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J. Biol. Chem., Vol. 277, Issue 52, 50557-50563, December 27, 2002

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Identification and Characterization of a Novel Secretory Granule Calcium-binding Protein from the Early Branching Eukaryote Giardia lamblia^*,

María C. Touz^§, Natalia Gottig^§, Theodore E. Nash, and Hugo D. Lujan^§¶

From the ^§ Catedra de Bioquímica y Biología Molecular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, CP 5000 Córdoba, Argentina and the  Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, March 15, 2002, and in revised form, August 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Giardia lamblia is a flagellate protozoan that infects humans and other mammals and the most frequently isolated intestinal parasite worldwide. Giardia trophozoites undergo essential biological changes to survive outside the intestine of their host by differentiating into infective cysts. Cyst formation, or encystation, is considered one of the most primitive adaptive responses developed by eukaryotes early in evolution and crucial for the transmission of the parasite among susceptible hosts. During this process, proteins that will assemble into the extracellular cyst wall (CWP1 and CWP2) are transported to the cell surface within encystation-specific secretory vesicles (ESVs) by a developmentally regulated secretory pathway. Cyst wall proteins (CWPs) are maintained as a dense material inside the ESVs, but after exocytosis, they form the fibrillar matrix of the cyst wall. Little is known about the molecular mechanisms involved in granule biogenesis and discharge in Giardia, as well as the assembly of the extracellular wall. In this work, we provide evidences that a novel 54-kDa protein that exclusively localizes to the ESVs is induced during encystation similar to CWPs, proteolytically processed during granule maturation, and able to bind calcium in vitro. The gene encoding this molecule predicts a novel protein (called gGSP for G. lamblia Granule-specific Protein) without homology to any other protein reported in public databases. Nevertheless, it possesses characteristics of calcium-sequestering molecules of higher eukaryotes. Inhibition of gGSP expression abolishes cyst wall formation, suggesting that this secretory granule protein regulates Ca²⁺-dependent degranulation of ESVs during cyst wall formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The protozoan Giardia lamblia colonizes the upper small intestine of several vertebrate hosts and is one on the most common causes of intestinal disease worldwide. This parasitic organism has a simple life cycle, alternating between the disease-causing trophozoite and the environmentally resistant cyst, which is responsible for the transmission of the disease among susceptible hosts. After the reception of the stimulus that triggers encystation, G. lamblia undergoes several metabolic and morphologic changes. These comprise the synthesis, processing, and transport of leucine-rich repeat-containing proteins that will be finally secreted and assembled at the cell exterior to form a protective cyst wall (1, 2). From their synthesis in the endoplasmic reticulum (ER),¹ cyst wall proteins (CWPs) must be transported to the cell membrane in a very effective and controlled manner because the survival of the parasite outside the intestine of the host depends on the success of this process (3). Unfortunately, the knowledge of the transport machinery in Giardia is limited, mostly due to the fact that trophozoites lack essential secretory organelles, such as morphologically identifiable Golgi apparatus and secretory granules (4, 5). However, constitutive protein secretion in Giardia is exemplified by the continuous transport to the plasma membrane and release into the culture medium of variant-specific surface proteins (VSP) (6, 7), whereas regulated secretion apparently occurs only during encystation (3, 8). The biogenesis of the Golgi complex and of typical secretory granules containing cyst wall materials, called encystation-specific secretory vesicles (ESVs), has been reported to occur at early stages of trophozoite differentiation into cyst (8-10).

In higher eukaryotes, proteins that will be secreted are assembled and glycosylated during their transport from the ER, through the Golgi apparatus, to the plasma membrane (11). Regulated secretion of hormones and peptides from endocrine and exocrine cells, as well as other parasitic organisms (12, 13), occurs via a regulated secretory pathway and involves the storage of secretory proteins into secretory granules until an appropriate stimulus triggers the release of the content of the granule to the cell exterior (11, 14-16). Since Giardia derives from the earliest branch of the eukaryotic line of descent (17), the study of the biogenesis of secretory organelles that occurs during differentiation might provide new insights into the minimal machinery required for protein transport by the regulated secretory pathway in eukaryotes (1-3). Moreover, the ability to control the biogenesis of the Golgi apparatus (5) and secretory granules (8) in Giardia by just varying the composition of the culture medium (18) offers an excellent opportunity to investigate the physiological mechanisms underlying granule formation, maturation, and discharge (1, 2).

During encystation, expression of molecules implicated in the regulated secretory pathway increases several times, and this distinctive characteristic can be use to identify and characterize developmentally regulated molecules (1, 2). In previous works, using monoclonal antibodies (mAb) generated against purified cyst walls, two proteins that localize within ESVs in encysting trophozoites and to the cyst wall of mature cysts were identified and characterized (CWP1 and CWP2) (9, 10). CWP1 and CWP2 are acidic proteins of 26 and 39 kDa, respectively, that contain typical eukaryotic signal peptides and five tandem copies of leucine-rich repeats and form a stable complex between them, likely mediated by disulfide bonds (1). Besides CWPs, several other molecules that are similarly induced during encystation are known to participate in the intracellular transport and secretion of cyst wall materials: chaperones such as BiP/GRP78 (19, 20) and protein disulfide isomerase 2 (PDI-2) (21, 22)²; cathepsin C (23); proteins of the SNARE complex including syntaxin 1 and 2, NSF, SNAP (soluble NSF attachment protein), and vesicle-associated membrane protein (VAMP)³; as well as others still undefined.⁴

It is known that a rise in intracellular Ca²⁺ concentration is necessary to induce regulated secretion in higher eukaryotes (24, 25). Calcium-induced aggregation of secretory proteins has been proposed to be necessary for sorting and storage of secretory proteins into secretory granules of endocrine and exocrine cells (26). Since secretory granules contain large amounts of Ca²⁺, calcium function has also been attributed to the packaging and processing of the contents of the secretory granule (27). The presence of calcium-binding proteins along the secretory pathway is thought to be necessary for the temporal control of signaling processes acting as Ca²⁺ buffers or Ca²⁺-sequestering proteins (27-29). Nevertheless, the role of calcium and secretory granule calcium-binding proteins during the biogenesis of secretory granules and regulated secretion remains unclear.

In this work, using two different mAbs generated against purified secretory granules of encysting Giardia trophozoites, we identified a novel acidic protein of 54 kDa (gGSP for G. lamblia granule-specific protein), which localizes to the lumen of ESVs, is proteolytically cleaved before cyst wall formation, and is able to bind calcium. In addition, inhibition of gGSP expression blocks cyst wall formation, suggesting that this secretory granule protein is essential for calcium-mediated exocytosis during encystation of this important human pathogen.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G. lamblia Cultivation and Encystation in Vitro-- Trophozoites of the isolate WB clone 1267 (30) were cultured in TYI-S-33 medium supplemented with 10% adult bovine serum and 0.5 mg/ml bovine bile (growth medium) as described (31). Encystation of trophozoite was accomplished by the method described by Boucher and Gillin (32). Trophozoites were recovered from the medium, and cysts were collected and resuspended in tap water (32). Mature cysts were counted in a Coulter Z₁ cell counter.

Purification of Encystation-specific Secretory Vesicles and Generation of Specific Monoclonal Antibodies-- ESVs were purified essentially as described (9). The biochemical characterization of the granule fractions was done by immunoreactivity with the mAb specific for CWPs as an indication of the secretory granule enrichment, by acid phosphatase activity as an indication of lysosomal contamination, and by immunoreactivity with the mAb 9C9 (specific for BiP) as an indication of ER contamination (19) and mAb 5C1 (specific for VSP1267) as plasma membrane contamination (9). Monoclonal antibodies against purified ESVs were produced by standard procedures as described previously (9). Culture supernatants of antibody-secreting hybridomas were screened for specific reactivity with ESVs of encysting trophozoites by indirect immunofluorescence assays. Two antibodies, mAbs 7E7 (IgG₂a) and 9C3 (IgG₁), that bind specifically to Giardia secretory granules (see below) were purified from ascites and labeled as reported (9).

Immunoblotting Analysis-- Proteins were incubated in sample buffer with 2-mercaptoethanol and fractionated by SDS-PAGE in 4-12% gradient gels. Electrophoretic transfer of proteins to nitrocellulose was performed as reported (9, 23). Filters were blocked with 3% defatted milk/0.1% Tween/Tris-buffered saline and then incubated with mAbs (1:1000 dilution). Following incubation with alkaline phosphatase-conjugated goat-anti-mouse IgGs at 1:2000 dilution, Giardia proteins were visualized by development with alkaline phosphatase color development reagent (Bio-Rad). In particular experiments, alkaline phosphatase-labeled mAbs were used.

Immunofluorescence Analysis-- Cells cultured in grown medium, pre-encystation medium, or encystation medium were harvested and processed as described previously (9, 23). Slides were then incubated with fluorescein-conjugated 7D2 mAb (specific for CWP2 (9)) and rhodamine-conjugated 9C3 and 7E7 mAbs (specific for gGSP). The specimens were mounted and viewed on a Leica IRME fluorescence microscope, and images were captured with the Leica DC250 camera and processed with the Leica QFluoro Software (Leica Microsystems).

Phase Separation of Integral Membrane Proteins in Triton X-114 Solution-- Purified ESVs were dissolved in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1.0% Triton X-114 at 0 °C and subjected to detergent and aqueous phase partitioning as described previously (33).

Cloning and Sequencing of gGSP-- Nucleic acids were purified and analyzed by standard methods as described (9). A cDNA expression library constructed in gt22a using polyadenylated RNA from encysting cells (9) was screened with mAbs 7E7 and 9C3 by standard procedures (34). Positive clones were purified, and the reactivity of the fusion proteins was verified by immunoblotting using mAbs 7E7 and 9C3. The cDNA inserts from three clones 30, 37 (mAbs 7E7), and 41 (mAb 9C3) were amplified by PCR using primers flanking the cloning site of the  vector, cloned, and sequenced. For Southern hybridization, 10 µg of G. lamblia genomic DNA was digested with EcoRI, Sau3AI, and HindIII. Both Southern and Northern hybridizations were performed as reported previously (9, 23). Primers generated from extreme sequences of inserts in clones 30, 37, and 41 were used to amplify genomic Giardia DNA. Fragments were cloned into pBlueScript SKII⁺, sequenced, and used as probes for the screening of a genomic DNA library (23). Analysis of the DNA sequence was performed with the computer program DNAStar (Lasergene). Signal sequence prediction was done using SignalIP (35). SMART-Simple Modular Architecture Research Tools (36) was used for searching for protein domains and patterns. Homology searches and other structural predictions were performed with software available at ExPASy (www.expasy.ch/).

Immunoprecipitation-- Trophozoites induced to encyst for 24 h (6 × 10⁸ cells) were harvested and lysed in 1 ml of lysis buffer as reported (9). Immunoprecipitation using mAb 9C3 and 7E7 was as described previously (9). To investigate the Ca²⁺ binding capability of the different forms of gGSP, polyacrylamide gels after non-reduced SDS-PAGE were stained with Stains-all (27, 37). Additionally, immunoprecipitated gGSP was blotted onto polyvinylidene difluoride membranes (Millipore) using a Bio-Rad slot blot apparatus and subjected to ⁴⁵Ca²⁺ overlay assays as described (27).

gGSP Antisense Silencing-- For gGSP functional analysis, sense primer Gf (5'-GTTGATATCATGTTGCGGAAGTTTCGGTCATTC-3') containing an EcoRV site and Gr antisense primer (5'-GGACCATGGTATTATTTCTATGGTCAGTTTTAT-3') with a NcoI site (restriction sites in boldface) were used to amplify by PCR the entire gsp open reading frame. The 1374-bp band was purified, restricted, and cloned into the vector PtubH7HApac (23). In this way, the gsp gene was inserted inside Ptub-HApac inversely, giving the gsp antisense vector that was used for the inhibition of gGSP expression. As a control, the same vector was used to express an HA-tagged version of gGSP, resulting in PtubGSPHApac. Sequences were confirmed by dye terminator cycle sequencing (Beckman Coulter). Transfection of G. lamblia trophozoites was done by electroporation as described previously (23), and cells carrying the antisense construct were selected under puromycin pressure (41). Western and Northern blotting was used to analyze gGSP and CWP2 expression.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of mAb 7E7 and 9C3-- The mAbs 7E7 and 9C3 were generated using purified encystation-specific secretory vesicles (ESVs) as immunogen (Ref. 9 and Supplementary Fig. 1) and using hybridomas screened for reactivity to ESVs of encysting trophozoites. An immunoblotting assay showed that mAb 9C3 recognizes 54- and 30-kDa polypeptides in encysting trophozoites (Fig. 1), whereas mAb 7E7 only recognizes a 30-kDa protein (Supplementary Fig. 2). The expression of such proteins was not observed in non-encysting or pre-encysting cells, but their expression level increases during encystation in vitro coincident with the expression of Giardia cyst wall proteins (Supplementary Fig. 2). When mAbs 7E7 or 9C3 were used in immunofluorescence analysis, they labeled ESVs of encysting trophozoites (Fig. 2A). In contrast to antibodies that recognize CWP2 (mAb 7D2), mAb 7E7 and 9C3 did not label the cyst wall (Fig. 2B), indicating that they detect ESV-specific antigens. Additional evidence that CWPs and this novel antigen colocalize to ESVs comes from the almost exact localization pattern observed for both polypeptides in fractions obtained after sucrose isopycnic centrifugation of the 30,000 × g particulate fraction of encysting Giardia (Fig. 2C). The fact that those proteins were present only in secretory granules prompted us to analyze whether they were soluble or membrane-associated. ESVs were purified from 24-h encysting Giardia and proteins subjected to a detergent and aqueous phase separation using Triton X-114. Results showed that those proteins partition to the aqueous phase and therefore are not part of the ESV membrane (Supplementary Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Monoclonal antibody 9C3 recognizes proteins developmentally induced during encystation. Two bands of 54 and 30 kDa were observed by immunodetection with mAb 9C3 only in encysting trophozoites (arrowheads). The mAb 7E7 only recognized the 30-kDa band (not shown). N, trophozoites in growth medium (non-encysting trophozoites); PE, trophozoites in pre-encystation medium; ET, trophozoites in encystation medium for 24 h. Molecular size standards are shown at the left (MW).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Monoclonal antibody 9C3 detects proteins in secretory granules of encysting trophozoites but not in cyst wall. Immunofluorescence images of encysting trophozoites encysted for 24 h (A) and cyst (B). Fluorescein-conjugated mAb 7D2 (specific for CWP2) and rhodamine-conjugated mAb 9C3 (specific for gGSP) were used. In the center panel of A, the merged image shows the identical localization of CWP2 and gGSP to secretory granules of encysting trophozoites. Similar results were always observed with mAb 7E7. DIC, differential interference contrast. Dot blot experiments using 16 fractions obtained after sucrose isopycnic centrifugation of the 30,000 × g fraction of encysting Giardia trophozoites (9) are shown in C. CWP2 and gGSP show an almost identical pattern, indicating that both proteins colocalize to ESVs in encysting cells.

Screening of cDNA Expression Library Showed That the 54- and 30-kDa Giardia Secretory Granule Proteins Are Products of the Same Gene-- We performed a screening of cDNA expression library using mAbs 7E7 and 9C3. Positive clones were purified, and the resultant fusion proteins were verified by immunoblotting using the same mAbs. The cDNA inserts from three clones, 30, 37 (mAbs 7E7), and 41 (mAb 9C3), were amplified and sequenced (Fig 3). Analysis of these sequences demonstrated the presence of common regions in clones 30 and 37 (Fig. 3). Additionally, Southern blot experiments using the inserts present in clones 30 and 41 showed identical hybridization patterns (Fig. 4), indicating that they could be fragments of the same gene. To address this issue, PCR and screening of a gDNA library from Giardia isolate WB clone 1267 (9) allowed us to obtain the complete sequence of the gene encoding the protein recognized by both antibodies, plus 5'- and 3'-untranslated regions (Fig. 3). gGSP (accessible in GenBank^TM under accession number AF293411) has an open reading frame of 1374 nt including the TAA stop codon, which is followed by two bases before the putative Giardia polyadenylation signal ATTAAAA. Like almost all Giardia genes, gGSP lacks introns (38, 39) (Fig. 3). The translated nucleotide sequence encoded an acidic, leucine-rich 54-kDa protein composed by 480 amino acids where the 25-amino acid N-terminal hydrophobic region corresponds to the signal peptide. The deduced protein also shows a sequence rich in basic amino acid (RRLRLVPQRKSRRRIDKRKR, amino acids 265-292). Interestingly, in higher eukaryotes, basic domains in secretory proteins are a target for proteolytic processing by furin, a serine-like protease that is localized in the trans-Golgi network (40, 41). The location of this domain within the protein predicts that once processed by that protease, gGSP could appear as 54 and 30 kDa, as was shown by Western blotting (Fig. 1 and Supplementary Fig. 2). Alignment of the full-length gGSP sequence with those obtained after the initial screening of the cDNA library indicates that the fragments recognized by the mAb 7E7 are at the N terminus of the basic domain, whereas the region recognized by mAb 9C3 is at the C terminus (Fig. 3).

View larger version (114K): [in this window] [in a new window]   Fig. 3.   Structure of Giardia GSP and the deduced amino acid sequence. The full-length nucleotide sequence of the gGSP, including 5'- and 3'-untranslated regions, is shown. The open reading frame is depicted in upper case, and the 5'- and 3'-untranslated regions are depicted in lower case. Nucleotide sequences in blue (nt 238-1044), orange (nt 397-866) and green (nt 1214-1297) denote the cDNA sequences obtained from clones 30, 37, and 41, respectively. The start codon is underlined, and the stop codon (labeled with an asterisk) is located 2 bases before the putative polyadenylation motif (red) starts. The deduced amino acid sequence is marked in yellow, whereas the signal peptide is shown in gray. The calsequestrin motif is underlined, the Asp/Glu-rich C-terminal domain is double underlined, and the putative furin cleavage site is illustrated in boldface letters. Nucleotide and amino acid positions are depicted at the right.

View larger version (77K): [in this window] [in a new window]   Fig. 4.   Southern blot indicates that gGSP is present as a single copy gene in Giardia genome. Ten µg of genomic Giardia DNA were cut with Eco RI (a), Saul 3AI (b), and Hind III (c). The inserts of clones 30 and 41 (nt 397-866 and 1214-1297 of the gGSP gene) and the control clone 126 (nt 241-421 of the syntaxin 1 gene) were the probes used.

Based on the Western blot results (Fig. 1 and Supplementary Fig. 2), sequence analysis of the cDNA clones identified by each mAb (Fig. 3), and sequential immunoprecipitations (not shown), mAb 7E7 seems to react only with the processed 23-kDa N-terminal domain of gGSP and not with the complete protein, as is the case for mAb 9C3, which recognizes the C terminus and, therefore, detects the full-length 54-kDa protein and the processed 30-kDa C-terminal domain. Apparently, on both reduced and non-reduced trophozoite protein extracts, the epitope recognized by mAb 7E7 is only accessible to the antibody once the protein is proteolytically processed.

BLAST homology searching (42) revealed that this protein does not have significantly homology with any other eukaryotic or prokaryotic protein deposited in public databases. Nevertheless, detailed analysis of its sequences shows a domain with high homology to proteins involved in calcium regulation, such as a calsequentrin domain between the amino acids 130 and 167 (36) and a C-terminal region enriched in glutamic and aspartic acids typical of some families of calcium-binding proteins (43, 44) (Fig. 3).

Transcripts Encoding gGSP Increase during Encystation-- Southern hybridization studies of the identified gene showed that a single copy gene encodes this protein (Fig. 4). To analyze the expression of gGSP mRNA, Northern hybridization was performed using total RNA obtained from trophozoites at different stages of encystation. Results indicate that the steady-state level of mRNA increases during the differentiation of Giardia, in contrast to constant expression of syntaxin 1 (GenBank^TM accession number AF293409).² Syntaxins are proteins that are also involved in intracellular vesicular transport but, in contrast to gGSP, their transcripts do not vary significantly during differentiation (Fig. 5). Therefore, the increase in expression observed during the encystation must be regulated at the level of the transcription or mRNA stability.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   The expression of gGSP mRNA increases during encystation. Hybridization of 10 µg of total RNA from trophozoites induced to encyst for different time periods. In the upper panel, the assay was done using the insert of clone 30 as probe. In the lower panel, the insert of clone 126 (syntaxin 1) was used as control for constitutive expression.

Giardia GSP Has Calcium Binding Properties-- The dye Stains-all was used to demonstrate the calcium binding properties of isolated gGSP. With this dye, calcium-binding proteins stain blue, whereas others proteins either are not labeled or stain pink (27, 37). This technique was used previously for detection of calcium-binding proteins such as calreticulin (43) and calsequestrin (37). Giardia gGSP was isolated from encysting trophozoite protein extracts by immunoprecipitation using mAb 9C3, and calcium-binding proteins were then detected with Stains-all. The dye detected two blue bands of 54 and 30 kDa after SDS-PAGE, in agreement with the Western-blot analysis of gGSP (Fig. 6A). Moreover, ⁴⁵Ca²⁺ overlay assays performed on slot-blotted immunoprecipitated gGSP demonstrated that this protein binds calcium in vitro (Fig. 6B). Mock immunoprecipitations using either unrelated antibodies or secondary antibodies further support the specific calcium binding capability of gGSP. In these experiments, the differential binding profiles of mAbs 7E7 and 9C3 (determined by Western blotting and screening of the cDNA library; see above) indicated that the C-terminal Asp/Glu-rich domain of gGSP contains the preferential calcium-binding region of the protein.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Giardia GSP is a novel Ca2+-binding protein. In A, immunoblot of 24 h encysting trophozoites using alkaline phosphatase-labeled mAb 9C3 showed the 54- and 30-kDa bands of gGSP (WB). Stain-all labeling of the two gGSP mAb 9C3-immunoprecipitated forms stain blue after SDS-PAGE (SA). In B, calcium overlay assay on slot-blotted immunoprecipitated gGSP indicates that this protein is able to bind 45Ca2+ by its C-terminal domain. mAb 7E7 and 9C3 were used as controls. Only the proteins immunoprecipitated by mAb 9C3 (which contain the C-terminal region of gGSP) are strongly labeled with 45Ca2+, whereas the protein immunoprecipitated by mAb 7E7 (N-terminal 27-kDa domain) weakly binds the cation.

Inhibition of GSP Expression Avoids Cyst Formation-- To determine the role of gGSP during encystation, Giardia clone WB/1267 was transfected with a vector carrying the full-length antisense sequence of gsp. Cells were selected with puromycin (41), and clones C11, E11, and F11 were chosen among 25 other positive clones for subsequent analyses. Immunoblotting (Table I and Fig. 7) and immunofluorescence assays (not shown) using mAb 9C3 confirmed the complete inhibition of GSP expression by the antisense construct. Results summarized in Table I show that when these clones were cultured in growth medium, neither cell viability nor proliferation was affected, when compared with either untransfected trophozoites or trophozoites transfected with an HA-tagged sense gsp. After 48 h in encystation medium, trophozoites expressing antisense gsp did not form cyst (Table I). In these cells, CWP2 was expressed and incorporated into ESVs similar to untransfected trophozoites (Table I and Fig. 7), but the granules did not release their content to the cell exterior to form the cyst wall. These results indicated that gGSP is involved in the latest steps of encystation, likely regulating secretory granule discharge.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Inhibition of gGSP expression. Untransfected (UT) G. lamblia trophozoites (top panel), trophozoites transfected with the tagged gGSP (gGSP-HA), and cells transfected with antisense gsp (C11, E11, and F11) were induced to encyst, harvested, and transferred to nitrocellulose membranes for Western blot assay. For gGSP, specific mAb 9C3 was used to determine the inhibition of gGSP expression (54 and 30 kDa) by the antisense construct. Monoclonal antibody 7D2 was used to determine CWP2 expression (39 and 26 kDa) in encysting trophozoites.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In specialized eukaryotic cells, the biogenesis of secretory granules could be separated into three distinct events (24): (a) aggregation of regulated secretory proteins and sorting to the trans-Golgi network; (b) budding of immature secretory granules from the trans-Golgi network; and (c) maturation of granules during the transport toward the plasma membrane. Regulated secretory proteins form a vesicle core that was described as a dynamic aggregate (45, 46). After their synthesis in the ER, regulated secretory proteins are sorted and segregated into secretory granules as soluble proteins; inside the vesicles, these proteins are converted to a temporarily insoluble form and finally reassembled after exocytosis, adopting a most stable structure. In this process, the completion of each step depends on the interaction between the proteins inside the vesicles and others that participate in the regulated secretory pathway such as chaperones, SNAREs, ARFs (ADP ribosylation factors), and coat proteins, among others (16, 47-53). The best known proteins acting in this process are proteases, such as prohormone convertases, that are in charge of processing secretory proteins inside the vesicles. In most of cells that produce secretory granules, the success of the proteolytic action seems to be crucial for retention of the proteins in the regulated secretory pathway (54).

In this work, we identified and characterized a protein that possesses distinctive characteristics when compared with other previously reported encystation-induced proteins in the intestinal parasite G. lamblia. This is due to the fact that it is only present in secretory vesicles of encysting trophozoites and not in the cyst wall. Monoclonal antibodies showed that the expression of this protein increases during encystation similarly to CWP1 (10) and CWP2 (9), proteins that are transported within ESVs before they assemble into the cyst wall (1, 2). Transcript levels also increase during encystation as was seen in assays using total RNA of trophozoites cultured in encystation medium for different periods. Screening of a complementary DNA library using mAbs and subsequent cloning and sequencing allowed us to obtain the complete sequence of the gene that codifies a 54-kDa protein with an pI of 5.2. The analysis of gGSP showed a typical eukaryotic signal peptide for sorting the protein to the secretory pathway. No transmembrane regions were detected in the amino acid sequence deduced from the gene, a fact confirmed by Triton X-114 partitioning. No significant homology was observed between gGSP and other proteins reported on GenBank^TM using BLASTP (42). However, gGSP showed one domain highly homologous to those present in calcium-binding protein of the calcequestrin type (55), as well as an ED-rich putative calcium-binding region at the C terminus. In fact, immunoprecipitated gGSP was stained using the metachromatic cationic carbocyanine dye Stains-all, and ⁴⁵Ca²⁺ overlay assays demonstrated the calcium binding properties of gGSP. In addition, the inhibition of gGSP expression completely avoided cyst formation but not CWP expression or ESV biogenesis, denoting that gGSP is essential for cyst wall formation. Only one calcium-binding protein was described previously in Giardia: caltractin/centrin, a prominent component of the basal body of higher cells and a member of the EF-hand superfamily of calcium-binding proteins (56).

Calcium function during protein secretion in other microorganisms has been studied. In Tetrahymena thermophila and Paramecium tetraurelia, for example, the vesicle core synthesis occurs by assembly of soluble proteins into an insoluble, well organized lattice (46, 57). It was proposed that in both microorganisms, this process involves binding of extracellular ionic calcium to regulated secretory proteins (46, 58). Verbsky and Turkewitz (46) postulate that the proregion of Tetrahymena Grl1p (Granule lattice protein 1) may prevent folding of proprotein into the specific conformation inside the immature granules. A function of the proregion might be to allow transport through the calcium-rich early secretory pathway. Once in the mature granule, protein processing results in generation of polypeptides that adopt a metastable structure within the core lattice. In Giardia, structure and processing of CWP2 suggest that it might act similarly during granule maturation.

Yoo and Albanesi (55) reported the effect of inositol 1,4,5-trisphosphate that induces calcium release from secretory vesicles and describe the importance of these vesicles as an intracellular calcium store. They assume that the high Ca²⁺ buffering capacity of secretory vesicles is due to intravesicle proteins with high Ca²⁺ binding capacity because of their calsequestrin-like properties. Similar results were found for secretory granules of higher eukaryotic cells, such as for zymogen-secreting pancreatic acinar cells (59) and for radish vacuoles (27). During Giardia encystation, it has been shown that the addition of calcium to the culture medium facilitates encystation of Giardia trophozoites and postulated that the calcium per se could have a direct effect on cyst wall formation (1). Furthermore, experiments using a Ca²⁺-ionophore also indicate that this cation is essential for secretory granule release.³ These facts, besides the characteristics of gGSP, support the presence in Giardia of secretory mechanisms similar to those proposed for Paramecium and Tetrahymena (27). In the regulated secretory pathway that develops when Giardia encysts, gGSP might function as a calcium sensor within the ESVs and, therefore, might regulate granule discharge at the time of cyst wall formation.

Based on the data presented here and those in previous reports from different laboratories, we propose the following model for secretory granules biogenesis and transport in Giardia, which includes proteins known to be differentially expressed during encystation: synthesis of proteins that will be part of the cyst wall, CWP1 and CWP2 (9, 10), and others implicated in the regulated secretory process (1) begins once cells receive the stimulus that triggers the encystation (18). After their synthesis, CWP1 and CWP2 form a stable complex with each other that might be interacting with a putative receptor at the membranes of the ER or at a post-ER compartment. This interaction could allow the complex to concentrate at specific regions of these membranes, a necessary step for granule biogenesis (1). The correct folding of proteins in the ER could be mediated by the action of chaperones such as BiP (19) and Giardia protein disulfide isomerase 2 (21)⁴; these chaperones may also assist in the complex formation of cyst wall proteins. In this phase of encystation, the structure of CWP1-CWP2 receptor could maintain these proteins in a soluble form during the transport across early secretory compartments enriched in Ca²⁺ (60, 61). During CWP1-CWP2 transit within ESVs toward the plasma membrane, CWP2 is processed by the encystation-specific cysteine proteinase (23). The presence of gGSP inside ESVs suggests an essential role for this molecule as a Ca²⁺ storage protein, avoiding the premature assembly of cyst wall proteins inside the secretory vesicles and controlling the exocytic mechanisms by Ca²⁺ release. Upon exocytosis, cyst wall proteins may then form the filamentous portion of the cyst wall. Further characterization of the molecular interactions occurring among the already known proteins that participate in this regulated secretory pathway (BiP, PDI-2, encystation-specific cysteine protease (ESCP), SNAREs, gGSP, CWPs), as well as other molecules yet to be discovered, will allow not only a better understanding of the basic machinery for protein transport in Giardia but also of the evolution of the adaptive mechanisms developed by eukaryotes early in evolution.

	FOOTNOTES

^* This work was supported by the Agencia Nacional para la Promocion de la Ciencia y la Tecnologia (ANPCYT), Fundacion Antorchas, Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), and the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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/EBI Data Bank with accession number(s) AF293411 and AF293409.

The on-line version of this article (available at http://www.jbc.org) contains three supplementary figures illustrating the purification of ESVs from encysting Giardia trophozoites; Western blot analysis of Giardia trophozoites showing that protein recognized by the mAb is induced during encystation similar to CWPs; and Western blots showing that gGSP is in a soluble form inside secretory vesicles.

^¶ To whom correspondence should be addressed: Cátedra de Bioquímica y Biología Molecular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Pabellón Argentina 2^° piso, Ciudad Universitaria, CP 5000, Córdoba, Argentina. Tel./Fax: 54-351-433-3024; E-mail: hlujan@biomed.uncor.edu.

Published, JBC Papers in Press, September 26, 2002, DOI 10.1074/jbc.M202558200

² M. C. Touz, M. J. Nores, N. Gottig, and H. D. Lujan, unpublished results.

³ H. D. Lujan, M. C. Touz, and N. Gottig, unpublished observations.

⁴ M. J. Nores and H. D. Lujan, unpublished results.

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; CWP, cyst wall protein; mAb, monoclonal antibody; ESV, encystation-specific secretory vesicle; gGSP, G. lamblia granule-specific protein; gsp, G. lamblia granule-specific protein gene; NSF, N-ethylmaleimide-sensitive factor; SNARE, soluble NSF attachment protein receptors; HA, hemagglutinin; nt, nucleotides.

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