From the
Giardia lamblia, an intestinal parasite of humans and
other vertebrates, undergoes surface antigenic variation by modulating
the expression of different variant-specific surface proteins (VSP).
VSPs are cysteine-rich surface proteins that bind zinc and other heavy
metals in vitro. We developed an immunoaffinity
chromatographic method to purify a VSP in order to determine its
biochemical properties. The sequences of two different proteolytic
fragments agreed with the sequence deduced from the cloned gene, and
amino-terminal sequence indicated the removal of a 14-residue signal
peptide, consistent with the transport of VSP to the cell surface. The
protein is not glycosylated and has an isoelectric point of 5.3. X-ray
microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc
concentration in Giardia cells was found to be 0.43
mM and the iron concentration 0.80 mM when compared
with standard samples (zinc) or calculated from a known physical
constants (iron). We propose that metal coordination stabilizes VSPs,
rendering them resistant to proteolytic attack in the upper small
intestine. Moreover, the ability to bind ions by Giardia may
play a role in nutritional deficiency and/or malabsorption in heavily
infected persons.
Giardia lamblia is a binucleate flagellated protozoan
that causes acute and chronic intestinal infections in humans and other
vertebrates
(1) . Infections due to Giardia are among
the most common human infections worldwide, and in developing countries
most children are continually infected or reinfected. Clinical
manifestations vary from asymptomatic infection to severe diarrhea and
malabsorption
(1, 2) .
The entire surface of a
Giardia trophozoite, including the adhesive disk and flagella,
is covered by a protein coat
(3, 4) composed of a single
variant-specific surface protein
(VSP)
With some notable
exceptions
(11, 12, 13, 14) , biochemical
properties of VSPs have been deduced from amino acid sequences
predicted from cloned cDNAs or genes. Analysis of these sequences
showed that VSPs are rich in cysteine (typically 12 mol %;
Fig. 1
), which is found primarily in
Cys-X-X-Cys tetrapeptides distributed nonrandomly
throughout the polypeptide
(13) . Sequence comparisons indicated
that, in contrast to the variability observed among amino-terminal
regions, carboxyl-terminal primary structure, including the putative
transmembrane domain, is highly conserved among VSPs (Ref. 15 and
Fig. 1
). Although all VSPs contain at least one consensus
N-linked glycosylation site, the glycosylation status of these
molecules is unknown. In addition, Nash
(5) identified
zinc-binding motifs in VSPs and suggested that the sequestration of
zinc by VSPs could contribute to some of the symptoms of clinical
giardiasis and zinc malnutrition. Recently, the ability of these
proteins to bind
Here we report a simple method for the purification of a
VSP from the Giardia isolate GS/M, clone H7, by immunoaffinity
chromatography. Our results verify the expression of the protein
predicted from the cloned VSP H7 cDNA and indicate that VSP H7 is
processed by cleavage of a 14-residue amino-terminal signal peptide.
The purified protein contains the COOH-terminal pentapeptide conserved
among all VSPs and, furthermore, is not glycosylated. Consistent with
our previous metal-binding studies, we confirm that native VSP binds
zinc and iron, most likely by coordination to cysteinyl sulfurs.
The mAb G10/4 (IgG
To purify the VSP, 1 ml of
Giardia-soluble proteins in buffer A (about 100 mg) was loaded
at a flow rate of 0.5 ml/h onto the affinity column (5-ml column
volume, 10-mm diameter) pre-equilibrated with 100 ml of buffer A. The
column was washed under the same conditions with 200 ml of buffer A,
and bound VSP was eluted with buffer B (0.10 M glycine, pH
3.0, 0.05% n-butyl alcohol, 0.1% Thesit, 5 µg/ml
leupeptin, 100 µM TLCK, 100 µM
phenylmethylsulfonyl fluoride). Proteins in the fractionated eluate
were detected spectrophotometrically at 280 nm, and fractions
containing the VSP were pooled. Pooled samples were concentrated, the
buffer exchanged for PBS-0.01% Thesit using Centricon 10 concentrators
(Amicon), and stored at -70 °C.
For subsequent analyses,
VSP was precipitated by addition of ice-cold acetone to a final
concentration of 50% (v:v), washed once with acetone at -20
°C, and air-dried. Precipitated samples were resuspended in 0.1%
SDS at a final concentration of approximately 1 mg/ml, determined by
Bio-Rad DC protein assay calibrated with bovine serum albumin as a
standard.
The surface location, abundance, and capacity for antigenic
variability of VSPs suggest an important role for these proteins in the
interaction between the parasite and immune and nonimmune components of
the host small
intestine
(3, 4, 5, 12, 13, 14) .
To better understand the function of these proteins, we purified VSP
H7, expressed by the human G. lamblia isolate GS/M, clone H7,
by detergent extraction and one-step immunoaffinity chromatography. We
constructed an immunoaffinity column by coupling mAb G10/4
(16) to glutaraldehyde-activated Trisacryl
We previously suggested that the
absolute conservation of primary structure at the carboxyl termini of
VSPs reflects a selection for an important biological
function
(15) . However, no published data indicate that either
the COOH-terminal pentapeptide or the putative transmembrane domain are
present on membrane-associated VSP. In order to assess the presence of
the conserved carboxyl-terminal pentapeptide in VSP H7, and perhaps
generate a panreactive VSP antiserum, we raised specific antibodies to
synthetic peptides corresponding to the seven carboxyl-terminal VSP
residues. Among total trophozoite proteins, hyperimmune antipeptide
sera specifically recognized a species of comparable mobility to that
detected with mAb G10/4 (Fig. 3, compare lane 8 with
lane 2). In contrast to mAb G10/4, which recognizes a VSP H7
epitope sensitive to reduction, the antipeptide sera required reduction
of the sample for reactivity (Fig. 3, compare lanes 6 and 8). The identity of this species as VSP H7 was
confirmed by analysis of the purified protein (Fig. 3, lanes
10 and 16). These data indicate that the conserved
COOH-terminal pentapeptide, which contains two charged residues and is
unlikely to be buried in the lipid bilayer, is present on VSP H7. VSPs
are not modified by the addition of a glycosylphosphatidylinositol
anchor
(32) or by prenylation.
The energy-dispersive spectra of
Giardia and VSP samples ( Fig. 4and 5, respectively)
show elemental peaks with x-ray energy of 2500 eV and greater exhibited
directly. Since these experiments were not performed in vacuum,
elements with energies lower than 6000 eV were attenuated. The major
elements in Giardia were zinc, iron, and potassium, with small
quantities of manganese and nickel. In purified VSP the major elements
were zinc and iron. For Giardia cell pellets, the observed
fluorescent count rate of the Zn K-(
In order
to determine the zinc ion environment in Giardia, zinc EXAFS
data were separately collected on zinc meso-tetraphenylporphyrin
(ZnTPP) and zinc sulfide (ZnS) models to provide amplitude and phase
shift information, so as to calibrate the data with respect to
interatomic distances and coordination number (30). The data were
manipulated to remove the contribution from the absorption edge and
these ``background subtracted'' data are shown in
Fig. 6
. Data were Fourier-transformed to convert to a radial
distribution function, and the Giardia Fourier-transformed
data are shown in Fig. 7. The Fourier-transformed data show the
neighboring atoms of the central metal environment directly. That is,
the back-scattering of the atoms in the metal ion vicinity are
exhibited. The main peak shows the atoms directly bounded to zinc.
However, the Fourier-transformed data only show the relative radial
distances of these neighbor atoms. The actual distances of the liganded
atoms are at the observed relative radial distance plus a phase shift,
which depends on the chemical nature of the back-scattering atom. The
first shell peak for Giardia is seen in Fig. 7to be at
2.0 Å. The first shell peak for the ZnTPP, which has four
nitrogens coordinating to zinc at an actual average distance of 2.04
Å, was 1.8 Å (not shown). The first shell peak for the zinc
sulfide model, which has four sulfur atoms coordinated at 2.35 Å,
was observed at 2.0 Å (not shown). Thus, the Giardia environment at first glance closely resembles the zinc sulfide
model. There was very little contribution beyond the first shell except
for a clear peak near the limit of observation (5 Å).
The
Fourier-filtered data of the Giardia sample were fit to
Fourier-filtered model data. Data analysis with fixed coordination
numbers was carried out as described previously using the Bell
Laboratories EXAFS package on a 386 PC
(34) . We compared several
fits with different sulfur:nitrogen (S:N) coordination numbers held
fixed in the fitting procedure. Possible Zn-S and Zn-N distances can be
easily resolved; however, nitrogen and oxygen are generally not
distinguishable. This fitting can define a family of structures
consistent with the EXAFS data. However, without additional information
on the zinc site structure, a unique solution cannot be provided. We
measured the distances, Debye-Waller factors, and energy shift values
of these scatterers by Fourier filtering, back-transforming, and
nonlinear least squares fitting of the Giardia data and
comparing the data with
models
(29, 30, 34, 35, 36, 37, 38, 39) .
The model data are reduced, transformed, and back-transformed
identically to the unknown data, so that artifacts of the manipulations
cancel out. Data fitting ranges were 4-11.75 Å
Previous
investigations of the copper protein stellacyanin and the zinc site of
retroviruses illustrated the difficulties in establishing the
individual sulfur and nitrogen coordination numbers in cases where both
are present, and the total coordination number is four or
five
(30, 39) . This is due to the cross-correlation of
parameters in the fit, especially the coordination number and the
Debye-Waller factor. The relationship of Debye-Waller factors and
amplitudes for the EXAFS equation is such that a more positive
Debye-Waller factor tends to reduce the amplitude, while increasing the
coordination number has the opposite effect. This is an explanation for
how a mixture of site types could be modeled by a single class of sites
in the fit, while still having chemically reasonable Debye-Waller
factors.
Previous EXAFS studies of the (Cys)
Recently, a number of signals characteristic of
iron-sulfur clusters have been found in Giardia, and
ferrodoxin was purified from this parasite
(43) . Since
Giardia lacks mitochondria and hydrogenosomes, previous
studies suggested the importance of iron-binding proteins in electron
transport pathways
(1, 2, 44) . VSP H7 contains
three iron-binding motifs
(-CX
Giardia trophozoites thrive in the proteolytic and lipolytic milieu of the
upper small intestine. We
(5, 13) and others
(12, 14) have proposed that the coordination of metals by VSPs could
stabilize these proteins, thereby rendering them resistant to
proteolytic attack and maintaining the integrity of the cell surface.
In this report we provide compelling support for this model, which
should serve as a new basis for the rational design of effective
therapeutic agents for this important disease. In addition, the ability
of VSP to bind zinc suggests an important role for these surface
proteins in the pathophysiology of the giardiasis. VSP could bind
metals and inhibit important intestinal enzymes such as
carboxypeptidases
(46) . Similarly, VSP metal binding could
inhibit zinc or iron absorption contributing to metal
deficiency
(13, 46) .
A
number of fitting solutions were attempted to model the Giardia EXAFS data. Three results are shown to illustrate the overall
fitting. A single-atom type was sufficient to model the data.
Parameters in bold in the table are chemically unreasonable (30, 39).
Attempts to model the site with mixed zinc plus sulfur coordination
were unsuccessful except for the use of 0.5 nitrogen atoms in the third
solution above. The cross-correlation of coordination number and
Debye-Waller factors is such that increased application of the
amplitude of a nitrogen model (i.e. as the coordination number
assigned to nitrogen) results in an unreasonable Debye-Waller factor as
that contribution approaches 20% of the ligands (one out of five). It
is tacitly assumed that the data could also be adequately fit with
zinc-chlorine scatterers, since sulfur and chlorine differ in Z value by only 1.
We thank Dr. G. Ashwell for carbohydrate analysis, J.
T. Conrad for technical assistance, and Dr. R. Fischetti for assistance
in the calculation of the iron concentration.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(5) . During trophozoite growth
this VSP may be replaced with another antigenically distinct
VSP
(6) . This surface antigenic variation occurs spontaneously
in vitro (7), i.e. in the absence of known selective
pressure, and has been demonstrated in experimental infections of
humans
(8) and laboratory animals
(9) . The potential
antigenic variability is encoded in the VSP gene repertoire, the
minimum size of which has been estimated as 133
(10) . The
mechanism by which Giardia modulates the expression of
different VSP genes, and thereby effects antigenic variation, is
unknown.
Zn in vitro was reported by two
independent groups
(13, 14) . Like other metal-binding
proteins, VSPs can bind different cations, including
Cd
, Co
, Cu
,
Fe
, and Mn
(13, 14) .
Unlike other similar metal-binding proteins, VSPs are localized on the
cell surface.
Figure 1:
Amino acid
sequence of VSP H7 deduced from a cloned cDNA and peptides sequenced
from purified VSP H7. The amino acid sequence of VSP H7 as deduced from
a cloned cDNA (GenBank accession number M80480) is shown with
cysteine residues shaded. Underlining indicates
peptide sequences determined from purified VSP H7. Bold type designates consensus N-linked glycosylation sites. A
box surrounds the putative transmembrane domain, and
asterisks designate residues conserved among the six deduced
VSP sequences (VSP A6, VSP 1267, VSP H7, CRP 72, TSP 11, and TSA 417;
GenBank accession numbers M63966, M80480, M83933, M95814, and M33641,
respectively) as determined by the GCG program
PILEUP.
The function of VSPs is unknown, but the features
conserved among VSPs suggest that these proteins play an important role
in Giardia biology. Direct biochemical analyses of purified,
native VSP would greatly enhance our understanding of VSP structure and
function.
Parasites
The origin of the G. lamblia GS/M isolate and derivation of the H7 clone has been
reported
(16) . Trophozoites were cultured axenically at 37
°C in Diamond's medium TYI-S-33 supplemented with 10% adult
bovine serum and 0.5 mg of bovine bile/ml
(17) . Cultures were
grown to confluence in 8-ml glass tubes. The medium and nonadherent
trophozoites were discarded and replaced with the same volume of
ice-cold phosphate-buffered saline containing 1 mM of
L-cysteine and 20 µg/ml of bathocuproine sulfonate
(PBS-Cys)
(18) . The tubes were chilled for 15 min, inverted 10
times, and the trophozoites counted with a Coulter model ZB1 electronic
counter (Coulter Electronic, Hialeah, FL). Surface antigenic
homogeneity of the harvested cells was deemed greater than 95% by
indirect immunofluorescence using mAb G10/4 (16).
VSP Purification
All steps in the purification
procedure were performed at 4 °C. Trophozoites (5-6
10
cells) were washed twice with ice-cold PBS-Cys and
lysed in 1 ml of buffer A (0.10 M sodium carbonate, pH 7.2,
0.05% n-butyl alcohol, 0.1% Thesit, 5 µg/ml leupeptin, 100
µM TLCK, 100 µM phenylmethylsulfonyl
fluoride) by incubation at 4 °C for 2 h. Cell ghosts were
sedimented at 14,000
g for 60 min at 4 °C, and the
supernatant stored at -70 °C until used (within 1 week).
)
(16) was purified from
ascites by chromatography on protein G Plus-agarose (Pierce). The IgG
fraction (50 mg of protein at 10 mg/ml in 0.2 M carbonate
buffer, pH 8.0) was immobilized on 5 ml of glutaraldehyde-activated
Trisacryl
beads (IBF) according to the manufacturer's
instructions. The column was then washed with 100 ml of PBS and stored
in same buffer containing 0.02% sodium azide at 4 °C up to 3 months
without loss of activity.
Amino Acid Sequence Analysis
Amino-terminal
sequences were determined from purified VSP H7 spotted onto PVDF
membrane (Applied Biosystems, Inc.) by Edman degradation on an
automated Applied Biosystems 477A protein sequencer equipped with an
on-line phenylthiohydantoin analyzer. Internal sequences from VSP H7
were determined by digestion of the purified protein with
endoproteinase Lys-C and separation of peptides by narrowbore reversed
phase chromatography on a Hewlett-Packard 1090 HPLC/1040A diode array
detector using a Vydac C-18 column (2.1 150 mm). Peptides were
eluted with a gradient of 0.057% trifluoroacetic acid/acetonitrile, at
a flow rate of 150 µl/m. Gradient conditions were 5% B at 0 min to
33% B at 63 min, 60% B at 95 min and finally 80% B at 105 min. Peaks
defined by an absorbance at 210 nm were collected and stored at -20
°C before peptide sequence analysis. Amino-terminal sequences were
determined as described above.
Carbohydrate Analyses
Carbohydrate analyses were
performed on purified VSP and control buffer B (treated as the sample).
Purified VSP (10 µg/200 µl) was dried under a stream of
N, 100 µl of 4 M trifluoroacetic acid added to
the white residue, and heated at 100 °C for 4 h. Then, 10 µg of
VSP hydrolysate was injected into a pellicular anion exchange column
(PA-1, 4
250 mm) as described
(19) .
Nucleotide and Amino Acid Sequence Analysis
DNA
Strider 1.2
(20) , AnalyzeSignalase 2.0.3
(21) ,
BLASTP
(22) , and programs in the Genetics Computer Group package
(23) running on the NIH Convex System were used to analyze the
data.
Peptide Synthesis and Conjugation
VSP
carboxyl-terminal peptides made on an Applied Biosystems, Inc. model
430 peptide synthesizer using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry were provided by
the Biological Resources Branch, NIAID. Peptides were purified by
reversed phase HPLC on Vydac C-18 columns and their identities verified
by sequence and compositional analyses. Peptides A (H-FLCRGKA-OH) and B
(H-CFLC-acetoimidomethyl-RGKA-OH) were conjugated individually to both
keyhole limpet hemocyanin (Sigma) and bovine serum albumin (Pentex)
using
N-iodoacetyl--alanine-N-hydroxysuccinimide
(
)
and
-maleimidobutyric acid
N-hydroxysuccinimide ester (24), respectively. After dialysis
against PBS, keyhole limpet hemocyanin conjugates were sterilized by
passage through Millex-HV 0.45-µm filter units (Millipore) before
injection into rabbits.
Antiserum Preparation
Polyclonal rabbit antiserum
raised against GS/M H7 trophozoites was described
previously
(16) . Polyclonal antisera specific for the conserved
VSP carboxyl terminus were raised in female New Zealand White rabbits
by immunization with keyhole limpet hemocyanin conjugates of peptides A
or B. Rabbits were injected initially with 1 mg of conjugate in the
Ribi adjuvant system and boosted twice at 4-week intervals with 0.5 mg
of conjugate; antigen was administered according to the suggestions of
the manufacturer. Peptide-specific reactivity of hyperimmune sera was
established by enzyme-linked immunosorbent assay using plates coated
with the bovine serum albumin-peptide conjugates.
Electrophoretic and Immunoblot Analysis
One- and
two-dimensional polyacrylamide gel electrophoreses
(25) were
performed with a Bio-Rad minigel apparatus as recommended by the
manufacturer. The ampholite combination for the isoelectric focusing
dimension of the two-dimensional gels was 20% pH 3-10, 80% pH
4.0-6.0 (Bio-Lyte, Bio-Rad), to obtain maximal resolution at the
predicted pI of the protein
(10) . Gels were stained with either
Coomassie Brilliant Blue or Bio-Rad Silver Stain Plus according to the
manufacturer's instructions. Electrophoretic transfer of proteins
to nitrocellulose was at 30 V for 10 h in 20 mM Tris, 150
mM glycine, 20% methanol
(26) . Filters were blocked
with 3% defatted milk, 0.1% Tween 20, PBS for 1 h. VSP H7 was
visualized either by reacting with the mouse mAb G10/4, the rabbit
polyclonal antisera anti GS/M isolate, or the rabbit polyclonal
antisera against the COOH-terminal peptides. Polyclonal and mAb
antibodies were used at the dilution specified in the figure legend.
EXAFS and X-ray Microanalysis
Data were collected
at the National Synchrotron Light Source on beamline X-9B using a
Si[111] crystal monochromator. The x-ray EXAFS data of the Zn
K-() signal from Giardia cells were collected using a
13-element energy resolving Germanium detector
(27) . The x-ray
microanalysis data, performed for cells and purified VSP H7, were
collected from the center element of the array. The incident x-ray beam
energy was set at 10.5 keV, and the energy-dispersive x-ray
fluorescence spectra were recorded by a Canberra Ge detector and a
multichannel analyzer. The detector was placed at an angle of 90°
to the incident x-ray beam so that incoherent scattering from the
highly polarized synchrotron beam is minimized. Data were collected at
low temperature as for EXAFS. The x-ray microanalysis spectra shown in
Fig. 4
and Fig. 5are the observed counts with a correction
for the background made by subtracting a spectrum of a pure water
sample. The spectrum of a series of zinc acetate samples was also
collected in the same geometry and with the same path lengths (1 mm) to
create a calibration curve to determine the concentration of zinc in
the Giardia samples. This allowed us to determine the zinc
concentration fairly accurately (0.43 mM), with an error
mostly determined by the unknown density of the Giardia sample
in paste form. The iron concentration could be estimated based on the
ratio of zinc/iron counts if the following three corrections were made:
1) differences in zinc/iron cross-sections, 2) higher fluorescence
yields for zinc, 3) less reabsorption of zinc fluorescence by the
medium (which was assumed to be water in this case). Thus, although the
ratio of zinc/iron-integrated counts was 2.5/1 for the Giardia pellet, the actual ratio of concentrations is lower due to the
above three factors. Briefly, the fluorescence probability for zinc is
0.48, for iron it is 0.34, the zinc and iron cross-sections are 2.33
and 1.409, and the attenuation coefficients for Zn K-(
) and iron
K-(
) are 7.349 and 18.35cm
(28) ,
respectively. Thus, the net integral of iron counts substantially
underestimates the actual concentration by a factor of 4.6 (this also
takes into account losses due to 100 mm of air and the 0.3-mm beryllium
window of the Germanium detector). Thus, for the Giardia pellet the ratios of counts for zinc/iron of 2.5/1, the elemental
ratio is 1/1.85. The methods for collecting zinc EXAFS data have been
published previously
(29, 30) . Data were acquired at 150
K using a helium Displex. For the data shown in Fig. 6and
Fig. 7
, 16 scans with internal total count rates of 40,000
channel
s
or less were co-added.
The Fourier-transformed data were filtered with a window from 1.40 to
2.7 Å
and back-transformed. The fitting range
was 4-11.75 Å
.
Figure 4:
X-ray microanalysis of intact Giardia cells, incident x-ray beam at 10.5 keV. The zinc and iron elements
produce the major x-ray fluorescence seen. Small amounts of manganese
and nickel are observed.
Figure 5:
X-ray microanalysis of purified VSPs,
incident x-ray beam at 10.5 keV.
Figure 6:
Background subtracted
k weighted EXAFS data of intact Giardia. This spectrum is the sum of 16 individual
scans.
Figure 7:
Fourier transform of Fig. 6. The first
shell peak is observed at 2.0 Å, the same position as the Fourier
transform of a zinc sulfide model.
(IBF). This
hydrophilic support, obtained by the polymerization of
N-acryloyl-2-amino-2-hydroxymethyl-1,3-propanediol, together
with use of 0.1% Thesit in the chromatographic buffers, allowed the
purification of VSP H7 at a yield of approximately 13%. Two-dimensional
SDS-PAGE and silver staining revealed high degree of purification
(Fig. 2C). The purity of the preparation was also
confirmed by Western blot analysis using polyclonal antisera against
GS/M-H7 trophozoites (Fig. 2D); no other proteins were
detected. mAb G10/4 detected the same single species (not shown).
Relative to standards, VSP H7 migrated at
55 kDa with a pI of 5.3
(Fig. 2, C and D). These determinations agree
well with the M
of 55,344 and a pI of 5.28
calculated for the deduced VSP H7 amino acid sequence, assuming removal
of a 14-residue amino-terminal signal peptide predicted by the
algorithm of von Heijne
(31) . Cleavage of this putative signal
peptide was verified by the amino-terminal sequence of the purified
protein (Fig. 1). In addition, the sequences of two internal
proteolytic fragments of the purified VSP were identical to those
predicted from the gene (Fig. 1). When compared against a
nonredundant data base that included PDB, SwissProt, PIR, and GenPept
as well as updates (as of April 3, 1995), peptide 1 (Fig. 1,
residues 93 through 110) identified only VSP H7 while peptide 2
(Fig. 1, residues 394 through 406) identified VSP H7 as well as
the corresponding regions of other VSPs with identities ranging from 53
to 70%. Considered together, these data provide compelling evidence
that the immunoaffinity purified protein corresponds to VSP H7.
Figure 2:
Two-dimensional SDS-PAGE and
immunoblotting analysis of total trophozoite proteins and purified VSP
H7. Silver-stained two-dimensional gels of total cell proteins
(A), detergent extract (B), and purified VSP H7
(C) obtained as was described under ``Experimental
Procedures.'' Immunoblot (D) of the purified VSP H7 using
a rabbit antiserum raised against GS/H7 trophozoites (1:1000 dilution).
Top labels indicate isoelectric points and right labels indicate
relative mobility of the standards in kDa
(Bio-Rad).
The
mobility of VSP H7 in SDS-PAGE and its calculated pI suggest that the
protein is monomeric and lacks significant post-translational
modification. Consistent with this suggestion, and despite the presence
of two potential sites of N-glycosylation (Fig. 1,
bold type), carbohydrate analysis indicated that VSP H7 is not
glycosylated (results not shown).
(
)
We infer,
therefore, that this protein is anchored in the plasma membrane by the
putative transmembrane domain.
Figure 3:
Comparative immunoblot analysis of total
trophozoite proteins and purified VSP H7 using COOH-terminal
peptide-specific antisera. Immunodetection by mAb G10/4 (lanes 2,
4, 10, 12), control ascites (lanes 1, 3, 9, 11),
preimmune rabbit serum (lanes 5, 7, 13, 15), and immune rabbit
anti-COOH-terminal peptide serum (lanes 6, 8, 14, 16), in
total trophozoite proteins (lanes 1-8) or in purified
VSP H7 (lanes 9-16). Reduction of the sample by
-mercaptoethanol is indicated at the
bottom.
Previous reports
(13, 14) indicated that VSPs bind zinc, iron, and other similar
metal ions in vitro. To determine the levels and types of
metal ion present in Giardia and specifically in VSPs, we
performed x-ray microanalyses on both samples and collected EXAFS data
on Giardia cells.
) peak was compared with the
signal from defined samples examined in a frozen water matrix. As
discussed under ``Experimental Procedures,'' the zinc
concentration was determined by comparison with standard samples and
the iron concentration was calculated based on known physical constants
(28). The measured zinc concentration was 0.43 mM and the
calculated iron concentration was 0.80 mM. The levels of these
metals in relation to copper were unusually high
(33) . Also, the
similarities in the x-ray microanalysis results with respect to ratios
of transition metal ions raises the possibility that our results in
Giardia are due to the metal content of the VSP.
and R-space windows for the Fourier filter were generally in the
range of 1.4-2.7 Å for the Giardia data and the
models. With these windows and this data range, over six degrees of
freedom are available, so that a two-atom type fit, with coordination
numbers fixed, can be supported. However, as can be seen, one atom type
fits are sufficient to adequately model the data (Fig. 8). The
distance errors are estimated by a number of methods described
previously
(35, 36) . Variations in the individual Zn-S
distances were not resolved by this procedure, data of this k-range
were unable to resolve contributions that are separated by 0.1 Å
or less
(35, 36) .
Figure 8:
Fourier-filtered and back-transformed data
of Fig. 7 compared with simulated fit data of Table I, the one atom fit
with 4 ZnS atoms at 2.35 Å distance. Solid circles represent back-transformed data; squares indicate
simulation.
shows the results of a
series of one- and two-atom fitting procedures. A major uncertainty in
this analysis is a distinct knowledge about the heterogeneity of zinc
sites for the organism. Zinc enzymes are certainly present in
Giardia; however, most zinc enzymes demonstrate mostly
nitrogen coordination (40). Nevertheless, the data can be modeled
adequately by a single type of site with all sulfur ligands. An
adequate fit to the data was provided by 3.8 ± 0.4 sulfur
ligands at 2.35 ± 0.01 Å distance from zinc. The
Fourier-filtered data compared with the fit is shown in Fig. 8.
Applying other fixed coordination numbers; including 3-4 sulfurs
per 1 nitrogen, resulted in unreasonable Debye-Waller factors or energy
shift values for the fit. A reasonable fit was found with a fixed
nitrogen contribution of 0.5. However, this reflects the
cross-correlation of nitrogen and in the
fit
(30, 39, 40) . This additional nitrogen
contribution does not improve the fit to sufficiently warrant the
additional parameters that are required in the modeling.
zinc finger
from the glucocorticoid receptor show minimal back-scattering from the
methene carbons of the cysteine
(41) . This may be due to
disorder in the positions of these carbon atoms. Thus, we do not expect
a strong signal from these carbons in a biological back-scattering
environment. EXAFS studies of the zinc environment of TF-IIIA (which
has two histidine residues), and zinc fingers from retroviruses (which
have one histidine in the zinc finger site), clearly show higher shell
signals due to the pyrrole carbons of the
histidine
(30, 42) . However, the failure to observe such
signals in this sample is not conclusive evidence of lack of histidine
coordination
(29) , due to potential disorder of the site. Thus,
the zinc environment of Giardia is highly unusual and is
presumptively linked to the metal content of the VSP rather than to
cellular enzymes.
CX
CX
C-,
Fig. 1
), which likely explains the presence of iron in the native
protein. This motif as well other iron-binding motifs
(45) are
present in all the known VSP sequences. Preliminary experiments showed
that iron chelators added to Giardia culture medium are toxic
to the parasite.
(
)
Nevertheless, the role of VSP
in the uptake, reserve, or metabolism of iron by Giardia trophozoites remains to be established.
Table:
Fitting solutions for Giardia EXAFS data
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.