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Volume 270, Number 49, Issue of December 8, 1995 pp. 29307-29313
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Novel Giardia lamblia Cyst Wall Protein with Leucine-rich Repeats
IMPLICATIONS FOR SECRETORY GRANULE FORMATION AND PROTEIN ASSEMBLY INTO THE CYST WALL (*)

(Received for publication, August 22, 1995; and in revised form, October 4, 1995)

Hugo D. Luján (1), (§),  Michael R. Mowatt (1),  John T. Conrad (1),  Blair Bowers (2),  Theodore E. Nash (1)

From the  (1)Laboratory of Parasitic Diseases, NIAID and the (2)Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Giardia lamblia trophozoites, like most intestinal parasitic protozoa, undergo fundamental biological changes to survive outside the intestine of their mammalian host by differentiating into infective cysts. This complex process entails the coordinated production, processing, and transport of cyst wall constituents for assembly into a protective cyst wall. Yet, little is known about this process and the identity of cyst wall constituents. We previously identified a 26-kDa cyst wall protein, CWP1. In the present work, using monoclonal antibodies to cyst wall antigens, we cloned the gene that encodes a novel 39-kDa cyst wall protein, CWP2. Expression of CWP1 and CWP2 was induced during encystation with identical kinetics. Soon after synthesis, these two proteins combine to form a stable complex, which is concentrated within the encystation-specific secretory granules before incorporation into the cyst wall. Both proteins contain five tandem copies of a 24-residue leucine-rich repeat, a motif implicated in protein-protein interactions. Unlike CWP1, CWP2 has an extremely basic 121-residue COOH-terminal extension that might be involved in the sorting of these proteins to the secretory granules.

INTRODUCTION

Giardia lamblia is one of the most common protozoan parasites of man and other vertebrates. Giardia exists in two developmental forms, trophozoites and cysts. Trophozoites, the motile dividing stage, inhabit the upper small intestine and are responsible for the epidemic and endemic diarrhea caused by this organism. Cysts, the infective form of the parasite, develop in the intestine and are excreted in the feces. Cyst formation, or encystation, is essential for the survival of Giardia outside the host intestine and for the transmission of the parasite among susceptible hosts (reviewed in (1) and (2) ).

Giardia constitutes the earliest branching lineage among eukaryotes(3, 4) , and encystation may represent an adaptive response that eukaryotes developed early in evolution to survive harmful conditions. Encystation ultimately results in the assembly of a protective cyst wall, which confers resistance to environmental factors, including hypotonic lysis(1, 2) . The mechanism of cyst wall formation is unknown, but its assembly is preceded by concerted developmental changes in the trophozoite including the synthesis, packaging, and release of secretory components destined for the cyst wall(5, 6) . During encystation, biosynthetic and molecular sorting capacities are induced and culminate in the appearance of the encystation-specific vesicles (ESVs), (^1)which transport cyst wall components to the plasma membrane for release to the cell exterior(5, 6) . Ultrastructural studies indicated that the rigid cyst wall consists of interconnected filamentous components (7, 8, 9) resistant to treatment by amyloglucosidase, SDS, and proteinases(10, 11) .

The molecular constituents of the cyst wall are largely undefined although, immunochemically, this extracellular structure contains antigens whose synthesis is induced in encysting trophozoites(6, 9, 12, 13, 14, 15) . Furthermore, galactosamine and N-acetylgalactosamine are undetectable in nonencysting trophozoites, but enzymes required for galactosamine and N-acetylgalactosamine synthesis and metabolism are induced during encystation (10, 16, 17, 18) and presumably account for the abundance of N-acetylgalactosamine in the cyst wall(17) . Among the molecules that comprise the cyst wall, only one protein has been defined by cloning and sequencing its corresponding gene(6) . The gene CWP1 predicts an acidic and leucine-rich protein of M(r) 26,000 likely targeted to the secretory pathway by an amino-terminal signal peptide. The accumulation of CWP1 in a disulfide-linked form in encysting trophozoites and its five tandemly arrayed 24-residue leucine-rich repeats (LRRs) suggest that this protein is a constituent of the fibrillar component of the cyst wall(6) .

LRRs are found in a functionally diverse group of proteins related by the ability to participate in protein-protein interactions(19, 20) . LRRs are believed to confer conformational flexibility upon proteins in which they reside, thereby promoting protein-protein interactions (19, 20, 21) . The repeats in CWP1 are characteristic of the extracellular domains of some cell surface adhesive proteins and receptor-like protein kinases, as well as secreted proteins of the extracellular matrix(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) .

The differentiation of Giardia trophozoites to cysts constitutes an important and novel model system for studying gene regulation(6) , organelle biogenesis(5, 18) , and the biosynthesis and assembly of proteins into an extracellular superstructure(6) . Ultimately, the understanding of these processes will also facilitate the design of new therapeutic agents against this important human pathogen.

In this work, we show that two monoclonal antibodies (mAbs), generated against purified encystation-specific secretory vesicles and purified cyst walls, recognize a novel cyst wall protein, CWP2, which contains five tandem copies of a LRR. Expression of both CWP2 and CWP1 is induced coordinately during encystation. Soon after their synthesis, the two proteins form a stable complex and colocalize in the ESVs before release to form the cyst wall. Implications of the structure of these proteins in the biogenesis of secretory granules and in the formation of the cyst wall are discussed.

EXPERIMENTAL PROCEDURES

Giardia Cultivation and Encystation in Vitro

Trophozoites of the G. lamblia isolate WB, clone WB/1267(31) , were cultured in TYI-S-33 medium supplemented with 10% adult bovine serum and 0.5 mg/ml bovine bile (growth medium) as described(32) . Encystation of trophozoite monolayers was accomplished by the method described by Boucher and Gillin(33) .

Purification of Encystation-specific Vesicles and Cyst Walls

To purify ESVs, Giardia trophozoites were induced to encyst, harvested, homogenized, and fractionated as reported (18) . After the primary isopycnic centrifugation of the homogenate, approximately 17 fractions (400 µl each) were collected from the bottom of the gradient(18) . To detect ESVs in the sucrose gradient, a 20-µl aliquot from each fraction was analyzed by immunoblotting using the mAb 5-3C(34) , which is specific for CWP1(6) . Fractions containing CWP1 were pooled and washed with 13 ml of 0.25 M sucrose containing 10 mM sodium phosphate by centrifugation at 100,000 times g for 1 h in a Beckman type 40 rotor. ESV pellets were resuspended in 0.5 ml of 0.25 M sucrose in phosphate and loaded on a preformed Percoll gradient consisting of 1 ml each of 10, 15, 20, 25, 30, 35, 40, and 45% of a Percoll stock solution (90% Percoll in 0.25 M sucrose, 10 mM sodium phosphate). The gradient was centrifuged for 2 h in a Beckman SW 40 rotor at 20,000 times g at 4 °C; 1-ml fractions were then collected from the bottom, washed, and analyzed as described above. The fractions containing ESVs were pooled, processed for electron microscopy as described below (Fig. 1A), and assayed for malate dehydrogenase, alkaline phosphatase, and acid phosphatase activities (18) as measurement of contamination. The preparation of ESVs appeared approximately 80% pure by these biochemical criteria (results not shown)

Figure 1: Reactivity of monoclonal antibody 7D2 against Giardia preparations. Immunoblot analysis (B and D) of total cell proteins (under reducing conditions) of either encysting trophozoites, purified preparation of ESVs or cyst walls, using mAb 7D2 (vesicles and cyst wall preparations are shown in A and C; magnification times 22,200 and times 240, respectively). 7D2 recognized two species (39 and 26 kDa) in purified encystation-specific vesicles, but only the 26-kDa form in purified cyst walls.


To purify cyst walls, Giardia cysts generated in vitro were collected from the supernatant medium of cells cultured for 3 days in encystation medium by centrifugation at 1000 times g for 5 min. Cells were washed twice with PBS, treated with distilled water for 12 h at 4 °C, and then centrifuged at 250 times g for 5 min at 4 °C. The pellet, resuspended in 5 ml of water, was then layered atop 10 ml of 1 M sucrose and centrifuged at 250 times g for 5 min at 4 °C. The material obtained from the water phase was centrifuged and the pellet frozen-thawed 10 times, centrifuged again, and the resulting pellet resuspended in 5 ml of water. The suspension was sonicated 50 times (30 s, 20 A, in a Tekmar Sonic Disruptor, at 4 °C), loaded on top of 10 ml of 0.5 M sucrose, and centrifuged at 250 times g for 5 min at 4 °C. Unbroken cysts and debris remained in the pellet while purified cyst walls were obtained from the supernatant (Fig. 1C).

Production of Monoclonal Antibodies

Six-week-old female BALB/c mice (National Institutes of Health Frederick Cancer Research Facility) were immunized subcutaneously with either 200 µg of a purified preparation of ESVs or cyst walls emulsified in Ribi adjuvant system (Ribi Immunochem Research, Inc.) as recommended by the manufacturer. Mice were boosted subcutaneously after 21 days with 200 µg of the same preparation, and 20 days later boosted intravenously with 100 µg of the antigen preparation. Three days later, the mice were euthanized and the spleen cells used for fusion to SP2/0 myeloma cells. Hybridomas secreting antibodies were screened by indirect immunofluorescence (6, 18) with nonencysting trophozoites, encysting trophozoites, and in vitro derived Giardia cysts. Selected hybridomas were cloned by limiting dilution. Ascites were generated as described(35, 36) , cleared by centrifugation at 4000 times g, 15 min, filter-sterilized using 0.45-µm pore size filters, and saved at -70 °C. The mAb isotype and light chain composition were determined by the mouse monoclonal antibody isotyping kit (dipstick format) from Life Technologies, Inc.

Immunofluorescence and Immunoblot Analysis

For immunofluorescence analysis, cells cultured in either growth medium, pre-encystation medium, or encystation medium were harvested and processed as described previously(18) . Slides were then incubated for 1 h with both fluorescein conjugated mAb 5-3C (34) and rhodamine-conjugated mAb 7D2 (Ig G(1)) and washed as described above. mAbs were purified from ascites (37) and labeled directly as reported(38) . The specimens were mounted in Vectashield (Vector Laboratories) and viewed on Bio-Rad laser scanning confocal microscope.

Immunoblot analyses were done essentially as described(6, 18) .

Electron Microscopic Immunolabeling

Encysting G. lamblia were fixed by dilution of the encystation medium with an equal volume of 5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 6.8. Fixation, post-fixation, embedding in LRWhite (London Resin Co. Ltd.) were performed as reported(6) . Thin sections on nickel grids were blocked for 1 h in 1% bovine serum albumin in PBS (BSA/PBS) and then incubated for 2 h in a 1:1000 dilution of mAb 7D2 ascites in BSA/PBS followed by a thorough rinse in PBS. Sections were then incubated for 1 h in goat anti-mouse IgG coupled to 10-nm gold particles (BioCell, Ted Pella) and rinsed in PBS and distilled water. The grids were stained with uranyl acetate and lead citrate and carbon-coated before examination. Controls omitting the primary antibody or using purified nonimmune mouse IgG(1) (Southern Biotechnology Associates, Inc.) as the primary antibody showed no label.

Biosynthetic Labeling and Immunoprecipitation

Trophozoite monolayers in 15-ml glass tubes (approximately 15 times 10^8 cells) were induced to encyst as described above. After 24 h in complete encystation medium, cells were preincubated for 15 min in encystation labeling medium (Dulbecco's modified Eagle's medium lacking methionine pH 7.8, 10% dialyzed calf serum, 0.5 mML-cysteine, 20 µg/ml bathocuproine sulfonate, 5 mM lactic acid hemi-calcium salt, and porcine bile 0.25 mg/ml) before the addition of [S]methionine (TransS-label, ICN) at a concentration of 250 µCi/ml, and pulse labeled for 5 min at 37 °C. Next, labeling medium was decanted and the attached trophozoites washed twice with prewarmed complete encystation medium containing 2 mM of cold methionine. Tubes were chilled on ice for 15 min and the trophozoites collected by centrifugation at 1000 times g for 5 min. Cells were washed twice in PBS and then lysed in 500 µl of lysis buffer (50 mM sodium phosphate, 150 mM NaCl, 5 mM KCl, 5 mM MgCl, 1% Triton X-100, 0.5% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 20 µg/ml leupeptin, 5 µg/ml E64, 5 mM phenylmethylsulfonyl fluoride, 5 mM) Nalpha-p-tosyl-L-lysine chloromethyl ketone for 1 h at 4 °C. Lysates were sedimented at 16,000 times g for 5 min and the supernatant precleared in the presence of Protein G Plus-agarose beads (Oncogene) only. Subsequent nonimmune or immune precipitations were performed for 2 h at 4 °C using 50 µl of mAb-protein G beads complexes (50% suspension in lysis buffer) per 100 µl of cell lysate. Under these conditions, the efficiency of the immunoprecipitation was always greater that 80%, as judged by sequential precipitations. In some experiments, sequential immunoprecipitations were performed to determine the composition of the complexes obtained in the primary precipitation. Immune complexes were washed twice with lysis buffer and solubilized in electrophoresis sample buffer, boiled for 5 min, and analyzed by SDS-polyacrylamide gel electrophoresis in 4-20% gradient gels (Bio-Rad) under reducing conditions. Gels were fixed in 7.5% acetic acid, 20% methanol for 15 min, soaked in Enlightning (DuPont NEN) for 30 min, dried, and analyzed by fluorography.

Nucleic Acid Purification and Hybridization Analysis

Nucleic acids were extracted and analyzed by standard methods as described previously(6) . Polyadenylated RNA was purified from total cellular RNA with the PolyATtract® mRNA isolation system (Promega Corp.). P-End-labeled antisense oligonucleotides oMM133 (CWP2 nts 213 through 194; GenBank(TM) U28965), oMM103 (G. lamblia CWP1 nts 272 through 253, GenBank(TM) U09330), oMM79 (G. lamblia triose-phosphate isomerase nts 289 through 270, GenBank(TM) L02120), and GDH9B (G. lamblia glutamate dehydrogenase nts 1080 through 1061, GenBank(TM) M84604) were used in RNA hybridization studies.

Library Construction, Screening, and Subcloning

A cDNA expression library was constructed in gt22A using polyadenylated RNA from encysting Giardia trophozoites. SuperScript II reverse transcriptase was used to perform first strand cDNA synthesis by extension of an oligo(dT) primer modified to allow directional cloning of cDNA in the bacteriophage vector (Life Technologies, Inc.). Approximately 400,000 recombinant plaques from the amplified library were screened with mAb 7D2. Positive plaques were purified to homogeneity, and reactivities of the beta-galactosidase fusion proteins were verified by immunoblotting against mAb 7D2 and isotype-matched mAbs of different specificity, including the CWP1-specific mAb 5-3C (6) . The cDNA insert from one clone, c122, was amplified using Taq polymerase and primers that flank the cloning site of gt22A; the product was partially sequenced (see below) and cloned in pGEM-T (Promega Corp.) to yield the plasmid pMM100. The agarose gel-purified insert of pMM100 was radiolabeled with [alpha-P]dCTP by extension of random hexamers (39) and used to screen a WB/1267 Sau3AI partial genomic library constructed in FIXII(40) . A 3.8-kilobase pair BamHI fragment from genomic clone C8 was subcloned in pGEM-4 (Promega) to yield plasmid pMM109.

DNA Sequence Determination and Analysis

DNA sequences were determined from double-stranded recombinant plasmid templates using Sequenase version 2.0 and from amplified DNA using the Sequenase PCR product sequencing kit (U. S. Biochemical Corp.). Sequences were generated from pMM109 and pMM100 by ``primer walking'' on both strands using oligonucleotides designed from the newly derived sequences and made on an Applied Biosystems DNA synthesizer model 392. The identity of each reported nucleotide was determined at least twice on each strand.

DNA Strider 1.2(41) , AnalyzeSignalase 2.0.3(42) , BLASTP(43) , and programs in the GCG package (44) running on the National Institutes of Health Convex System were used to analyze and format the data.

RESULTS

Production of Monoclonal Antibodies Specific for Giardia Cyst Wall Constituents

To study the components of the Giardia cyst wall, as well as other secretory granule molecules expressed during encystation, we developed mAbs to these organelles. Since the purification of neither encystation-specific secretory vesicles nor cyst walls had been reported previously, we first developed methods to purify these structures (Fig. 1, A and C). Subsequently, we used these materials as immunogens to produce mAbs in mice. Among the mAbs tested in indirect immunofluorescence assays, seven demonstrated comparable reactivity with cysts produced in vitro or derived from infected gerbils. (^2)Immunoblotting revealed that mAb 7D2 (generated against cyst walls) and mAb 8G8 (generated against ESVs) bind the same antigen, the expression of which is induced during encystation; however, this antigen is distinct from CWP1 (see below). Because mAb 7D2 exhibited greater affinity for the reduced and denatured form of the protein than did 8G8, we decided to use 7D2 for further detailed analysis of its corresponding antigen, which we called CWP2. Immunoblot analysis performed on the samples used for the immunizations showed that mAb 7D2 recognizes CWP2 as a single 26-kDa band in purified cyst walls (Fig. 1D), but reacts with bands of 26 and 39 kDa in the purified ESV preparation (Fig. 1B). These results suggest that a 13-kDa fragment is removed from the 39-kDa CWP2 precursor before, and/or during, its incorporation into the cyst wall.

Subcellular Localization of CWP2

To confirm the reactivity of mAb 7D2 with the Giardia cyst wall and to establish the subcellular location of CWP2 in encysting trophozoites, we performed immunoelectron microscopic analysis. CWP2 is concentrated in the ESVs of encysting trophozoites (Fig. 2, a and c) prior to its incorporation into the cyst wall (Fig. 2b). No labeling of encysting trophozoites or cysts was observed if nonspecific mouse IgG(1) was used in place of mAb 7D2.

Figure 2: CWP2 is concentrated in the encystation-specific vesicles before its incorporation into the cyst wall. Immunoelectron microscopic detection of CWP2 in encysting trophozoites (a, c) and a cyst (b) using the mAb 7D2. a, an area of an encysting trophozoite revealing CWP2 localization in large electron-dense encystation-specific vesicles. b, a portion of a 24-h in vitro derived cyst showing gold label throughout the cyst wall that surrounds the trophozoite. Lysosome-like peripheral vacuoles are also observed. c, electron-dense encystation-specific vesicles containing CWP2 form from a cleft (arrow). Glycogen, which is abundant in encysting trophozoites and cysts, was extracted by the immunostaining procedure. Bars represent 1 µm.


Our previous observation that CWP1 is concentrated in the ESVs prompted us to determine whether CWP1 and CWP2 were present in the same ESVs of encysting trophozoites. To address this issue, we performed laser scanning confocal immunofluorescence microscopy on encysting trophozoites labeled simultaneously with rhodamine-conjugated mAb 7D2 and fluorescein isothiocyanate-conjugated mAb 5-3C. This analysis showed that CWP1 and CWP2 consistently colocalize within the ESVs of encysting trophozoites and to the cyst wall of in vitro derived cysts (results not shown).

Coordinate Induction of CWP1 and CWP2 Expression during Encystation

We studied the kinetics of CWP2 expression during encystation by immunoblot analysis of total trophozoite proteins. Under reducing conditions, mAb 7D2 detected CWP2 as an antigen of 39 kDa, the expression of which is strongly induced during encystation with kinetics identical to those exhibited by CWP1 (Fig. 3, compare left panels). Like mAb 5-3C, which detected antigens of 35 and 23 kDa in addition to the 26-kDa CWP1 late in encystation, mAb 7D2 detected 26- and 23-kDa species, as well as the predominant 39-kDa CWP2, late in encystation if samples were reduced before electrophoresis (Fig. 3, left panels). Under nonreducing conditions, both mAbs detected a 65-kDa species that was expressed during encystation with kinetics identical to the reduced antigens (Fig. 3, compare left and right panels). We consistently observed smearing above the 65-kDa antigen(s) detected by the two different mAbs (Fig. 3, right panels).

Figure 3: The expression of CWP1 and CWP2 is coordinately regulated during encystation in vitro. Immunoblot analysis of reduced or nonreduced total trophozoite proteins using mAb 7D2, specific for CWP2 (bottom panels), or mAb 5-3C, specific for CWP1 (top panels). Lanes: A, trophozoites cultured in growth medium; B, trophozoites cultured in pre-encystation medium; C-H, trophozoites cultured in encystation medium for 1, 2, 3, 4, 12, and 24 h, respectively. Mobilities of protein size standards are indicated on the left.


Formation of a Stable CWP1-CWP2 Complex in Encysting Trophozoites

The ability of CWP1 and CWP2 to form disulfide bonds (Fig. 3) and their colocalization in the ESVs and the cyst wall prompted us to investigate the possibility that the 65-kDa species observed in immunoblots of nonreduced encysting trophozoite proteins represents a complex of the two cyst wall proteins. We addressed this issue by immunoprecipitation analysis of encysting trophozoites labeled for 5 min with [S]methionine. Cells were harvested and extracts for precipitation were prepared immediately after the 5-min pulse labeling reaction. In contrast to their clearly distinguishable patterns of reactivity in immunoblots, mAbs 5-3C and 7D2 exhibited identical immunoprecipitation profiles: 26 and 39 kDa (Fig. 4, lanes A and B, respectively). Control precipitations employing isotype-matched unrelated mAbs showed no precipitation (results not shown). When supernatants from the precipitations shown in lanes A and B of Fig. 4were subsequently precipitated with the opposite mAb, the same two bands were detected, but at significantly reduced levels (Fig. 4, lanes C and D). Because mAb 5-3C does not bind recombinant CWP2 and, likewise, mAb 7D2 does not bind recombinant CWP1 (results not shown), these data indicate that CWP1 and CWP2 form a stable complex with each other within 5 min of their synthesis.

Figure 4: CWP1 and CWP2 form a stable complex soon after their synthesis. Immunoprecipitation analysis of encysting trophozoites metabolically labeled with [S]methionine for 5 min. Prior to SDS-polyacrylamide gel electrophoresis and fluorography, precipitations were performed individually with mAb 5-3C (A) or 7D2 (B) and sequentially with 5-3C then 7D2 (C) or 7D2 then 5-3C (D).


Developmental Regulation of CWP2 mRNA

We used mAb 7D2 to screen a cDNA expression library prepared from encysting trophozoite mRNA. Two identical clones were selected that contained inserts of approximately 1200 base pairs, one of which was completely sequenced. The reading frame of the cDNA fragment was coincident with the beta-galactosidase gene of the expression vector. In addition, immunoblotting of crude preparations of the recombinant protein verified its reactivity with mAb 7D2 and established its inability to bind mAb 5-3C (result not shown). An antisense oligonucleotide derived from the DNA sequence was used as a hybridization probe to evaluate the expression of CWP2 mRNA during encystation. The probe, which identifies a single copy gene in genomic Southern hybridizations (data not shown), detects a single transcript of 1320 nt in trophozoites cultured in encystation medium for 7 h (Fig. 5, left panel). Subsequent hybridization of this filter with an oligonucleotide probe specific for CWP1 transcripts shows that both CWP mRNAs are of comparable abundance in encysting trophozoites. Long autoradiographic exposures indicated that both mRNAs are present at low levels in nonencysting and pre-encysting cells (data not shown). In contrast to the indistinguishable patterns of developmental regulation exhibited by the CWP gene transcripts, the steady-state levels of mRNAs that encode two metabolically important enzymes, glutamate dehydrogenase and triose-phosphate isomerase, vary no more that 2-fold during encystation (Fig. 5, right panel).

Figure 5: The steady-state level of CWP2 mRNA increases dramatically during encystation in vitro. Hybridization analysis of total RNA (10 µg/lane) from nonencysting (N) and pre-encysting (P) trophozoites or trophozoites encysted in vitro for 7 h (E) fractionated by 1.4% agarose, 0.22 M formaldehyde gel electrophoresis. Left panel, hybridization with antisense oligonucleotide probes for CWP1 and CWP2. Right panel, duplicate filter hybridized with antisense oligonucleotide probes for glutamate dehydrogenase (GDH) and triose-phosphate isomerase (TIM). Final post-hybridization washes were performed in 2 times SSC, 0.1% SDS at 50 °C. Individual transcripts are denoted by arrows, and RNA size markers (nucleotide) are indicated between the panels. The autoradiogram shown in the right panel was exposed four times as long as the one on the left.


Structure of CWP2 mRNA

Sequence determination indicated that the cDNA clone lacked an initiation codon but terminated in a polyadenylate tract of 42 nt, suggesting that it represented a cDNA fragment truncated at its 5` end. To complete the sequence, we obtained the single copy gene from a G. lamblia genomic library. The DNA sequence of the cloned gene, which we called CWP2, described an open reading frame of 1089 nt that extended the cDNA sequence by 26 base pairs to include the putative initiation codon (Fig. 6, nts 1 through 3). Primer extension analysis performed on an aliquot of the same RNA used for Northern hybridization (Fig. 5, ``encysting''; data not shown) supports the notion that translation initiates at position 1, since the 5` limit of the mRNA maps to position -7 (Fig. 6). A short 5`-untranslated region is a feature of CWP1 mRNA (6) and Giardia mRNAs in general(1) . Polyadenylation of the primary CWP2 transcript occurs at position 1145, 7 nt from the heptanucleotide AGTAAAC, which conforms to the motif found consistently between the termination codon and polyadenylation site of Giardia mRNAs(1) . Addition of a polyadenylate tract of 150 nt to the transcribed gene sequence would yield a CWP2 mRNA of 1295 nt, in good agreement with the size determined by hybridization.

Figure 6: Nucleotide and amino acid sequence deduced from CWP2, the gene that encodes the G. lamblia cyst wall protein CWP2. Position 1 is the first nucleotide of the putative initiation codon. The original cDNA clone, c122, spanned nts 27 through 1145. The + indicates the 5` limit of the CWP2 mRNA determined by primer extension, underlining delimits the predicted signal peptide, bold type shows LRRs, overlining indicates the putative Giardia polyadenylation signal, and the asterisk signifies the site of polyadenylate addition to CWP2 mRNA. These sequences appear in GenBank(TM) under accession number U28965.


Structure of CWP2 and Comparison with LRR-containing proteins, including CWP1

The 1089-nt open reading frame of CWP2 describes a polypeptide of M(r) 39,264 that contains five tandem copies of a 24-residue LRR (Fig. 6, bold type). When compared against a nonredundant data base that included PDB, SwissProt, PIR, and GenPept, the deduced CWP2 amino acid sequence identified CWP1 and several other proteins, including the tomato Cf-9 protein(22) , two Arabidopsis receptor-like kinases(23, 24) , plant inhibitors of fungal polygalacturonase(25) , a maize pollen extension-like protein(26) , an extracellular matrix protein specifically expressed in Antirrhinum flowers (27) , and stage-specific Leishmania surface antigens(28, 29) . Inspection of the BLAST alignments from this comparison showed that all these proteins contained 24-residue LRRs. In fact, except for CWP1, the similarity between CWP2 and these proteins was restricted to the LRR regions.

Among the proteins identified by similarity to CWP2, CWP1 (M(r) 26,027) is most closely related. Both proteins include hydrophobic amino-terminal signal peptides that likely target them to the secretory pathway ( Fig. 6and Fig. 7). In addition, the central region of both CWPs consists of 5 tandem LRRs (Fig. 7, cross-hatched boxes). Most strikingly, in the 241-residue overlap between the two proteins, they share positional amino acid sequence identity of 61%, largely due to the LRR region and the domain that immediately precedes it (Fig. 7). Both proteins possess a cysteine-rich domain (CWP1 16 mol % and CWP2 12 mol %) next to the LRR domain (Fig. 7). CWP2 is distinguished from CWP1 by a 121-residue carboxyl-terminal extension that is rich in basic amino acids. This extension accounts for the differences in M(r) and pI calculated for the two proteins: removal of this M(r) 13,060 peptide would yield a CWP2 fragment of M(r) 26,204 with a pI of 3.69 (Fig. 7).

Figure 7: The two closely related secretory proteins, CWP1 and CWP2, contain leucine-rich repeats but are distinguished by a strongly basic carboxyl-terminal tail. Schematic depiction of CWP1 and CWP2 based on their deduced amino acid sequences. The checkered boxes signify candidate signal peptides, cross-hatched boxes indicate tandemly arrayed leucine-rich repeats, stippled boxes show cysteine-rich regions, and shading denotes the basic 121-residue carboxyl-terminal tail of CWP2. Positional amino acid sequence identities between corresponding domains of the two proteins are indicated as are the isoelectric points of the individual proteins and substituent peptides.


DISCUSSION

The biosynthesis and assembly of eukaryotic extracellular superstructures such as the plant (45) and fungal cell walls(46, 47) , and the cyst wall of medically important intestinal pathogens(1, 48, 49) , are not completely understood. In this work, using a combination of biochemical, immunochemical, and molecular genetic approaches, we identified a novel protein constituent of the G. lamblia cyst wall, CWP2. The structural and biochemical properties of the CWPs revealed by this study have profound implications for the assembly of the cyst wall, and when considered in the context of intracellular protein transport, this new information also has intriguing ramifications for the biogenesis of the ESVs in encysting trophozoites and for the biogenesis of secretory granules of eukaryotic cells, in general.

The only defined protein constituents of the Giardia cyst wall, CWP1 and CWP2, are closely related in primary structure. The two proteins possess hydrophobic amino-terminal signal peptides that likely target them to the secretory pathway in encysting trophozoites. In addition, the high degree of positional amino acid sequence identity between the CWPs results from conservation of structural elements: a conserved amino-terminal domain precedes a LRR core, which is followed in turn by a cysteine-rich region (Fig. 7). Besides being structurally similar, both proteins are induced with identical kinetics during encystation and colocalize to the encystation-specific vesicles and cyst wall. Our studies suggest that the coordinated production, localization, and transport of CWP1 and CWP2 are necessary because both cyst wall proteins form a heterocomplex, the stability of which is sensitive to reduction.

The LRR consensus sequences of the Giardia CWPs most closely resemble those found in the extracellular domain of plant transmembrane and extracellular matrix proteins(22, 23, 24, 25, 26, 27) . These LRRs are characterized by absolutely conserved glycine and proline residues, a feature that distinguishes these 24-residue LRRs from other 24-residue LRRs, including small proteoglycans of mammalian extracellular matrix (50) . In both Giardia cyst wall proteins, the LRR domain is centrally located. This structural organization is also found in porcine ribonuclease inhibitor, for which the structure has been solved both free from and complexed with bovine ribonuclease(19, 20, 21) . As in ribonuclease inhibitor, the LRR regions of the CWPs may serve as flexible domains that facilitate the interaction of the amino- and carboxyl-terminal flanking regions. Alternatively, LRRs may play a more direct role in the interaction between the proteins.

Although CWP1 and CWP2 are closely related, CWP2 is distinguished from CWP1 by a 121-residue carboxyl-terminal extension. In purified ESVs, CWP2 was mainly found as a 39-kDa protein (26 kDa from the CWP1-like region plus 13 kDa from the basic tail); however, in the purified cyst wall, only a 26-kDa fragment could be found, indicating that proteolytic processing of CWP2 occurred before its incorporation into the cyst wall. The alkaline nature of this tail (pI = 12.23) predicts a high net positive charge at physiological pH, suggesting an electrostatic predilection for anionic molecule(s), e.g. acidic proteins or perhaps even acidic phospholipids(51, 52, 53) . Assuming cleavage of the amino-terminal signal peptide, the absence of a hydrophobic transmembrane region on either protein suggests that anionic receptors for CWP2 might be luminally disposed molecules associated with the membrane of the endoplasmic reticulum or a post-endoplasmic reticulum compartment. Oligomerization or aggregation of CWPs could result in ESV formation. As shown in Fig. 2c, electron-dense secretory materials aggregate within membrane-bound clefts(5) . These aggregates appear to grow up by direct addition of newly synthesized cyst wall proteins to form large ESVs. The formation of ESVs could be a direct consequence of the synthesis of the CWPs, especially CWP2, and their trafficking through the developmentally induced secretory pathway of encysting trophozoites.

Mechanisms of protein transport and secretion in Giardia are not well understood. Although several lines of evidence support the notion that a Golgi apparatus exists in Giardia trophozoites (18, 54) , no direct evidence unequivocally establishes the existence of this important protein-sorting organelle in Giardia. In higher eukaryotic cells, secretory granules form in the trans-Golgi network(55, 56) , where secretory proteins condense into a core that buds to form an immature secretory granule(55, 56, 57, 58) . In Giardia, however, it is unclear whether the ESVs form from an as yet uncharacterized trans-Golgi or by condensation within the endoplasmic reticulum(59) . Immunoelectron microscopy indicates that after their synthesis cyst wall antigens(5, 54) , including CWP1 (6) and CWP2 (Fig. 2), are located within a flattened cisterna which grows up to form a large (>1-µm diameter) membrane-bounded ESV. The solubility of CWPs in the ESVs is unknown, but the electron-dense nature of these vesicles suggests a tightly packed or highly condensed arrangement of their contents. No filamentous structures are present in the ESVs, suggesting that some mechanism for preventing premature formation of filaments within ESVs must exist (e.g. pH, molecular chaperones, calcium concentration)(56) . Presumably, filament formation is coordinated with the release of the ESV contents to the cell exterior.

Using Gas Chromatography/Mass Spectrometry, Manning (10) identified galactosamine as the predominant sugar associated with the filamentous component of the Giardia cyst wall and provided compelling data that refuted the presence of chitin as a major structural component(12, 60) . The abundance of GalNac, considered with the insolubility of the cyst wall, suggested its presence in a polymerized form in this structure. CWP1 and CWP2 each contain a single N-glycosylation site: in the second LRR of CWP1 and in COOH-terminal tail of CWP2. No published evidence supports the existence of N-glycosylation in Giardia. In fact, tunicamycin, at concentrations that block N-glycosylation in mammalian cells, did not block cyst wall formation. (^3)Moreover, although the primary structure of a trophozoite variant-specific surface protein includes two potential N-glycosylation sites, carbohydrate analysis of the purified protein showed that it is not glycosylated (61) . The profusion of galactosamine and GalNAc in the cyst wall, the abundance of potential sites of O-glycosylation in the CWPs (CWP1 and CWP2 are rich in serine and threonine; together, these two amino acids comprise 14% of the residues in each protein), their altered mobility in SDS-polyacrylamide gel electrophoresis late in encystation, and the induction of galactosamine and N-acetylgalactosamine transferase activities in encysting cells (18) suggest that the CWPs may be glycosylated. Direct biochemical characterization of purified cyst wall proteins will clarify their glycosylation status.

As shown in this work, the ability to induce Giardia encystation in vitro makes this organism an excellent model to study the formation and regulation of secretory granules and the biosynthesis and assembly of extracellular components. Further elucidation of the biological mechanisms employed by Giardia, which derives from the most primitive branch of the eukaryotic line of descent(3, 4) , will allow us to understand the evolution of fundamental eukaryotic cellular processes, such as signal transduction, control of transcription and translation, vesicular transport, and extracellular matrix formation.

FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U28965[GenBank].

§
To whom correspondence should be addressed: National Institutes of Health, Bldg. 4, Rm. 126, Bethesda, MD 20892-0425. Tel.: 301-496-6920; Fax: 301-402-2689; hlujan@4.niaid.nih.gov.

(^1)
The abbreviations used are: ESV(s), encystation-specific vesicle(s); CWP1, cyst wall protein 1; CWP1, the gene that encodes CWP1; CWP2, cyst wall protein 2; CWP2, the gene that encodes CWP2; LRR(s), leucine-rich repeat(s); mAb, monoclonal antibody; nt(s), nucleotide(s); PBS, phosphate-buffered saline; BSA, bovine serum albumin.

(^2)
H. D. Luján, unpublished results.

(^3)
H. D. Luján, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. J. Yee and Dr. D. Dwyer for critical reading of this manuscript.

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