the most abundant glycoprotein of amebic cyst walls (jacob ... · marta frisardi,1 sudip k. ghosh,1...

INFECTION AND IMMUNITY,0019-9567/00/$04.0010

July 2000, p. 4217–4224 Vol. 68, No. 7

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob)Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

MARTA FRISARDI,1 SUDIP K. GHOSH,1 JESSICA FIELD,1 KATRINA VAN DELLEN,1 RICK ROGERS,2

PHILLIPS ROBBINS,3 AND JOHN SAMUELSON1*

Department of Immunology and Infectious Diseases1 and BioMedical Imaging Institute,2 Harvard School of PublicHealth, and Department of Cell Biology, Boston University School of Dental Medicine,3 Boston, Massachusetts

Received 31 January 2000/Returned for modification 1 March 2000/Accepted 25 March 2000

The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study anextraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional proteingels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecularmass of ;100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDaprotein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but nothomologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins thatsurround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecylsulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars ofintact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which werequadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles.A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria andmucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membranegalactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains.

Entamoeba histolytica is a luminal protozoan parasite, whichis a frequent cause of dysentery and liver abscess in persons indeveloping countries that cannot prevent its fecal-oral spread(37). E. histolytica is part of a family of microaerophilic ame-bae, which reside as commensals in the human colon (En-tamoeba dispar and Entamoeba coli) or survive as free-livingorganisms in garbage (Entamoeba moscovshii) (12). The infec-tious stage of amebae is the chitin-walled, quadranucleate cyst,which is resistant to stomach acids (2, 15). Because it is notpossible to discriminate cysts of E. histolytica and E. dispar bymicroscopy, cysts identified in clinical samples are called “E.histolytica/E. dispar” (51).

Since E. histolytica parasites do not encyst in axenic culture,cyst formation has been studied using the reptilian pathogenEntamoeba invadens, which also forms a quadranucleate cyst(15). E. invadens, which is more closely related to E. histolyticathan to the human commensal E. coli, converts to cysts within2 days when deprived of glucose (38, 40). Amebic cyst wallproteins include 100- and 150-kDa glycoproteins, which bindwheat germ agglutinin (WGA), and uncharacterized antigens,which react with monoclonal anti-cyst antibodies (7, 49). Chitin(b-1,4-linked N-acetylglucosamine [GlcNAc]) is also present inamebic cyst walls (2). Although neither chitin synthase norchitinase is present in amebic trophozoites, both enzymes areexpressed by encysting parasites (3, 10, 47). Amebic chitinases,which have catalytic domains like those of nematode, insect,and plant chitinases, are present in hundreds of small cyst-specific secretory vesicles (4, 11, 19, 23, 39, 46).

Amebae have a plasma membrane Gal/GalNAc lectin, whichmay also be involved in encystation (8, 9). This lectin, whichhas been best characterized on the surface of E. histolyticatrophozoites, binds to galactose or N-acetylgalactosamine

(GalNAc) on bacteria, red blood cells and epithelial cells, ormucin-coated beads (13, 20). The E. histolytica Gal/GalNAclectin is composed of a large 170-kDa subunit, which has atransmembrane domain near its C terminus, and a small 35-kDa subunit, which has a glycosylphosphatidylinositol anchorat its C terminus (29, 31, 36, 44). An E. invadens gene encodinga homologue of the Gal/GalNAc lectin small subunit has beencloned (GenBank accession number AF016642). Since galac-tose but not GalNAc inhibits the aggregation and encystationof E. invadens parasites in vitro, it may be more accurate torefer to the E. invadens plasma membrane “galactose lectin”(8). It has been suggested that galactose exerts its effect onaggregation and encystation by blocking signal transductionmediated by the galactose lectin (9). Previously, inside-outsignaling by cytosolic domains of the Gal/GalNAc lectin wasshown to be important for epithelial cell adherence and amebicvirulence (48).

In this study, two-dimensional protein gels identified anabundant E. invadens cyst wall glycoprotein, which was calledJacob because it contained a ladder-like series of Cys-rich,chitin-binding domains. Jacob also contained sugars, whichwere recognized by the galactose lectin on encysting E. inva-dens, such that wall-less cysts formed in the presence of excessgalactose.

MATERIALS AND METHODS

Preparations of cysts and trophozoites. The IP-1 strain of E. invadens wasgrown at 25°C in axenic culture in TYI-SS medium (15). E. invadens encystationwas induced by placing parasites for 48 h in low-glucose (LG) medium, which hasreduced osmolarity, glucose, and serum levels with respect to TYI-SS medium(38). Cysts walls were identified by staining with 2 mg of Calcofluor per ml, whichbinds to chitin and emits a blue fluorescence when excited with UV light (5).Chitin and other carbohydrates in cyst walls were also stained with 50 mg each offluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA), FITC-ri-cin, or tetramethylrhodamine isothiocyanate-conjugated (TRITC)-WGA per mlfor 60 min at room temperature in phosphate-buffered saline (PBS) and washedfour times. Cyst nuclei were identified by permeabilizing cysts with 0.1% sodiumdodecyl sulfate (SDS) and staining with 1 mM Sytox green.

In an attempt to inhibit cyst wall formation, E. invadens parasites were placedin LG medium containing 100 mM lactose, galactose, GalNAc, or mannose and

* Corresponding author. Mailing address: Department of Immunol-ogy and Infectious Diseases, Harvard School of Public Health, 665Huntington Ave., Boston, MA 02115. Phone: (617) 432-4670. Fax:(617) 738-4914. E-mail: [email protected].

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incubated for 2 to 4 days at room temperature. Wall-less cysts, which wereformed in the presence of galactose, were motile, had four nuclei, and containedJacob and chitinase in secretory vesicles (see below) but lacked a chitin wall.Because a rabbit anti-E. histolytica Gal/GalNAc lectin antibody did not bind to E.invadens trophozoites, the E. invadens galactose lectin was indirectly detected ontrophozoites and wall-less cysts by incubating them with green fluorescent pro-tein-labeled bacteria with or without galactose (45). Wall-less cysts were alsoincubated in the absence of galactose with mucin-coated beads or uncoatedbeads (negative control) (20).

Two-dimensional SDS-PAGE of purified cyst walls. To purify cyst walls, weseparated cysts from adherent trophozoites by decanting unchilled flasks. Resid-ual trophozoites were lysed by five short pulses with a sonicator. Cysts wereconcentrated by centrifugation and resuspended in PBS plus 100 mM E-64 toinhibit amebic cysteine proteases. The cysts were broken by extensive sonication($50 pulses), and cyst walls were separated from cytosol, membranes, nuclei, andintact cysts by centrifugation through two 60% sucrose cushions (2). Cyst wallpreparations, which were checked by phase-contrast microscopy, fluorescence

FIG. 1. Transmission electron micrographs of E. invadens cyst walls. (A)Intact E. invadens cysts have an electron-dense wall, which overlies secretoryvacuoles and electron densities along the plasma membrane. (B) Cyst wallspurified by two sucrose gradients contain electron-dense material between chitinfibrils. (C) Chitin fibrils and little other electron-dense material remain in cystwalls after boiling in SDS. Bar, 200 nm. Magnification, 34,500.

FIG. 2. Two-dimensional gels of E. invadens cyst wall proteins. (A and B)Silver stains show that Jacob, which is by far the most abundant cyst wall protein(arrow in panel A), is absent from trophozoites (B). (C and D) Western blotsshow that ConA (C) and ricin (D), visualized with acid phosphatase, bind toJacob and numerous other cyst wall glycoproteins. (E) In contrast, anti-Jacobantibodies, visualized by chemiluminescence, bind to Jacob and larger andsmaller proteins with the same charge. Purified E. histolytica Gal/GalNAc lectin,which was detected with anti-lectin antibodies, binds to Jacob excised from aPonceau-stained two-dimensional gel (inset in panel E). A negative control, inwhich the Gal/GalNAc lectin was omitted, did not bind the anti-Gal/GalNAcantibodies (data not shown).

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microscopy with Calcofluor, and transmission electron microscopy (TEM), con-tained less than one intact cyst per 100 walls and no trophozoites.

Purified cyst walls were boiled in 1% SDS–5% b-mercaptoethanol (2-ME),and the supernatant of a microcentrifuge centrifugation was mixed with lysisbuffer (540 mg of urea per ml, 2% Triton X-100, 2% 2-ME, 2% ampholines 3 to10, 100 mg of E-64 per ml) and electrophoresed on two-dimensional gels (35).Precast gels (Pharmacia Biotech AB, Uppsala, Sweden) contained amphollytesfrom pH 3 to pH 10 in the first (isoelectric-focusing) dimension and a gradientof acrylamide from 10 to 20% in the second (SDS-polyacrylamide gel electro-phoresis [PAGE]) dimension. The gels were stained with Coomassie blue orsilver (to identify less abundant cyst wall proteins). The most abundant cyst wallglycoprotein (Jacob), which was acidic and was 100 kDa, was excised from aCoomassie blue-stained gel and digested with trypsin. Tryptic peptides wereseparated by high-pressure liquid chromatography, and N-terminal sequenceswere obtained by Edman degradation (32). Alternatively, two-dimensional pro-tein gels of cyst wall proteins were transferred to polyvinylidene difluoride(PVDF) filters, and Jacob, identified with Ponceau-S., was excised for N-terminalsequencing. Some PVDF membranes were blocked with powdered milk andtreated with anti-Jacob antibodies or the E. histolytica Gal/GalNAc lectin (asdescribed below). Other PVDF membranes were also incubated with biotinyl-ated ConA, ricin, WGA, or Sambucus nigra agglutinin (all 4 mg/ml in PBS),washed, and developed with avidin conjugated to alkaline phosphatase.

Cloning of the E. invadens jacob gene. A segment of the E. invadens jacob genewas isolated from DNA of E. invadens IP-1 using PCR and degenerate primersto N-terminal sequences of tryptic Jacob peptides (24). A degenerate senseprimer, CA(AG)TA(CT)TT(CT)GA(AG)TG(CT)(AT)(CG)(AT)AA(CT)AC,

was to QYFECSNT, while an antisense primer, AC(AG)TA(AG)TA(CT)TG(AG)AA(AG)TC(AG)TG, was to HDFQYYV. The jacob PCR product, which was488 bp long, was cloned in TA vector and sequenced by dideoxy methods. Thejacob PCR product was used to identify jacob gDNA clones from an E. invadensIP-1 strain DNA gDNA library (38). Like other E. invadens genes, the E.invadens jacob coding sequence contained no introns and had a 51% A1Tcontent in the third position (11, 19, 38). The E. invadens Jacob protein wascompared with proteins in the GenBank and EST databases and with productsof unfinished microbial genomes by using BLAST (1). N-terminal signal se-quences were predicted and potential transmembrane segments identified usingwell-established algorithms (16, 34).

Production of anti-Jacob antibodies, Western blots, and indirect immunoflu-orescence microscopy. E. invadens Jacob was excised from 10 two-dimensionalprotein gels, mixed with complete Freund’s adjuvant, and injected into rabbits.The rabbits were boosted at 3 and 6 weeks with Jacob from five gels each inincomplete Freund’s adjuvant. Western blots of two-dimensional gels of cyst wallproteins were incubated for 60 min at 25°C with anti-Jacob serum diluted 1:1,000in PBS. Filters were washed and incubated in anti-rabbit antibodies conjugatedto peroxidase (1:2,000 dilution), which was detected using chemiluminescentreagents.

To localize Jacob on the surface of E. invadens cysts, parasites were fixed with2% paraformaldehyde for 10 min at 4°C, washed in PBS, and immunostained for60 min at 37°C with rabbit anti-Jacob sera, diluted 1:100 in PBS containing 1 mgof bovine serum albumin per ml. To localize Jacob within secretory vesicles ofencysting E. invadens parasites, amebae were permeabilized by incubation with0.1% Triton X-100 for 5 min at room temperature and then immunostained withrabbit anti-Jacob, which was also diluted 1:100. The organisms were washed fourtimes and immunodecorated for 60 min with a Texas red-conjugated goat anti-rabbit antiserum. As a negative control, parasites were stained with preimmunerabbit serum. As a positive control, encysting parasites were stained with TRITC-WGA, which binds chitin. Alternatively, parasites were incubated with a rabbitanti-E. invadens chitinase antibody, which was previously made to a multianti-genic peptide containing chitinase repeats (19). Nuclei were stained with Sytoxgreen, and parasites were observed with a Leica NT-TCS confocal microscopefitted with argon and krypton lasers. Three-dimensional reconstructions weremade from a series of optical sections, which were made at 0.5- to 1-mm intervals.

TEM and immuno-EM of encysting parasites. Cysts and purified cyst wallswere fixed for 10 min in 1% paraformaldehyde, postfixed in 1% osmium tetrox-ide, stained en block with uranyl acetate, dehydrated, and embedded in Epon.Sections, which were 60 nm thick, were stained on the grid with uranyl acetateand lead citrate. To visualize Jacob on the surface of cysts or wall-less cysts,encysting parasites were fixed in paraformaldehyde, incubated with anti-Jacobantibodies as described for fluorescence microscopy, and then incubated withStaphylococcus protein A, which had been conjugated to 10-nm-diameter goldparticles. A negative control included preimmune rabbit serum. Parasites werewashed and prepared for TEM as described above. Alternatively, parasites werefixed in 2% paraformaldehyde, infiltrated with 2.3 M sucrose, and frozen. Ultra-thin sections were incubated with the anti-Jacob serum and protein A-goldcomplex. These parasites were prepared for TEM in the absence of osmiumtetroxide, so that the membranes remained unstained.

Methods to demonstrate binding of Jacob to chitin. To obtain soluble forms ofJacob, E. invadens parasites were encysted for 24 h and cytosolic extracts weremade by sonicating parasites in the presence of E-64. These extracts wereincubated with SDS-treated cyst walls, chitin beads, or GlcNAc beads, whichwere washed and then incubated with rabbit anti-Jacob antibodies and immu-nodecorated as described above. Negative controls included cytosolic extractsfrom E. invadens trophozoites, nonimmune sera, and Sepharose or agarosebeads. In addition, extracts of E. histolytica bound to chitin beads were stainedwith antibodies to alcohol dehydrogenase 1 or Ariel (negative controls) (22, 28).Amebic proteins binding to chitin beads were also eluted with 1% SDS–5%2-ME and run on one-dimensional SDS-PAGE. Gels were transferred to nitro-cellulose filters and incubated with anti-Jacob antibodies, which were detected

FIG. 3. Fluorescence micrographs of E. invadens cysts and trophozoites. (Aand B) FITC-ConA (arrowheads in panel A) and FITC-ricin (B) stained thesurface of cysts (c) much more intensely than the surface of trophozoites (t). (C)Similarly, anti-Jacob antibodies, made to the native Jacob protein, bound to thesurface of cysts but not to trophozoites. (D) The E. histolytica Gal/GalNAc lectin,which was detected with anti-lectin antibodies, also bound to E. invadens cystwalls but not to the surface of trophozoites.

FIG. 4. Primary structure of E. invadens Jacob, in single-letter code. An asterisk marks the stop codon. The signal sequence, proven by N-terminal sequencing ofintact Jacob, is underlined twice, while the N-terminal sequences of Jacob tryptic peptides are each underlined once. PCR primers for cloning the E. invadens jacobgene were made to QYFECSNT and HDFQYYV. A possible site of Asn-linked glycosylation (NDT) is marked with a wavy underline. Conserved Cys residues inputative chitin-binding domains, which are aligned, are marked in bold.

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with chemiluminescent reagents. A positive control included cyst wall proteins,which were run in a parallel lane.

Methods to demonstrate binding of the Gal/GalNAc lectin to Jacob. Bindingof the E. histolytica Gal/GalNAc lectin to cyst walls and to Jacob was demon-strated in three ways. First, E. histolytica HM-1 parasites were stained with anonspecific cytosolic stain, CFDA SE (Molecular Probes, Eugene, Oreg.) andincubated for 3 h at 37°C with intact E. invadens cysts, which were stained withCalcofluor. Phagocytosis of cysts by trophozoites was determined by fluorescencemicroscopy. Negative controls included trophozoites incubated with cyst wallsafter treatment with SDS to remove Jacob and other glycoproteins, as well asincubation of trophozoites with cyst walls in the presence of excess galactose.Second, E. invadens cysts were incubated with 5 mg of purified Gal/GalNAc lectinfrom HM-1 trophozoites per ml (36). Cysts were washed, and bound lectin wasdetected with a 1:500 dilution of rabbit anti-lectin antibodies (29), which wereimmunodecorated as described for anti-Jacob antibodies. Negative controls in-cluded replacement with SDS-treated cyst walls, addition of excess galactose toGal/GalNAc lectin, omission of the lectin, or incubation with nonimmune rabbitserum. Third, a Western blot of a two-dimensional gel of Jacob was incubatedwith the Gal/GalNAc lectin, washed, and incubated with anti-lectin antibodies,which were detected with chemiluminescent reagents. A negative control wasincluded in which the Gal/GalNAc lectin was omitted.

Nucleotide sequence accession number. The nucleotide and predicted aminoacid sequence of the jacob gene have been submitted to GenBank with accessionnumber AF175527.

RESULTSThe most abundant cyst wall protein is an acidic, 100-kDa

glycoprotein (Jacob). The walls of E. invadens cysts were elec-tron dense and had a uniform thickness of ;100 nm (Fig. 1A).Electron-dense material was also present in secretory vesiclesand along the plasma membrane. Purified cyst walls, whichwere prepared on sucrose gradients after sonication of cysts,closely resembled the walls of intact cysts (Fig. 1B). After thecyst walls were boiled in SDS and 2-ME, only fibrils, whichwere less tightly bound to each other, remained (Fig. 1C).These fibrils are presumably made of chitin, because theystained with Calcofluor (data not shown) (2).

More than a dozen cyst wall proteins (Fig. 2A), which wereabsent from trophozoites (Fig. 2B), were identified by two-dimensional SDS-PAGE (35). Some of these cyst wall proteinsformed multiple spots, which were the same size but had dif-ferent isolectric points. The most abundant cyst wall-specificprotein (Jacob) was acidic and had an apparent molecularmass of ;100 kDa. Jacob and other amebic cyst wall proteinswere glycoproteins, which bound ConA (Fig. 2C). Jacob alsobound ricin (Fig. 2D) and WGA (data not shown) but did notbind S. nigra agglutinin. Rabbit antibodies to purified Jacobbound to the 100-kDa spot and to higher- and lower-molecu-lar-mass spots (Fig. 2E), which had the same isoelectric pointas Jacob but were less abundant. Fluorescence microscopy wasused to confirm the Western blot findings. ConA (Fig. 3A) andricin (Fig. 3B) bound much more extensively to the surface ofcysts than to the surface of trophozoites, while anti-Jacob an-tibodies bound to cysts but not to trophozoites (Fig. 3C).

Jacob contains five Cys-rich domains, separated by acidicspacers. N-terminal sequences of tryptic peptides of Jacobwere used to design degenerative oligonucleotide primers toobtain a segment of the E. invadens jacob gene by PCR (24).The jacob PCR product was in turn used to obtain the entirecoding region of the jacob gene of E. invadens, which predicteda 405-amino-acid protein (Fig. 4). The formula weight of Jacob(Mr 45,122) was less than half the apparent molecular mass(100 kDa) of Jacob on two-dimensional protein gels (Fig. 2A).A signal sequence (MLSDILFGIAAA), which was identifiedby sequencing the N terminus of undigested Jacob, was cleavedat a site predicted by the -3,-1 rule as applied to eubacteriarather than eukaryotes (34). The predicted E. invadens Jacobalso contained N-terminal sequences of peptides obtainedfrom tryptic digests of Jacob. The amino acid composition ofthe predicted protein, which was rich in acidic (16%), basic(15%), and polar (43%) amino acids, matched that of Jacobexcised from the gel.

Jacob had one site of possible N-linked glycosylation(Asn33) and numerous Ser and Thr residues for possible O-linked glycosylation (21) (Fig. 4). Jacob did not have any pre-dicted transmembrane segments (16). Instead, it was com-posed of five similar domains, each of which contained six Cysresidues spaced 7, 12, 9, 5, and 12 amino acids apart. Chitin-binding domains of peritrophins and fungal, nematode, andinsect chitinases have mirror-image spacing of Cys residues,which are 12, 5, 9, 12, and 7 amino acids apart (all 61) (17, 23,39, 46). Jacob repeat domains also contained conserved aro-matic amino acids (Tyr, Phe, and Trp), which may be involvedin binding sugars, as described for WGA (52). Between theJacob repeat domains were acidic domains, which were similarin their composition but not their sequence to repeats in ame-bic chitinases, Ser-rich E. histolytica proteins, or Ariel surfaceprotein (11, 28, 41). There were no proteins homologous toamebic Jacob in GenBank, EST, or unfinished microbial ge-nome databases, although BLAST with Jacob identified thelarge subunit of the E. histolytica Gal/GalNAc lectin and Gi-

FIG. 5. Confocal micrographs of encysting E. invadens stained with anti-Jacob antibodies (red in panels A through E), anti-chitinase antibodies (red inpanel F) and Sytox green (nuclear stain in panels A through F). Jacob waspresent in multiple places on the surface of encysting parasites with one nucleus(in section [A] and three-dimensional composite [B]) and became more dense onparasites with four nuclei (in section [C] and composite [D]). Jacob was presentin numerous relatively large secretory vesicles (in composite [E]) that surroundthe nuclei of encysting parasites, which were permeabilized before labeling.Chitinase (in composite [F]) was present within hundreds of smaller secretoryvesicles of an encysting parasite. Bars, 5 mm.



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ardia lamblia variable surface proteins, which are also Cys rich(1, 29, 33, 44).

Jacob, which is released from multiple loci to the surface ofencysting parasites, is a chitin-binding lectin. Jacob mRNAs,detected by reverse transcription-PCR, were abundant in ex-tracts of E. invadens cysts but were weakly present in extractsof trophozoites, which inevitably contain some encysting par-asites (data not shown). Encystation-specific expression of Ja-cob was similar to but more abundant than that of chitinase(11). Anti-Jacob antibodies showed that Jacob was secretedfrom numerous foci onto the surface of encysting parasiteswith one nucleus (Fig. 5A and B) and continued until cysts hadfour nuclei (Fig. 5C and D). Jacob was present as electron-dense material in clumps between and on the surface of chitinfibrils (Fig. 6A and C). Jacob was also present in numerouslarge secretory vesicles in encysting parasites (Fig. 5E and 6D).These Jacob-associated secretory vesicles were larger thanthose visualized with anti-chitinase antibodies (Fig. 5F). It is

possible that small chitinase-containing vesicles are lysosomes,which are released during excystation.

An extract of encysting E. invadens, which contained solubleJacob, was incubated with purified cyst walls that had beentreated with SDS to remove their proteins. Jacob, which wasdetected with anti-Jacob antibodies, bound to chitin remainingin SDS-treated cysts walls (data not shown). Anti-Jacob anti-bodies did not bind to SDS-treated cyst walls, which had notbeen pretreated with extracts of encysting parasites. Jacob alsobound to chitin beads (Fig. 7). Jacob did not bind to Sepharoseor agarose beads, while abundant E. histolytica proteins includ-ing alcohol dehydrogenase 1 and Ariel did not bind to eitherSDS-treated cyst walls or chitin beads (22, 28). These resultssuggest that the Cys-rich domains of Jacob, which resemblethose of peritrophins and chitinase, are chitin-binding domains(17, 23, 39, 46).

Wall-less cysts are produced when amebae encyst in thepresence of galactose. We confirmed here the previous obser-

FIG. 6. Immuno-EM of anti-Jacob antibodies binding to cysts and wall-less cysts. (A) Anti-Jacob antibodies, visualized with gold particles, were present overelectron-dense material on the surface of encysting parasites, which were stained prior to fixation. (B) In contrast, wall-less cysts, which were made in the presence ofgalactose, lacked chitin and had a thin layer of electron-dense material that bound few anti-Jacob antibodies. (C and D) When encysting parasites were fixed andsectioned prior to staining, anti-Jacob antibodies bound to cyst walls and to numerous large secretory vesicles. Bars, 200 nm. Magnification, 34,500.



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vation that galactose is able to block aggregation and encysta-tion by amebae in vitro (8, 9). Remarkably, galactose-treatedparasites in encysting medium, which are referred to as wall-less cysts, were ameboid and quadranculeate, lacked Cal-cofluor-binding material on their surface, and were filled withnumerous secretory granules containing Jacob and chitinase(Fig. 6B and 8A to C). Quadranucleate, wall-less cysts werealso formed in the presence of lactose but not in the presenceof GalNAc or mannose (data not shown). In contrast, negative-control parasites, which were treated with galactose in normalculture medium, were mononucleate and lacked Jacob andchitinase (data not shown).

Galactose was demonstrated on the surface of cysts in threeways. First, purified E. histolytica plasma membrane Gal/GalNAclectin, which was detected with a monospecific rabbit antibodyto the E. histolytica Gal/GalNAc lectin, bound to intact E.invadens cysts but not to E. invadens trophozoites or to E.invadens cysts treated with SDS to remove cyst wall glycopro-teins (Fig. 3D). The anti-lectin antibody did not bind to cysts ofnegative controls in which the Gal/GalNAc lectin was omitted.Second, the E. histolytica Gal/GalNAc lectin, again detectedwith the anti-lectin antibody, bound to Western blots of Jacob(inset in Fig. 2E), while a negative control without the E.histolytica Gal/GalNAc lectin did not bind Jacob. These West-ern blots do not rule out the possibility that the Gal/GalNAclectin binds to other cyst wall glycoproteins. Third, E. histo-lytica trophozoites rapidly phagocytosed intact E. invadenscysts but not SDS-treated cysts, and phagocytosis was inhibitedby galactose (data not shown).

A putative E. invadens galactose lectin was shown on thesurface of quadranucleate, wall-less cysts by two indirect meth-ods. First, wall-less cysts phagocytosed bacteria labeled withgreen fluorescent protein (45), and the phagocytosis was in-hibited by galactose (Fig. 8E). Second, wall-less cysts phago-cytosed mucin-coated beads but not uncoated beads (Fig. 8F).Nonencysting E. invadens trophozoites also phagocytosed bac-teria and mucin-coated beads (data not shown), suggesting thegalactose lectin is constitutively expressed on E. invadens par-asites.

DISCUSSION

A model of amebic cyst wall construction. Our results sug-gest a two-lectin model of E. invadens cyst wall construction.First, the plasma membrane galactose lectin binds sugars onJacob and perhaps other encystation-specific secretory glyco-proteins that are part of the cyst wall (13, 29, 36, 44). Second,Jacob itself is a lectin with five Cys-rich domains, which bindchitin (3, 10, 47). Wall-less cysts are formed in the presence ofgalactose, because the galactose lectin no longer binds Jacoband other cyst wall glycoproteins and Jacob no longer bindschitin.

Weaknesses of this two-lectin model include the following.The gene encoding the large subunit of the E. invadens galac-tose lectin has not been identified. Conversely, the gene en-coding the E. histolytica homologue of Jacob has not beenidentified. Sugars on Jacob have not yet been analyzed. Thepossibility that galactose blocks secretion of Jacob and chiti-nase cannot be ruled out. It is also possible that the E. invadensgalactose lectin is involved in signal transduction during encys-tation, as has been suggested (9).

Examples of convergent and coincident evolution. Jacob ap-pears to be a chitin-binding lectin for three reasons. First,Jacob is localized to chitin-binding fibrils by immuno-EM. Sec-ond, Jacob binds chitin in SDS-treated cyst walls and bindschitin beads but does not bind to other carbohydrates such asSepharose or agarose. Third, Jacob contains five putative do-mains, each of which has six Cys residues with conserved spac-ing and numerous aromatic amino acids present in chitin-binding domains of peritrophins and fungal, nematode, andinsect chitinases (17, 23, 39, 46). Because the spacing betweenCys residues in repetitive domains of Jacob is the mirror-imageof those in lectin domains of peritrophins and chitinases, itappears that Jacob is related to these chitin-binding proteins bya common structure rather than a common ancestry (conver-gent evolution) (14). Convergent evolution is possible, becausechitin-binding domains are short (;50 amino acids) and con-tain fewer conserved residues than are present in the activesites of most enzymes.

The plasma membrane Gal/GalNAc lectin, which is impli-cated here in binding sugars on Jacob and cyst wall formation,is an important vaccine candidate on the surface of E. histo-lytica (29, 44). The Gal/GalNAc lectin is also an importantvirulence factor, because the amebic lectin binds sugars on hostepithelial cells and red blood cells (13, 36, 37), as well as onbacteria, which are the major energy source for parasites in thedistal colon (20). Although there is no fossil record to prove ordisprove our assertion, it is likely that cyst wall formation isancient and was present among free-living ancestors of ame-bae. Further invasion of host tissue is a reproductive dead endfor amebae, which are spread by chitin-walled cysts (37). Itseems likely, then, that the Gal/GalNAc lectin has been se-lected for its involvement in cyst wall formation and bacterialkilling and that its involvement in host cell killing is an exampleof coincident evolution (20). Similar arguments have beenmade for coincident evolution of other amebic virulence fac-tors including amebapores, cysteine proteinases, p21racA, andvacuolar ATPase (20, 25). Cysteine proteinases may also beimportant for cyst wall destruction during amebic excystation,as has been shown for excystation of Giardia (50).

Discrepancies with previous results. There are at least twoways in which our results are different from what might havebeen expected from the literature on amebae. First, it is likelythat wall-less cysts, which are formed in the presence of ga-lactose, did not miss the signal for encystation, as recentlysuggested (9), because they are quadranucleate and produce

FIG. 7. SDS-PAGE and Western blots of Jacob binding to chitin beads.Western blots with anti-Jacob antibodies to trophozoite proteins (T) before (2)and after (1) binding to chitin beads, total proteins from encysting parasites (E)before (2) and after (1) binding to chitin beads, and cyst wall proteins (C)(positive control) are shown. Two bands labeled with anti-Jacob antibodiescorrespond to 100-kDa and high-molecular-mass forms of Jacob identified ontwo-dimensional protein gels (Fig. 2). Molecular mass standards are marked onthe right.



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encystation-specific Jacob and chitinase in abundance (11).Indeed, wall-less cysts resemble quadranucleate ameboidforms, which appear when parasites excyst in vitro (our unpub-lished observations). It is possible that the quadranucleatewall-less cysts were missed in recent studies of galactose-in-duced inhibition of encystation, because the assay for cystsinvolved putting parasites in water and treating them withdetergent, which would lyse the wall-less cysts (8, 9). Second,although amebic lipophosphoglycans contain galactose, thesegalactose residues do not appear to be accessible on the sur-face of E. invadens or E. histolytica (42). E. histolytica tropho-zoites, which have the Gal/GalNAc lectin on their surface, donot adhere to and phagocytose each other (29, 36, 43). E.invadens and E. histolytica parasites fail to bind ricin by fluo-rescence microscopy, and both parasites are resistant to highconcentrations of ricin (our unpublished data). In contrast,Jacob, which is the first amebic glycoprotein identified thatcontains Gal or GalNAc (29, 43), is accessible on the surface of

encysting parasites, so that ricin binds to E. invadens cysts andE. histolytica trophozoites phagocytose cysts in a galactose-inhibitable manner.

The E. invadens cyst wall is more like the insect peritrophicmatrix than the walls of Giardia cysts or fungi. Like the peri-trophic matrix that lines the food bolus in the insect gut, the E.invadens cyst wall is composed of chitin and a lectin (peritro-phin and Jacob, respectively), which has five putative chitin-binding domains (17, 39). The E. invadens cyst walls and peri-trophic membranes have a similar thickness and appearance byTEM, and strong detergents are necessary to disrupt bothstructures. In contrast, the Giardia cyst wall contains polymersof GalNAc rather than GlcNAc, and the abundant Leu-richproteins of the Giardia cyst walls (CWP1 and CWP2) show nosimilarity in structure to Jacob or peritrophins (27, 30). LikeGiardia, the amebic cyst wall is synthesized simultaneouslyfrom numerous loci across the surface of parasites, while cystwalls of budding yeast or elongating fungal hyphae are secretedfrom particular loci (6, 18, 26). While chitin is a primary struc-tural component of the amebic cyst wall and peritrophic ma-trices, it is often used to shape fungal walls, which have acomplex architecture and composition (2, 5, 6). Future studieswill attempt to identify other cyst wall proteins and to deter-mine whether anti-Jacob antibodies may be used to discrimi-nate cysts of E. histolytica and E. dispar (49, 51).

ACKNOWLEDGMENTS

This work was supported in part by a National Institute of Allergyand Infectious Diseases Supplement to Promote Reentry into Biomed-ical and Behavioral Research Careers (M.F.) and by National Insti-tutes of Health grants AI-33492 (J.S.), GM-31318 (P.R.), and HL-330099 and HL-43510 to R.R.

We acknowledge the expert technical support of Jean Lai of theHarvard School of Public Health for confocal microscopy and imageanalysis and Maria Ericsson of the Harvard Medical School for TEMand immuno-EM. Thanks to William Lane of the MicrochemistryFacility at the Biological Laboratories of Harvard University for se-quencing N-terminal peptides. Thanks to of Daniel Eichinger of NewYork University Medical School for the E. invadens IP-1 strain DNAgDNA library. Thanks to Barbara Mann and Bill Petri of the Univer-sity of Virginia Medical School for purified E. histolytica Gal/GalNAclectin and rabbit anti-lectin antibodies.

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the most abundant glycoprotein of amebic cyst walls (jacob ... · marta frisardi,1 sudip k. ghosh,1...

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