich
Entamoeba histolytica protein in amebic phagocytosis of apoptotic host cells.
Infect. Immun. 76:959–966.
52. Tolstrup, J., E. Krause, E. Tannich, and I. Bruchhaus. 2007. Proteomic
analysis of Entamoeba histolytica. Parasitology 134:289–298.
53. Trombetta, E. S., and A. J. Parodi. 2003. Quality control and protein folding
in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19:649–676.
54. Van Dellen, K. L., A. Chatterjee, D. Ratner, P. E. Magnelli, J. Cipollo, M.
Steffen, P. W. Robbins, and J. Samuelson. 2006. Unique posttranslational
modifications of chitin-binding lectins of Entamoeba invadens cyst walls.
Eukaryot. Cell 5:836–848.
55. Wang, Y., S.-I. Wu, and W. S. Hancock. 2006. Approaches to the study of
N-linked glycoproteins in human plasma using lectin affinity chromatography
and nano-HPLC coupled to electrospray linear ion trap-Fourier transform
mass spectrometry. Glycobiology 16:514–523.
56. Yates, J. R., III, E. Carmack, L. Hays, A. J. Link, and J. K. Eng. 1999.
Automated protein identification using microcolumn liquid chromatogra-
phy-tandem mass spectrometry. Methods Mol. Biol. 112:553–569.
1668 CARPENTIERI ET AL.EUKARYOT. CELL