Experimental Parasitology 110 (2005) 363–373

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Entamoeba histolytica: Biochemical and molecular insights into the activities within microsomal fractions �

Milena Salgado a, Julio C. Villagómez-Castro b, Rocío Rocha-Rodríguez b,Myrna Sabanero-López b, Marco A. Ramos a,1, Alejandro Alagón a,

Everardo López-Romero b, Rosana Sánchez-López a,¤

a Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología (UNAM), Avenida Universidad 2001,Cuernavaca, Morelos 62210, Mexico

b Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Guanajuato 36000, Mexico

Received 24 November 2004; received in revised form 30 March 2005; accepted 2 April 2005

Abstract

One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway,particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isola-tion of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts,microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P Glc-NAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved fromphagolysosomal fractions. Sec61�-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized condi-tions for trophozoite homogenization and diVerential centrifugation that resulted in the separation of a 57,000g-sedimenting microsomalfraction containing EhSec61�-subunit, EhDPMS, and EhPDI (protein disulWde isomerase, a soluble marker of the lumen of the ER). Arelevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehprotea-some were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oli-gosaccharide was enriched with the identiWcation of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc2Man5core. No evidence of genes supporting further assembly steps was obtained at this time. 2005 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: ALG, asparagine-linked glycosylation; AP, acid phosphatase; DPMS, dolichol-P-mannose synthase; ER, endo-plasmic reticulum; Glc, glucose; GPI, glycosyl phosphatidylinositol; Man, mannose; �-NAcGase, �-N-acetyl-glucosaminidase; NAGPT, UDP-D-N-acetylglucosamine:dolichol-P N-acetylglucosamine-1-P transferase; NAGT, UDP-D-N-acetylglucosamine:dolichol-PP N-acetylglucosamine; PDI,protein disulWde isomerase; UDP-GlcNAc, UDP-N-acetylglucosamine; GFP, green Xuorescent protein; DHFR, dihydrofolate reductase

Keywords: Entamoeba; Endoplasmic reticulum; Microsomes; Dolichol-linked; Oligosaccharide; N-Glycosylation

� Nucleotide sequence data reported in this paper are available in theGenBank database under the Accession No: E. histolytica Sec61�-sub-unit, AY730760; E. histolytica, PDI, AY730725.

* Corresponding author. Fax: +52 777 3172388.E-mail address: rosana@ibt.unam.mx (R. Sánchez-López).

1 Present address: Facultad de Ciencias Químicas e Ingeniería,UABC, Calzada Tecnológico 14418, Tijuana, BC 22390, Mexico.

0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2005.04.002

1. Introduction

Eukaryotic cells contain highly specialized,membrane-bounded compartments fulWlling speciWcfunctions. Among them, the endomembrane system thatconstitutes the protein traYcking and the lipid biosyn-thetic pathways is the most prominent. It consists of the

364 M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373

endoplasmic reticulum (ER), pre-Golgi intermediates,the Golgi apparatus, and diVerent types of post-Golgi carriers and vesicles. The protein transportpathway accomplishes a multitude of interrelated func-tions including the translocation to the ER andtransport of de novo synthesized proteins, the N-linkedglycosylation and sorting of proteins to their Wnalcellular destinations , such as the lysosome,endosome, plasma membrane, and extracellular envi-ronment.

The Wrst events of protein N-linked glycosylationtake place in the ER. Accordingly, biosynthesis of thenucleotide-activated sugar precursors as well as theearly steps of assembly of the dolichol-linked oligosac-charide occur at the cytoplasmic side of the ER mem-brane whereas the further addition of four mannose andthree glucose residues takes place in the lumen of theER (Burda and Aebi, 1999). This results in the forma-tion of dolichol-PP-GlcNAc2Man9Glc3 intermediatewhich is transferred en bloc by the oligosaccharyltrans-ferase complex (OST) to a selected Asn-X-Ser/Thrsequence of nascent polypeptides (Knauer and Lehle,1999). N-Linked oligosaccharide is further modiWedand extended by the removal and addition of sugar resi-dues along the ER and Golgi transport to generatediVerent forms of N-glycans (Dean, 1999).

Entamoeba histolytica, the protozoan parasiteresponsible for human amebiasis, is estimated to causesevere disease in 48 million people, killing 70,000 eachyear [WHO/PAHO/UNESCO, 1997]. Since the earlystudies of Entamoeba cell biology, it became quite fas-cinating the fact that the nucleus is the only organelleidentiWed by electron microscopy. A cytosol abundantin apparently undiVerentiated vesicles and vacuoles ofvarious sizes but lacking other typical eukaryoticorganelles such as mitochondria, Golgi, and roughendoplasmic reticulum (rER) characterizes the ultra-structure of this parasite (Bakker-Grunwald and Wost-mann, 1993; Carrero and Laclette, 1996). Nevertheless,rER- and Golgi-like functions seem to be present in thetrophozoite. Amoebal virulence has been associated tothe active transport to the cell membrane of the Gal/GalNAc inhibitable lectin (Gilchrist and Petri Jr.,1999) and the secretion of cystein-proteases andamoebapore, among others (García-Rivera et al., 1999;Que and Reed, 1997; Leippe, 1997). Studies on theeVect of tunicamycin on trophozoites conWrmed N-gly-cosylation of the Gal/GalNAc inhibitable lectin (Mannet al., 1991). O-Glycosylation as well as the synthesis ofGPI-anchored proteins have also been reported(McCoy et al., 1993; Moody-Haupt et al., 2000; StanleyJr. et al., 1995). Brefeldin A-sensitive and -insensitiveprotein transport mechanisms seem to be present inE. histolytica trophozoites suggesting two diVerentsecretory pathways (Gosh et al., 1999; Manning-Celaet al., 2003). Despite all this evidence, amoebal subcel-

lular structures fullWlling ER- and Golgi-like functionshave not been conclusively demonstrated nor isolatedor characterized.

Two independent approaches were undertaken by ourgroups to investigate the existence of ER-functionalstructures in E. histolytica trophozoites. On one hand,enzymatic activities involved in the early reactions of thedolichol-linked oligosaccharide assembly were assessed.Accordingly, detergent-solubilized membranes of E. his-tolytica trophozoites displayed activities of DPMS,NAGPT, and NAGT (Vargas-Rodriguez et al., 1998;Villagómez-Castro et al., 1998). Another ER activity pre-sumptively involved in the N-glycan processing, such astype II-like �-glucosidase, which is responsible for theremoval of two �-1,3-linked glucosyl residues of the Glc-NAc2Man9Glc3 oligosaccharide in yeast and animal cells(Hersocovics, 1999a,b) was also detected in amoebamembranes (Zamarripa-Morales et al., 1999) and laterpuriWed and characterized (Bravo-Torres et al., 2004).

On the other hand, entamoebal genes coding forhighly conserved eukaryotic ER and Golgi residentproteins were cloned, thus providing the Wrst molecularevidence of functions related to these organelles inE. histolytica (Sánchez-López et al., 2000a). Theseincluded EhSRP54, which codes for a subunit of theSRP complex involved in the Wrst step of the secretorypathway, and the EhERD2 ortholog responsible for theretrieval of ER luminal proteins from post-ER com-partments (Ramos et al., 1997; Sanchez-Lopez et al.,1998). More recently, we have reported the isolation ofthe genes coding for a protein disulWde isomerase(EhPDI), a chaperone-like resident of the lumen of theER, which catalyzes the formation, breakage, and rear-rangements of disulWde bonds in nascent proteins(Ramos and Alagón, 2000), EhSTT3, a subunit ofthe OST complex (Gutiérrez et al., 2000), andEhSec61�-subunit, a core component of the ER proteintranslocation machinery (Sánchez-López et al., 2000b),respectively.

Here, we provide strong biochemical and molecularevidences supporting the presence of the machineryrequired for N-linked glycosylation of nascent proteinsin E. histolytica. Results of subcellular fractionationindicate that some of the enzymes involved in thedolichol pathway and the ER molecular markersEhSec61�-subunit, EhDPMS and EhPDI are all con-tained in membrane compartments with similar density.

2. Materials and methods

2.1. Strains and culture conditions

Trophozoites of E. histolytica, strain HM1:IMSS, weremaintained and propagated under axenic conditions inthe TYI-S-33 medium at 37 °C (Diamond et al., 1978).

M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373 365

2.2. Preparation of plasma membrane-free homogenate

Plasma membrane-free homogenates were obtainedfrom E. histolytica trophozoites by a modiWcation ofthe described by Aley et al. (1980). BrieXy, 1–4 £ 107 ice-chilled trophozoites were harvested at 280g at 4 °C,resuspended in PBS containing 10 mM MgCl2 and0.2 mg/ml concanavalin A and, after incubation on icefor 15 min, the suspension was centrifuged gently at200g for 1 min at 4 °C. The cell pellet was washed inPBS containing 0.25 mM MnCl2 and 0.5 mM CaCl2,resuspended in 10 mM Tris–HCl buVer, pH 7.5, con-taining 10 �M E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidine)butane] and incubated on ice. After 15min, cells were lysed by 25 strokes of a Potter–ElvehjemTissue Homogenizer w/PTFE pestle and the cellhomogenate was immediately layered on a two-stepgradient consisting of 8 ml of 0.5 M mannitol over a 4 mlcushion of 0.58 M sucrose that was spun at 250 g for30 min at 4 °C. Large plasma membrane fragments andother heavy debris that pelleted at the bottom of thegradient were discarded. The plasma membrane-freehomogenate was collected from the top of thegradient.

2.3. Lysis of trophozoites in slightly hypoosmotic conditions

This procedure is based on a modiWcation of themethod described by van Vliet et al. (1976). All opera-tions were carried out at 4 °C. BrieXy, midlog phase tro-phozoites were ice-chilled and resuspended at 107 cells/ml in BH buVer containing 200 mM sucrose, 1 mMEDTA, 10 mM NaHCO3, 5 mM PMSF, 0.5 mM E64,and a milieu slightly hypoosmotic (250 mOsmol/kg H2O)with respect to the trophozoite culture medium(356 mOsmol/kg H2O). Trophozoites were homogenizedby means of 10 strokes of a Potter–Elvehjem tissuehomogenizer w/PTFE pestle. Cell disruption was exam-ined by phase-contrast microscopy and sucrose concen-tration was immediately adjusted to 300 mM(362 mOsmol/kg H2O).

2.4. Sucrose gradient fractionation

The plasma membrane-free homogenate (10–30 mg)was layered on a continuous sucrose gradient (10–65%,w/v) that was centrifuged at »220,000g for 4h at 4 °C.Fractions (1.3 ml) were collected from the top to thebottom of the gradient and used immediately to determineprotein content, enzyme activities, and for slot blot analy-sis, as described below. Components of the microsomalfraction P57 were fractionated on a continuous sucrosegradient that was prepared and centrifuged as describedabove. Fractions (250�l) were collected from the top tothe bottom of the gradient and analyzed by Western blot.

2.5. Isolation of subcellular fractions by diVerential centrifugation

The trophozoite lysate (15–10 mg) obtained underslightly hypoosmotic conditions was centrifuged at 500gfor 10 min and the post-nuclear supernatant (PN) wassaved. The pellet containing nuclei, cell debris, andwhole amoeba cells was resuspended in BH buVer sup-plemented with 0.5% Nonidet P40 detergent and layeredon top of a 10 ml sucrose bed (1 M sucrose, 10 mM Tris–HCl, pH 8, and 1 mM EDTA). After centrifugation at6000g for 12 min, nuclei were recovered at the bottom ofthe tube and resuspended in 150–200 �l BH buVer (Nfraction). The post-nuclear supernatant was fractionatedby diVerential centrifugation as follows: 57,000g for30 min to obtain a S57 supernatant and a P57 pellet. TheS57 fraction was further centrifuged at 96,000g for90 min to separate cytosolic fractions (S96) from pellet(P96). P57 and P96 were resuspended in 250 and 100�lBH buVer, respectively. The distribution pattern of themolecular markers EhDPMS, EhSec61�-subunit,EhPDI, and Ehproteasome-� subunit in the subcellularfractions was assessed by Western blot analysis.

2.6. Enzyme assays

Activities of DPMS, NAGPT/NAGT, �-N-acetyl-glucosaminidase (�-NAcGase), acid phosphatase (AP),and �-glucosidase were measured using the correspondingsubstrates GDP-[14C]mannose, UDP-[14C]N-acetyl-glu-cosamine, 4-nitrophenyl N-acetyl-�-D-glucosaminide,4-nitrophenyl phosphate, and 4-methylumbelliferyl �-D-glucopyranoside essentially as described elsewhere(Anaya-Ruiz et al., 1997; Arias-Negrete et al., 1992;Bravo-Torres et al., 2004; Vargas-Rodriguez et al., 1998;Villagómez-Castro et al., 1998).

2.7. Antibodies

We have expressed 61 amino acids of the C-terminus ofEhSec61�-subunit (GenBank Accession No. AY730760)as a recombinant protein named His6£-DHFR-EhSec61�-subunit. This protein was used as immunogento raise rabbit antibodies against EhSec61�-subunit. Spe-ciWc antibodies were puriWed by aYnity chromatography onan immunoabsorbent of His6£-GFP-EhSec61�-subunitrecombinant protein coupled to CNBr-activated Sepharose4B. Antibodies against EhDPMS (TIGR IdentiWer number167.m00117) were produced in rabbits immunized with aHis6£-EhDPMS recombinant protein spanning 216 aminoacids of the hydrophilic N-terminus of EhDPMS and puri-Wed by aYnity chromatography on a column containing therecombinant protein. Anti-EhPDI antibodies were gener-ated by immunizing rabbits with a chimeric protein con-taining 187 amino acids of the C-terminus of EhPDI(GenBank Accession No. AY730725) fused to His6£-GFP

366 M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373

(His6£-GFP-EhPDI) and immunopuriWed by aYnity chro-matography on immunoabsorbent His6£-DHFR-EhPDIcoupled to CNBr-activated Sepharose 4B. Antibodiesagainst Ehproteasome-� subunit were prepared and puri-Wed as previously described by Sanchez et al. (2002).

2.8. Expression and analysis of proteins

Recombinant proteins were expressed in Escherichiacoli using the QIAexpress system (Qiagen). Western blotswere performed according to standard procedures. BrieXy,proteins were separated by SDS–PAGE (10% gel) underreducing conditions and electrotransferred to a nitrocellu-lose membrane (Bio-Rad). The membrane was blocked in0.1% non-fat milk, incubated with appropriate antibodies(at 0.1–2�g/ml) and developed with a goat anti-rabbitalkaline phosphatase conjugate (Zymed) and BCIP/NBT(Zymed). Slot blot analysis were performed by loading 5–10�l of sucrose gradient fractions (in 500�l of 25 mMTris–HCl, pH 6.8, containing 1.25% SDS, 5mM PMSF,0.5 mM E64, and 5% glycerol) onto a PVDF membrane(Immobilon P, Millipore) placed in a Slot blotter(PR648—Amersham Pharmacia Biotech). Protein bind-ing to the membrane was allowed for 1 h followed by lowvacuum Wltering. Membrane blocking and antibody incu-bation were performed according to standard Westernblot procedure. Densitometry of Western and slot blotmembranes was done using the NIH Image 1.62 softwarepackage. Results were plotted as percentage with respectto total signal detected on the membrane. Protein quanti-tation was done with the Micro BCA kit (PIERCE).

2.9. IdentiWcation of new E. histolytica genes by analysis in silico

Preliminary sequence data for E. histolytica genomeobtained by The Wellcome Trust Sanger Institute Patho-gen Sequencing Unit, in collaboration with Graham Clarkat the London School of Hygiene and Tropical Medicine(http://www.sanger.ac.uk/Projects/E_histolytica/) and bythe International Entamoeba Genome Sequencing Projectsupported by award from the National Institute ofAllergy and Infectious Diseases, National Institutes ofHealth (http://www.tigr.org/tdb/e2k1/eha1/) are available.Sequence searching was performed directly at their corre-sponding BLAST servers using amino acid sequences ofyeast proteins of interest as queries.

3. Results

3.1. Subcellular fractionation of ER-like enzymatic activities

Although it has been established that DPMS andNAGPT/NAGT activities in E. histolytica trophozoites

are associated to internal membranes, further fraction-ation to determine their subcellular distribution andtopological arrangement, as well as the analysis of thesequential activities of the lipid-oligosaccharide biosyn-thetic enzymes have remained undetermined. To evaluatethe resolution of structures containing amoebal sugar-donor enzymes and �-glucosidase activities, from otherinternal membranes, plasma membrane-free homoge-nates were fractionated by isopycnic centrifugation insucrose density gradients. The distribution of protein andthe monitored enzymes along the gradient is illustrated inFig. 1. DPMS and NAGPT/NAGT activities displayedan overlapping position, well resolved in fractions 16–22with a density range of 1.121–1.158 g/ml, and representing»80 and 82% of the total activity, respectively. Although�-glucosidase activity was present along the gradient,»56% of enzyme activity was recovered in a rather dis-crete peak at the top of the gradient (fractions 5–9; 1.054–1.082 g/ml) that may correspond to the cytosolic content.The remaining activity was poorly resolved.

To estimate the extent of resolution of the sucrosedensity gradient, we have also examined the distributionof acid phosphatase (AP) and �-N-acetyl-glucosamini-dase (�-NAcGase), a membrane-bound and a luminalenzyme, respectively, commonly used as markers forlysosomes in eukaryotes (Roerick et al., 1996; Spectoret al., 1998). About 22 and 48% of AP activity wasenriched in fractions 17–21 (1.128–1.151 g/ml) and 22–27 (1.157–1.112 g/ml), respectively. Centrifugationunder these conditions failed to resolve �-NAcGasewhich was detected all along the gradient, with a dis-crete peak of activity (28%) in fractions 7–11 (1.061–1.0935 g/ml). About 49% of activity remained poorlyresolved in fractions of higher density (17–27; 1.1282–1.1929 g/ml). This may be due to disruption of lyso-some-like structures and release of their content duringcell homogenization.

Taken together, our results clearly indicate that ER-like functional microsomes can be resolved from othercellular compartments based on their relative density.Further attempts to purify and better characterize thesemembrane vesicles were reinforced by the use of molecu-lar markers, as will be described later.

3.2. Fractionation of EhSec61�-subunit by sucrose density gradient

In prokaryotes and eukaryotes, proteins destined to thesecretory pathway are translocated to the periplasmic orthe ER membrane through an heterotrimeric membraneprotein complex called SecYEG or Sec61, respectively(Johnson and van Waes, 1999). In a previous study, wereported the cloning of the EhSec61�-subunit gene (Sán-chez-López et al., 2000b). Therefore, we considered it nec-essary to compare the distribution proWles of thismolecular marker and DPMS activity. The former was

M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373 367

detected with speciWc antibodies that recognized a singleband of the expected size (»40kDa) as judged by Westernblot analysis (see Section 3.3). A comparative proWle ofdistribution of EhSec61�-subunit and DPMS activity inthe gradient is shown in Fig. 2. Though EhSec61�-subunitwas detected from fraction 9 (1.083 g/ml) up to the bottomof the gradient and no sharp enrichment was observed, itspresence overlapped the peak of DPMS activity (fractions14–21) and then started to decrease. Although, we cannotrule out diVerences in density due to the loss of water ordiVusible material from cell structures containingEhSec61�-subunit, the wide density range of microsomescontaining EhSec61�-subunit is consistent with the heter-ogeneity of the signal recognized by EhSec61�-subunitantibodies in Wxed trophozoites (data not shown; Sanchezet al., 2005). Whether the EhSec61�-subunit detected cor-responded to active ER protein translocation machineries

(GreenWeld and High, 1999) or to unassembled, inactive,EhSec61�-subunit associated to diVerent cellular struc-tures remains to be explored.

3.3. Isolation of a microsomal fraction by diVerential centrifugation

To rule out the possibility that the diversity of micro-somes was due to non-speciWc fragmentation and re-organization of putative vesicles/vacuoles harboringEhSec61�-subunit, we proceeded to lyse the trophozoitesin a milieu slightly hypoosmotic with respect to the cellculture medium, as described by van Vliet et al. (1976).Shortly after breakage, osmolarity was adjusted and thehomogenate was fractionated by diVerential centrifuga-tion to separate four fractions. As shown in Fig. 3B, aconsiderable good preparation of a microsomal fraction

Fig. 1. Distribution proWle of diVerent enzyme activities in a continuous sucrose density gradient. A sample of plasma membrane-free homogenate ofE. histolytica trophozoites, routinely corresponding to 10–30 mg of protein, was layered on the top of a sucrose gradient (1–64%, w/v) that was centrifugedand fractionated in the conditions described in the text. Fractions (1.3 ml) were used to measure protein concentration and the indicated enzyme activities(closed circles), as well as sucrose density (open circles). For each enzyme, activity is expressed as percentage relative to the total enzyme activity present inthe gradient, respectively. Enzyme activities are as follows: DPMS, dolichol-P-mannose synthase; NAGPT/NAGT, UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase/UDP-D-GlcNAc:dolichol-PP GlcNAc transferase; �-glucosidase; acid phosphatase; and �-NAcGase, �-N-acetyl-glucosaminidase.

368 M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373

enriched in the ER-like marker EhSec61�-subunit wasobtained after pelleting the post-nuclear supernatant at»57,000g (P57 fraction). No signal was detected in thenuclear fraction nor the post-microsomal fractions (P96and S96). Given the isolation of a putative ER-equiva-lent fraction and taking advantage of the fact thatE. histolytica genome sequences databases are available,we cloned, in silico, the gene coding for EhDPMS, a pro-tein with a theoretical weight of 38.7 kDa (see Section3.5). On this background, speciWc antibodies againstEhDPMS were raised in order to extend the analysis toother ER molecular markers. A single band of »35 kDawas recognized by the anti-EhDPMS antibodies as indi-cated by Western blot analysis of a trophozoite lysate(see Fig. 3A). Expression and fractionation of EhPDI, athird ER-like marker (a protein with a predicted molecu-

Fig. 2. Distribution proWle of DPMS activity and EhSec61�-subunit ina sucrose density gradient. A sample of plasma membrane-freehomogenate of E. histolytica trophozoites was layered on the top of asucrose gradient (10–65%, w/v) that was centrifuged and fractionatedin the conditions described in the text. Fractions (1.3 ml) were used tomonitor density (open circles), as well as DPMS activity (closed cir-cles) and the EhSec61�-subunit (open triangles). DPMS activity isexpressed as percentage relative to the total enzyme activity present inthe gradient. EhSec61�-subunit, as detected by slot blot, is plotted aspercentage relative to the total amount present in the gradient, as mea-sured by densitometry.

lar mass of 35 kDa) was also investigated. As expected,EhDPMS and EhPDI (Figs. 3A and C) were retainedmainly in the microsomal fraction P57, leading to specu-late that dolichol pathway and ER-like functions suchtranslocation and folding of nascent proteins may becontained in the same microsomal fraction.

An interesting observation was the diVerent fraction-ation proWle of the 26S proteasome complex (typically2100 kDa), as detected by antibodies speciWc against theEhproteasome-� subunit (»25 kDa; Sanchez et al., 2002).Although Ehproteasome-� subunit was mainly recov-ered in the P96 fraction, it was not restricted to this frac-tion since a signiWcant signal was observed in thecytosolic fraction (S96; Fig. 3D). A poor signal was occa-sionally detected in P57, which may be due to contami-nation or unstability of the complex rather than a trueassociation to microsomes.

3.4. Fractionation of P57 on a sucrose density gradient

When the components of the microsomal fraction P57were separated on a sucrose density gradient, we observeda co-fractionation of EhSec61�-subunit and EhDPMS,overlapping a density range of 1.092–1.184 g/ml, where 80and 90% of the EhDPMS and EhSec61�-subunit signal,respectively, were concentrated between fractions 4 and10 (Fig. 4). EhPDI was not reproducibly detected in thegradient fractions, possibly due to a loss of soluble pro-teins during resuspension of fraction P57 and the subse-quent density fractionation (data not shown).

3.5. In silico search for genes related to the dolichol-linked oligosaccharide biosynthesis pathway

The structure and biosynthesis of the dolichol-linkedoligosaccharide core has been poorly studied in E. histoly-tica. To get an insight into the sequential steps involved inthis pathway, we applied standard bioinformatics proto-cols to retrieve putative genes from E. histolytica genomedatabases. Their relevance in the dolichol-linked oligosac-charide pathway is illustrated in Fig. 5 (marked with an

Fig. 3. DiVerential pelleting of ER molecular markers. A trophozoite lysate obtained in slightly hypoosmotic conditions was subjected to diVerentialcentrifugation. After each step, 40 �g protein of each sample were analyzed by Western blot probed with the corresponding speciWc antibodies. (A)anti-DPMS; (B) anti-EhSec61�-subunit; (C) anti-EhPDI; and (D) anti-Ehproteasome-� subunit. T, total lysate; N, nuclear fraction; P57 microsomalfraction (57,000g); P96, post-microsomal pellet (96,000g) and S96 soluble fraction. Molecular mass standards are in kDa.

M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373 369

Fig. 4. Analysis of EhDPMS and EhSec61�-subunit contained in the microsomal fraction (P57) and resolved in a sucrose density gradient. (A) West-ern blot analysis of fractions 1–13, probed with speciWc antibodies against either EhDPMS or EhSec61�-subunit. (B) Densitometry of EhDPMS(closed circles) and EhSec61�-subunit (open triangles) signals, as determined by Western blot, expressed as percentage of signal relative to the totalamount detected on the membrane. Open circles, density as g/ml.

Fig. 5. The biosynthesis of nucleotide-activated sugar donors and dolichol-linked oligosaccharide based on S. cerevisiae pathways. Sequential enzymes orgenes involved in individual reactions are as follows: NAGPT (ALG7, UDP-GlcNAc:dolichol-P-GlcNAc phosphotransferase; EC 2.7.8.15), NAGT(UDP-GlcNAc:dolichol-PP-GlcNAc transferase; EC 2.4.1.141-), ALG1 (GDP mannose:dolichol-PP-chitobiose mannosyltransferase; EC 2.4.1.142),ALG2 (GDP-mannose:dolichol-PP-GlcNAc2Man2 �1,3-mannosyltransferase; EC 2.4.1.132), glycosyltransferase (EC 2.4.1.-), ALG11 (GDP-man-nose:glycolipid �1,2-D-mannosyltransferase; EC 2.4.1.131), ALG3 (�-1,3-mannoysyltransferase; EC 2.4.1.130), ALG12 (�-1,6-mannosyltransferase; EC2.4.1.130), ALG9 (mannosyltransferase; EC 2.4.1.130), ALG6 (�-1,3-glucosyltransfease, EC 2.4.1.-), ALG8 (�-1,3-glycosyltransferase; EC 2.4.1.-), ALG10(�-1,2-glucosyltransferase; EC 2.4.1.-). AGM1 (phosphoacetylglucosamine mutase; EC 5.4.2.3), and UAP1 (UDP:GlcNAc pyrophosphorylase; EC2.7.7.23). ALG4 (SEC53, phosphomannomutase; EC 5.4.2.8), PSA1 (GDP:�-D-mannose pyrophosphorylase; EC 2.7.7.13), and DPM1 (DPMS, dolichol-P-mannose synthase; EC 2.4.1.83). PGM1 (phosphoglucomutase; EC 5.4.2.2), UGP1 (UDP:glucose pyrophosphorylase; EC 2.7.7.9), and ALG5 (DPGS,dolichol-P-glucose syntase; EC 2.4.1.117). E. histolytica enzymes and putative homologues of well-conserved genes are marked with an asterisk.

370 M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373

asterisk). Table 1 summarizes E values and TIGR Identi-Wer numbers of our Wndings.

Sequences coding for putative genes involved in thebiosynthesis of the nucleotide-activated donors UDP-N-acetyl-glucosamine, UDP-glucose, and GDP-mannosewere found in the E. histolytica genome. We have alsoidentiWed and expressed an amoebal EhDPMS gene cod-ing for a protein that shares 45 and 40% of amino acididentities with Leishmania mexicana and Saccharomycescerevisiae dolichol-P-mannose synthase, respectively(data not shown). Analysis of the EhDPMS proteinsequence also predicted two putative transmembrane(TM) domains at the C-terminus of the protein (data notshown). By analogy with L. mexicana and S. cerevisiaeorthologs (Ilgoutz et al., 1999; Zimmerman and Rob-bins, 1993), TM domains of EhDPMS may be responsi-ble for its attachment to the membrane (see Fig. 3A). Incontrast, mammalian dolichol-P-mannose synthaselacks the TM domain and requires the association with asmall hydrophobic protein (Dpm2) to be properly stabi-lized in the ER membrane (Maeda et al., 1998).

Though an exhaustive biochemical characterizationof nucleotide-activated sugar biosynthesis and sugartransfer to lipid carrier is still required, our resultsallowed us to postulate the stepwise assembly of doli-chol-PP-GlcNAc2Man5 core in E. histolytica. Our analy-sis retrieved putative homologues of the well-conservedyeast/mammalian/trypanosomatids genes NAGPT/ALG7, ALG1, ALG2, and ALG11 coding for nucleotide-activated sugar-dependent glycosyltransferases involvedin the initial steps of the oligosaccharide assembly andtypically associated to the cytosolic face of the ER(Fig. 5).

An unexpected Wnding was that no sequences relatedto luminal ER dolichol-P-monosaccharide-dependentmannosyltransferases (ALG3, ALG9, and ALG12), norglucosyltransferases (ALG6, ALG8, and ALG10)

involved in the later steps of the oligosaccharide corehave been found (see Fig. 5). However, we have observedthat the E. histolytica genome codes for a putative ER �-1,4-mannosyltransferase, PIG-M (Table 1). The Wrstmannose of the phosphatidyl-inositol glycan anchor(GPI) is added by PIG-M, using dolichol-P-mannose assugar donor (Maeda et al., 2001).

An interesting result was the absence of hits forsequences coding for a putative dolichol-P-glucose syn-thase (DPGS or Alg5p). In yeast and mammalian cells,DPGS/Alg5p is the glucosyltransferase required for thesynthesis of dolichol-P-glucose, the donor of the threeglucosyl residues added lately during the oligosaccharidecore assembly (Burda and Aebi, 1999). The lack of amoe-bal DGPS/ALG5 sequences led us to speculate that thestructure of the oligosaccharide core in E. histolytica maynot contain glucosyl residues, thus implying no need for aglucosyltransferase (see below). In other organisms suchas trypanosomatids which are defective in the formationof the dolichol-P-glucose, underglucosylated oligosac-charide cores are produced (Parodi, 1993).

4. Discussion

Basic concepts on the structural organization ofeukaryotic cells and function of organelles are basedmainly on studies performed in model organisms, such asyeast and mammalian cells. Years ago, we considered thatisolation of genes encoding well-conserved ER-residentproteins and assessment of ER-like enzymatic activitieswill lead us to the unequivocal identiWcation of ER-likestructures in Entamoeba trophozoites (Gutiérrez et al.,2000; Ramos et al., 1997; Ramos and Alagón, 2000; San-chez-Lopez et al., 1998, 2000b; Vargas-Rodriguez et al.,1998; Villagómez-Castro et al., 1998; Zamarripa-Moraleset al., 1999). Although our results provided strong

Table 1E. histolytica putative genes involved in the synthesis of nucleotide-activated sugar and dolichol-linked oligosaccharide core

a GenBank Accession No.b Dm, Drosophila melanogaster; Dd, Dictyostelium discoideum; Ath, Arabidopsis thaliana; Lm, Leishmania mexicana; Cp, Cryptosporidium parvum;

Sc, Saccharomyces cerevisiae.

Gene ontology TIGR identiWer number

E valueb Enzyme EC number Reference

UAP1 138.m00095 6.7e-61 (Dm) UDP-GlcNAc pyrophosphorylase 2.7.7.23 This studyPGM1 Y14444.1a 1e-142 (Dd) Phosphoglucomutase 5.4.2.2 Ortner et al. (1997)UGP1 106.m0013 1e-105 (Dd) UDP-glucose pyrophosphorylase 2.7.7.9 This studySEC53/ALG4 26.m00333 3e-65 (Ath) Phosphomannomutase 5.4.2.8 This studyDPM1 167.m00117 2e-46 (Lm) Dolichol-P-mannose synthase 2.4.1.83 This studyNAGPT/ALG7 142.m00140 1e-55 (Cp) UDP-N-GlcNAc:dolichol-P

GlcNAc-1-phosphotransferase2.7.8.15 This study

ALG1 192.m00078 1e-65 (Sc) GDP-mannose:dolichol-PP-chitobiose mannosyltransferase

2.4.1.142 This study

ALG2 90.m00172 6e-64 (Sc) GDP-mannose:dolichol-PP- GlcNAc2Man2 �1,3mannosyltransferase

2.4.1.132 This study

ALG11 3.m00560 1e-57 (Sc) GDP-mannose:glycolipid �1,2mannosyltransferase 2.4.1.- This study

M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373 371

evidences supporting a well-conserved molecular andenzymatic ER-like machinery, our main question is stillholding.

DPMS, NAGPT/NAGT, PDI, and the Sec61 com-plex are considered to be markers of the ER, based onfunctional/structural, as well as subcellular fractionationand indirect immunolocalization studies in a number ofhigher and lower eukaryotes (Burda and Aebi, 1999;Kamhi-Nesher et al., 2001). Confocal microscopy andimmunolocalization in Wxed E. histolytica trophozoites,revealed a punctate distribution of EhDPMS andEhSec61�-subunit, diVerent from the typical perinuclearsignal observed in almost every eukaryotic cell (data notshown; Sanchez et al., 2005). The literature describessimilar images regarding cytolocalization of proteinsinvolved in either phagocytosis/endocytosis (RabGTP-ases) (Aley et al., 1984; Rodríguez et al., 2000; Welteret al., 2002), ER-functions (BiP and PDI) (Gosh et al.,1999; Manning-Cela et al., 2003), post-ER compart-ments (ERD2, RabB, and �COP) (Manning-Cela et al.,2003; McGugan and Temesvari, 2003; Rodríguez et al.,2000) or post-Golgi vesicles (Rab8) (Juárez et al., 2001),all of them associated to a large number of vacuoles andvesicles throughout the cytoplasm of E. histolytica tro-phozoites. However, a systematic analysis of their rela-tive spatial distribution within the parasite remained achallenging question. Taken together, these observationsprompted us to consider a biochemical analysis based onthe subcellular fractionation of such a set of enzymaticand molecular ER markers.

Here, we describe experimental conditions thatallowed us to address the question of whether ER-related enzymes were present in the same microsomalfractions than other ER-molecular markers. Accord-ingly, we demonstrated that DPMS and NAGPT/NAGT activities and EhSec61�-subunit co-fractionatein a continuous sucrose gradient and appear to be asso-ciated to microsomes of similar density. In a previouswork, we showed the presence in E. histolytica of solubleand membrane-bound forms of �-glucosidase (Zamarr-ipa-Morales et al., 1999). Whereas the soluble enzyme ismost probably involved in the general carbohydratemetabolism than in N-glycan processing (Bravo-Torreset al., 2003) most of the properties of the membrane-associated enzyme are consistent with a processing, typeII-like �-glucosidase (Bravo-Torres et al., 2004). Here,56% of the �-glucosidase activity was recovered in apeak at the top of the gradient, most likely correspond-ing to the soluble form of the enzyme. The remainingactivity was not well resolved along the rest of the gradi-ent. Finally, results of AP distribution observed here arein good agreement with previous studies. Aley et al.(1980) reported that »70 and »15% of the AP activity inE. histolytica was recovered in the internal and crudeplasma membranes, respectively. Anaya-Ruiz et al.(1997) found that »85% of the E. histolytica AP activity

was associated to internal membranes whereas an inde-pendent study in Entamoeba invadens showed that 40,18, 11, and 15% of the activity was recovered in thephagolysosome, the crude plasma membrane, the micro-somes and the 100,000g supernatant, respectively (vanVliet et al., 1976). AP activity was retained in membranesof lysed phagolysosomes and banded at 33% sucrose(»1.1270 g/ml) in a continuous density gradient. In par-allel, »50 and »30% of the �-NAcGase activity wasdetected in the phagolysosomal and in the cytosolic frac-tions, respectively (van Vliet et al., 1976). Aley et al.(1980) also detected �-NAcGase activity in the solubleintra-lysosomal fraction. In our experimental conditions,�-NAcGase activity was, however, poorly resolved.

Assessment of membrane-bound enzyme activities byfractionation in sucrose density gradients turned to be alaborious and time-consuming routine. This motivated theisolation of microsomal fractions by diVerential centrifu-gation. EhDPMS, EhSec61�-subunit, and EhPDI markerswere conWned to the microsomal fraction isolated by two-step diVerential centrifugation of trophozoites lysed inslightly hypoosmotic conditions. Faint signals fromEhDPMS, EhSec61�-subunit, and EhPDI were occasion-ally detected in the nuclear fraction. However, when thepost-nuclear supernatant was spun at 57,000g, the threeER markers were contained in the P57 microsomal frac-tion. The post-microsomal material was further centri-fuged at higher speed (96,000g) in order to sedimentmacromolecular complexes and, eventually, putative smallmembranous structures or vesicles. Neither EhDPMS,EhSec61�-subunit, nor EhPDI were present in the P96fraction. S96 was also free of ER markers. On the otherhand, ER markers were resolved from the proteasomemacromolecular complex. Ehproteasome-� subunit wasenriched in, but not restricted to, the P96 fraction. A minorproportion of the Ehproteasome-� subunit signal wasdetected in fraction S96. As reported by Sanchez et al.(2002), we did not detect Ehproteasome-� subunit associ-ated to the nuclear fraction. In a previous study, Scholzeet al. (1996) described a proteasome-like activity containedin a large molecular weight complex isolated from a cyto-solic fraction (100,000g) of E. histolytica trophozoites.

Our results are in line with fractionation of microsomesfrom E. histolytica homogenates conducted by othergroups. Internal membranes, collected as a 40,000g pellet,supported the synthesis of E. histolytica proteophospho-glycan (PPG), an abundant cell-surface glycoconjugatewith a novel GPI-anchor (Gal1Man2GlcNAc-myo inosi-tol) linked to a highly acidic polypeptide. Further fraction-ation of internal membranes on a discontinuous sucrosegradient revealed that the ER-marker BiP was present inthe interphase between the 25 and 30% sucrose layers,whereas the interphase between the 30 and 35% sucroselayers, containing galactosyltransferase activity, wasenriched in enzymes involved in the in vitro synthesis ofPPG (Arya et al., 2003). On this background, it is thus

372 M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373

relevant to investigate the synthesis of the dolichol-likedoligosaccharide in microsomal fractions immunoprecipi-tated with either anti-EhDPMS or anti-EhSec61�-subunit.

In eukaryotic cells, it has been established that the syn-thesis of dolichol-P-monosaccharides and the transfer oftwo GlcNAc residues to dolichol-P by NAGPT/NAGTand Wve mannose residues (from GDP-mannose) to formdolichol-PP-GlcNAc2Man5 occur at the cytosolic face ofthe ER. Thereafter, dolichol-P-monosaccharides and dol-ichol-PP-GlcNAc2Man5 are somehow translocated intothe luminal side to resume the synthesis of the full-lengtholigosaccharide core. On the other hand, based on studiesin yeast, it has been proposed that for each individualstep in the biosynthesis of the dolichol-linked oligosac-charide, one speciWc and well conserved glycosyltransfer-ase exists (Oriol et al., 2002). In the case of E. histolytica,identiWcation of putative genes EhALG7, EhALG1,EhALG2, and EhALG11 led us to postulate the synthesisof the dolichol-PP-GlcNAc2Man5 precursor in this para-site. However, amoebal genes related to dolichol-P-monosaccharide-dependent glycosyltransferases involvedin successive steps of the dolichol-linked oligosaccharideassembly were not found.

According to the model proposed by Oriol et al.(2002) all glycosyltransferases using dolichol-P-mono-saccharide as donor substrate were originated by sev-eral events of gene duplication from a hypotheticalcommon ancestral gene. In this regard, an E. histolyticaputative �-1,4-mannosyltransferase identiWed in thisstudy (PIG-M) may be involved in the transfer of themannosyl residue from the dolichol-P-mannose sugardonor to GPI-anchor proteins. The analysis of theexpression of putative EhPIG-M and EhALG genes introphozoites, as well as in encysting Entamoeba specieswhere a high demand of cell wall synthesis has to be sat-isWed, will be the matter of future research.

In summary, we have set up a two-step procedure forthe isolation of an ER-microsomal fraction as an initialstep towards the biochemical, structural, and functionalcharacterization of the endomembrane system of E. his-tolytica trophozoites. We have also demonstrated thatpart of the dolichol-linked oligosaccharide pathway andputative ER-translocation and folding machineries arecontained in the same ER-microsomal fraction. Molecu-lar evidence provided by our in silico Wndings suggest thestepwise assembly of the dolichol-PP-GlcNAc2Man5core in E. histolytica trophozoites, a highly interestingaspect of the N-glycosylation process in this parasite.Characterization of the dolichol-PP-glycans structureremains as a challenging matter to explore in the future.

Acknowledgments

This work was supported by CONACyT (ConsejoNacional de Ciencia y Tecnología) Grants 33079-N and

27818-N, and Dirección General de Asuntos del Per-sonal Académico-UNAM Grant 208400. We thank Fel-ipe Olvera for technical assistance, Shirley Ainsworth forbibliographical assistance and Dr. George Odell for crit-ical reading of the manuscript, as well as Ricardo Ciria,Abel Linares, Arturo Ocadiz, Juan Manuel Hurtado,and Alma Martínez for computer support.

References

Aley, S.B., Scott, W.A., Cohn, Z.A., 1980. Plasma membrane of Ent-amoeba histolytica. Journal of Experimental Medicine 152, 391–404.

Aley, S.B., Cohn, Z.A., Scott, W.A., 1984. Endocytosis in Entamoebahistolytica. Journal of Experimental Medicine 160, 724–737.

Anaya-Ruiz, M., Rosales-Encina, J.L., Talamás-Rohana, P., 1997.Membrane acid phosphatase (MAP) from Entamoeba histolytica.Archives of Medical Research 28, 182–183.

Arias-Negrete, S., Sabanero-López, M., Villagómez-Castro, J.C., 1992.Biochemical analysis of Entamoeba histolytica HM1 strain resistantto complement lysis. Archives of Medical Research 23, 135–137.

Arya, R., Mehra, A., Bhattacharya, S., Vishwakarma, R.A., Bhattach-arya, A., 2003. Biosynthesis of Entamoeba histolytica proteophospho-glycan in vitro. Molecular and Biochemical Parasitology 126, 1–8.

Bakker-Grunwald, T., Wostmann, C., 1993. Entamoeba histolytica as amodel for the primitive eukaryotic cell. Parasitology Today 9, 27–31.

Bravo-Torres, J.C., Calvo-Méndez, C., Flores-Carreón, A., López-Romero, E., 2003. PuriWcation and biochemical characterization ofa soluble �-glucosidase from the parasite Entamoeba histolytica.Antonie van Leeuwenhoek 84, 169–178.

Bravo-Torres, J.C., Villagómez-Castro, J.C., Calvo-Méndez, C., Flores-Carreón, A., López-Romero, E., 2004. PuriWcation and biochemicalcharacterisation of a membrane-bound �-glucosidase from the par-asite Entamoeba histolytica. International Journal of Parasitology34, 455–462.

Burda, P., Aebi, M., 1999. The dolichol pathway of N-linked glycosyla-tion. Biochimica et Biophysica Acta 1426, 239–257.

Carrero, J.C., Laclette, J.P., 1996. Molecular biology of Entamoeba his-tolytica: a review. Archives of Medical Research 27, 403–412.

Dean, N., 1999. Asparagine-linked glycosylation in the yeast Golgi.Biochimica et Biophysica Acta 1426, 309–322.

Diamond, L.S., Harlow, D.R., Cunnick, C.C., 1978. A new medium forthe axenic cultivation of Entamoeba histolytica and other Ent-amoeba. Transactions of the Royal Society of Tropical Medicineand Hygiene 72, 431–432.

García-Rivera, G., Rodríguez, M.A., Ocadiz, R., Martínez-López,M.C., Arroyo, R., Gonzalez-Robles, A., 1999. Entamoeba histoly-tica: a novel cystein protease and an adhesin form the 112 kDa sur-face protein. Molecular Microbiology 33, 56–569.

Gilchrist, C.A., Petri Jr., W.A., 1999. Virulence factor of Entamoeba his-tolytica. Current Opinion in Microbiology 2, 433–437.

Gosh, S.K., Field, J., Frisardi, M., Rosenthal, B., Mai, Z., Rogers, R.,Samuelson, J., 1999. Chitinase secretion by encysting Entamoebainvadens and transfected Entamoeba histolytica trophozoites: locali-zation of secretory vesicles, endoplasmic reticulum and Golgi appa-ratus. Infection and Immunity 67, 3073–3081.

GreenWeld, J.A., High, S., 1999. The Sec61 complex is located in boththe ER and the ER-Golgi intermediate compartment. Journal ofCell Science 112, 1477–1486.

Gutiérrez, A., Sanchez-Lopez, R., Ramos, M.A., Alagón, A., 2000.Cloning of the Entamoeba hystolytica STT3 gene, a subunit of theoligosaccharyltransferase complex. Archives of Medical Research31, S162–S164.

Hersocovics, A., 1999a. Processing glycosidases of Saccharomyces cere-visiae. Biochimica et Biophysica Acta 1426, 275–285.

M. Salgado et al. / Experimental Parasitology 110 (2005) 363–373 373

Hersocovics, A., 1999b. Importance of glycosidases in mammalian gly-coprotein biosynthesis. Biochimica et Biophysica Acta 1473, 96–107.

Ilgoutz, S.C., Zawadski, J.L., Ralton, J., McConville, M.J., 1999. Evi-dence that free GPI glycolipids are essential for growth of Leish-mania mexicana. EMBO Journal 18, 2746–2755.

Johnson, A.E., van Waes, M.A., 1999. The translocon: a dynamic gate-way at the ER membrane. Annual Review of Cellular and Develop-mental Biology 15, 799–842.

Juárez, P., Sanchez-Lopez, R., Stock, R.P., Olvera, A., Ramos, M.A.,Alagón, A., 2001. Characterization of the Ehrab gene, a marker ofthe late stages of the secretory pathway of Entamoeba histolytica.Molecular and Biochemical Parasitology 116, 223–228.

Kamhi-Nesher, S., Shenkman, M., Tolchinsky, S., Vigodman-Fromm,S., Ehrlich, R., Lederkremer, G.Z., 2001. A novel quality controlcompartment derived from the endoplasmic reticulum. MolecularBiology of the Cell 10, 1711–1723.

Knauer, R., Lehle, L., 1999. The oligosaccharyltransferase complexfrom yeast. Biochimica et Biophysica Acta 1426, 259–273.

Leippe, M., 1997. Amoebapores. Parasitology Today 13, 178–183.Maeda, Y., Tomita, S., Watanabe, R., Ohishi, K., Kinoshita, T., 1998.

DPM2 regulates biosynthesis of dolichol phosphate-mannose inmammalian cells: correct subcellular localization and stabilizationof DPM1, and binding to dolichol phosphate. EMBO Journal 17,4920–4929.

Maeda, Y., Watanabe, R., Harris, C.L., Hong, Y., Ohishi, K., Kinosh-ita, K., Kinoshita, T., 2001. PIG-M transfers the Wrst mannose toglycosylphosphatidylinositol on the lumenal side of the ER. EMBOJournal 20, 250–261.

Mann, B.J., Torian, B.E., Vedvick, T.S., Petri Jr., W.A., 1991. Sequenceof a cysteine-rich galactose-speciWc lectin of Entamoeba histolytica.Proceedings of the National Academy of Science of the UnitedStates of America 88, 3248–3252.

Manning-Cela, R., Marquez, C., Franco, E., Talamás-Rohana, P.,Meza, I., 2003. BFA-sensitive and insensitive exocytic pathways inEntamoeba histolytica trophozoites: their relationship to pathogen-esis. Cellular Microbiology 5, 921–932.

McCoy, J.J., Mann, B.J., Vedvick, T.S., Pak, Y., Heimark, D.B., PetriJr., W.A., 1993. Structural analysis of the light subunit of the Ent-amoeba histolytica galactose-speciWc adherence lectin. Journal ofBiological Chemistry 268, 24223–24231.

McGugan, G.C., Temesvari, L.A., 2003. Characterization of a Rab11-like GTPase, EhRab11, of Entamoeba histolytica. Molecular andBiochemical Parasitology 129, 137–146.

Moody-Haupt, S., Patterson, J.H., Mirelman, D., McConville, M.J.,2000. The major surface antigens of Entamoeba histolytica tropho-zoites are GPI-anchored proteophosphoglycans. Journal of Molec-ular Biology 24, 409–420.

Ortner, S., Binder, M., Scheiner, O., Wiedermann, G., Duchene, M.,1997. Molecular and biochemical characterization of phosphoglu-comutases from Entamoeba histolytica and Entamoeba dispar.Molecular and Biochemical Parasitology 90, 121–129.

Oriol, R., Martinez-Duncker, I., Chantret, I., Mollicone, R., Codogno,P., 2002. Common origin and evolution of glycosyltransferasesusing dol-P-mannosaccharides as donor substrate. Molecular Biol-ogy and Evolution 19, 1451–1463.

Parodi, A.J., 1993. N-glycosylation in trypanosomatid protozoa. Gly-cobiology 3, 193–199.

Que, X., Reed, S.L., 1997. The role of extracellular cystein proteinasesin pathogenesis of Entamoeba histolytica invasion. ParasitologyToday 13, 190–194.

Ramos, M.A., Mercado, G.C., Salgado, L.M., Sanchez-Lopez, R., Stock,R.P., Lizardi, P.M., Alagón, A., 1997. Entamoeba histolytica contains agene encoding a homologue to the 54 kDa subunit of the signal recog-nition particle. Molecular and Biochemical Parasitology 88, 225–235.

Ramos, M.A., Alagón, A., 2000. Molecular cloning of a gene encodinga PDI-like protein from Entamoeba histolytica. Archives of MedicalResearch 31, S173–S175.

Rodríguez, M.A., García-Pérez, R.M., García-Rivera, G., López-Reyes,I., Mendoza, L., Ortiz-Navarrete, V., Orozco, E., 2000. An Ent-amoeba histolytica Rab-like encoding gene and protein: functionand cellular location. Molecular and Biochemical Parasitology 108,199–206.

Roerick, T., Wu, Y.J., Vavougios, G., Hou, Y., Hinek, A., Mahuran,D.J., 1996. IdentiWcation of functional domains within the � and �subunits of �-hexosaminidase A through the expression of ��fusion proteins. Biochemistry 35, 10894–10903.

Sánchez, R., Alagón, A., Stock, R.P., 2002. Entamoeba histolytica:intracellular distribution of the proteasome. Experimental Parasi-tology 102, 187–190.

Sánchez, R., Saralegui, A., Olivos-García, A., Scapolla, C., Damonte,G., Sanchez-Lopez, R., Alagón, A., Stock, R.P., 2005. Entamoebahistolytica: intracellular distribution of the Sec61� subunit of thesecretory pathway and down-regulation by antisense peptidenucleic acids. Experimental Parasitology 109, 241–251.

Sanchez-Lopez, R., Gama-Castro, S., Ramos, M.A., Merino, E.,Lizardi, P.M., Alagón, A., 1998. Cloning and expression of the Ent-amoeba histolytica ERD2 gene. Molecular and Biochemical Parasi-tology 92, 355–359.

Sánchez-López, R., Gutiérrez, A., Juárez, P., Olvera, A., Olvera, F.,Ramos, M.A., Sánchez, R., Saralegui, A., Stock, R.P., Alagón, A.,2000a. Molecular genetics of the secretory pathway in E. histoly-tica: an overview. Archives of Medical Research 31, S151–S152.

Sánchez-López, R., Siminovich, B., Alagón, A., 2000b. Entamoeba his-tolytica codes for a protein homologue of the Sec61 alpha subunit,a component of the endoplasmic reticulum translocon. Archives ofMedical Research 31, S168–S170.

Scholze, H., Frey, S., Cejka, Z., Bakker-Grunwald, T., 1996. Evidencefor the existence of both proteasomes and a novel high molecularweight peptidase in Entamoeba histolytica. Journal of BiologicalChemistry 271, 6212–6216.

Spector, D.L., Goldman, R.D., Leinwand, L.A., 1998. Cells: A Labora-tory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor.

Stanley Jr., S.L., Tian, K., Koester, J.P., Li, E., 1995. The serine-richEntamoeba histolytica protein is a phosphorylated membrane pro-tein containing O-linked terminal N-acetylgucosamine residues.Journal of Biological Chemistry 270, 4121–4136.

van Vliet, H.H.D.M., Spies, F., Linnemans, W.A.M., Klepke, A., Opden Kamp, J.A.F., van Deenen, L.L.M, 1976. Isolation and charac-terization of subcellular membranes of Entamoeba invadens. Jour-nal of Cell Biology 71, 357–369.

Vargas-Rodriguez, L., Villagómez-Castro, J.C., Flores-Carreón, A.,López-Romero, E., 1998. IdentiWcation and characterisation ofearly reactions of asparagine-linked oligosaccharide assembly inEntamoeba histolytica. International Journal of Parasitology 28,1333–1340.

Villagómez-Castro, J.C., Calvo-Méndez, C., Vargas-Rodríguez, L., Flo-res-Carreon, A., Lopez-Romero, E., 1998. Entamoeba histolytica:solubilization and biochemical characterization of dolichol phos-phate mannose synthase, an essential enzyme in glycoprotein bio-synthesis. Experimental Parasitology 88, 111–120.

Welter, B.H., Laughlin, R.C., Temesvari, L.A., 2002. Characterizationof a Rab7-like GTPase, EhRab7: a marker for the early stages ofendocytosis in Entamoeba histolytica. Molecular and BiochemicalParasitology 121, 254–264.

Zamarripa-Morales, S., Villagómez-Castro, J.C., Calvo-Méndez, C.,Flores-Carreón, A., López-Romero, E., 1999. Entamoebahistolytica: identiWcation and properties of membrane-bound

and soluble �-glucosidases. Experimental Parasitology 93, 109–115.

Zimmerman, J.W., Robbins, P.W., 1993. The hydrophobic domain ofdolichyl-phosphate-mannose synthase is not essential for enzymeactivity or growth in Saccharomyces cerevisiae. Journal of Biologi-cal Chemistry 268, 16746–16753.