(Received for publication, August 22, 1995; and in revised form, October 4, 1995)
From the
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.
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), ()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 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.
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
22,200 and
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 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
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
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
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).
Immunoblot analyses were done essentially as described(6, 18) .
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.
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).
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.
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).
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
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.
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.
Among the
proteins identified by similarity to CWP2, CWP1 (M 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
and pI calculated for the two proteins: removal of this M
13,060 peptide would yield a CWP2 fragment of M
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.
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. ()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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U28965[GenBank].