Eukaryotic Cell, October 2006, p. 1664-1673, Vol. 5, No. 10
1535-9778/06/$08.00+0 doi:10.1128/EC.00120-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Threonine-Rich Repeats Increase Fibronectin Binding in the Candida albicans Adhesin Als5p
Jason M. Rauceo,1,
Richard De Armond,2
Henry Otoo,1
Peter C. Kahn,3
Stephen A. Klotz,4
Nand K. Gaur,2 and
Peter N. Lipke1*
Department of Biology and the Center for Gene Structure and Function, Hunter College of the City University of New York, New York, New
York,1
Southern Arizona VA Health Care System, Tucson, Arizona,2
Department of Biochemistry and Microbiology, Cook College Rutgers University, New Brunswick, New Jersey,3
Department of Medicine, University of Arizona, Tucson,
Arizona4
Received 20 April 2006/
Accepted 10 August 2006
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ABSTRACT
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Commensal and pathogenic states of Candida albicans depend on cell
surface-expressed adhesins, including those of the Als family. Mature Als proteins consist of a 300-residue N-terminal region predicted to have an immunoglobulin (Ig)-like fold, a 104-residue conserved Thr-rich
region (T), a central domain of a variable number of tandem repeats
(TR) of a 36-residue Thr-rich sequence, and a heavily glycosylated
C-terminal Ser/Thr-rich stalk region, also of variable length
(N. K. Gaur and S. A. Klotz, Infect. Immun. 65:
5289-5294, 1997). Domain deletions in ALS5 were
expressed in Saccharomyces cerevisiae to excrete soluble
protein and for surface display. Far UV circular dichroism indicated
that soluble Ig-T showed a single negative peak at 212 nm, consistent
with previous data indicating that this region has high ß-sheet
content with very little
-helix. A truncation of Als5p with
six tandem repeats (Ig-T-TR6) gave spectra with additional
negative ellipticity at 200 nm and, at 227 to 240 nm, spectra
characteristic of a structure with a similar fraction of
ß-sheet but with additional structural elements as
well. Soluble Als5p Ig-T and Ig-T-TR6 fragments
bound to fibronectin in vitro, but the inclusion of the TR region
substantially increased affinity. Cellular adhesion assays with S.
cerevisiae showed that the Ig-T domain mediated adherence to
fibronectin and that TR repeats greatly increased cell-to-cell
aggregation. Thus, the TR region of Als5p modulated the structure of
the Ig-T region, augmented cell adhesion activity through increased
binding to mammalian ligands, and simultaneously promoted fungal
cell-cell
interactions.
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INTRODUCTION
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In Candida albicans, as in other microorganisms that associate
with animals, adherence to host tissues is a key factor in the
maintenance of commensal and pathogenic states. Strains with mutated
adhesin genes or with mutations in genes that regulate adhesins display
reduced virulence (1,
6,
37). The genes in the
ALS (agglutinin-like sequence) family encode cell surface
glycoproteins that mediate adherence to various host substrates.
Currently, there are eight known ALS genes, and the Als
proteins are the most widely expressed adhesins in C. albicans
(16,
37). Als1p, Als3p, and
Als5p have been extensively characterized, and Als1p and Als3p are
involved in C. albicans virulence
(6,
37). Als proteins show a
high degree of identity and similarity between each other, thus
predicting similar three-dimensional structures
(32). Each Als protein
has an N-terminal secretion signal of
20 residues, a
300-residue region with sequence and structures similar to
those of tandem immunoglobulin (Ig) domains (the Ig-like region; 55% to
90% identical across the family), and a 104-residue conserved
threonine-rich region (T) that has 90 to 98% similarity among Als
proteins. There is a central domain consisting of a variable number of
tandem copies of a Thr-rich 36-residue sequence (TR). There is also a
variable-length C-terminal Ser/Thr-rich region which has multiple N-
and O-glycosylation sites. In this region, the sequence variability is
highest between family members
(8,
17,
18). A 13- to 20-residue
C-terminal sequence signals the attachment point of a
glycosylphosphatidylinositol (GPI) anchor, which is processed to
cross-link the adhesins to the cell wall polysaccharide matrix
(21,
25).
Because
multiple Als adhesins are expressed on C. albicans, most of
our knowledge of the activities of individual Als proteins derives from
a Saccharomyces cerevisiae surface display model. In this
system, expression of Als5p results in binding of yeast cells
to a variety of substrates, such as extracellular
matrix-coated beads, human buccal epithelial cells, and
various peptide sequences
(9,
10,
22,
28,
32,
34). In an assay
measuring adherence to protein-coated magnetic beads, Als5p-mediated
adhesion is stable over a broad pH range, persists after vortexing, and
is reversibly inhibited when treated with urea or formamide
(9). The initial binding
of Als5p-expressing cells to ligand triggers a conformational change in
Als5p, leading to the formation of cellular aggregates
(30). Als5p-mediated
adhesion and aggregation are independent of metabolic activity and
signal transduction, but cellular aggregation is inhibited by agents
which perturb protein secondary structure or by H-bond disruptants
(10,
30).
Domain
deletions and swaps in the S. cerevisiae surface display model
show that the Ig-like region mediates much of the substrate binding.
Mutations or deletion of the Ig-like region of Als1p resulted in
inhibition of activity
(28). Adherence was
significantly reduced when the TR region was deleted
(28). Also, a swap of the
Ig-like regions of Als5p and Als6p showed that the binding specificity
of the chimeras mimicked the specificity of the Als protein from which
the Ig-T region came
(32).
Two studies
of the Ig-like region showed that this domain is a ß-sheet-rich
structure. Hoyer and Hecht reported a circular dichroism (CD) spectrum
characteristic of very high ß-sheet content in the Ig-like
region of Als5p excreted and purified from Pichia pastoris
(17). CD spectrometry and
Fourier transform infrared spectroscopy of a soluble Als1p Ig-T
fragment also showed an antiparallel ß-sheet structure, similar
to those of adhesins and invasins of the immunoglobulin superfamily
(17,
32). Comparative modeling
studies of Als proteins and the homologous S. cerevisiae
sexual adhesin
-agglutinin show that this region is compatible
with Ig-like structures
(13,
24,
32).
A small amount
of binding activity remains in Als1p if most of the Ig-like region
(residues 29 to 285) is deleted, and there is a reduction in activity
with deletion of the tandem repeats
(28). This reduction was
attributed to the loss of the extended peptide stalk that elevates the
active site away from the wall surface. Three arguments question this
interpretation. First, the S. cerevisiae sexual adhesins are
fully active on the cell surface with much shorter stalks. Second, the
aggregation activity of Als proteins appears to be proportional to the
number of tandem repeats and not the size of the stalk: the Als1p stalk
is shorter than Als5p, and the former has greater adherence, although
the length of the proteins is around 1,200 residues regardless of the
number of repeats. In addition, the lengths of Als proteins vary
between 900 and 2,000 residues, and forms of various lengths are active
(32). Third, the sequence
of the repeats is conserved, in contrast to known stalk sequences in
Als and other fungal adhesins
(16,
26). This conservation
argues for the preservation of a function strongly dependent on a
specific sequence or conformation. Therefore, we designed and tested
Als5p with and without the TR regions to determine whether there were
activities dependent on the presence of the TR
region.
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MATERIALS AND METHODS
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Construction of plasmid vectors.
The previously
described shuttle vector pGK114
(9) containing the
ALS5 open reading frame facilitated subcloning into the
pYES2.1/V5-His-TOPO cloning kit (Invitrogen), which adds the simian
virus 5 V5 epitope- and His6-encoding sequences just before
the stop codon in cloned open reading frames. Plasmid pGK114 was used
as a template for PCR amplifications. The PCR and sequencing
primers used in this study are listed in
Table 1, and the various plasmid constructs made are shown in Fig.
1.

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FIG. 1. Expressed
versions of Als5p. The regions of Als5p are marked: S, signal sequence
(residues 1 to 17); Ig, tandem Ig-like domains (residues 18 to 327); T,
conserved Thr-rich region (residues 328 to 431); TR, tandem Thr-rich
repeats (residues 432 to 664); Stalk, Ser-Thr-rich glycosylated stalk;
GPI, glycosylphosphatidylinositol addition signal; V, V5 epitope tag;
H, His6 tag. For the protein names, the N-terminal-expressed
residues are numbered, and "W" denotes that residues
650 to 1419 (stalk and GPI) are also expressed. The results of
immunofluorescence assays (IFA) for each cell-bound construct next to
each surface-bound construct are shown. The primary antibodies were
anti-Als1p for all constructs except Als5p1-17,432-662-W,
which was labeled with
anti-V5.
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For plasmids pRL02, pRL03, and pRL09, the
corresponding sequences were amplified and ligated into the pYES
vector. Following amplification in Escherichia coli XL10-Gold
cells (Stratagene), plasmid DNA was extracted using a QIAGEN plasmid
extraction kit according to the manufacturer's instructions and
analyzed by restriction enzyme digestion to verify correct insert
orientation. Plasmids containing the correct ALS5 insert
orientation were sequenced, amplified in E. coli, and
extracted using a QIAGEN maxiprep kit. Plasmid DNA was used to
transform S. cerevisiae W303-1B cells by the lithium acetate
protocol (11).
To
create plasmid pRL10, the ALS5 N-Terminal Ig-T region was
amplified by PCR. The 5' forward primer contained an EcoRI
endonuclease site, and the 3' reverse primer contained a
six-histidine tag for immunodetection, followed by a BglII endonuclease
site. Amplicons were ligated into pYES to generate plasmid RL10. In the
second PCR, the ALS5 C-terminal region containing the
Ser/Thr-rich stalk, GPI anchor, and stop codon was amplified. The
5' forward primer contained a BglII endonuclease site, and the
3' reverse primer contained an XhoI endonuclease site.
Amplicons were ligated to pYES vector to form plasmid pRL10'.
Plasmids pRL10 and pRL10' were amplified in E. coli
and purified. To generate a secretion signal, the chimeric shuttle
vector pGL01 was used. This vector is a modification of plasmid
p423GAL1 (ATCC), containing an invertase secretion
signal/
-agglutinin/green fluorescent protein (GFP) fusion
product regulated by a GAL1 promoter. The
-agglutinin/GFP fusion protein was shown to be localized at
high levels on the yeast cell surface when induced by galactose (M.
Gonzalez, personal communication). pGL01 was digested with EcoRI and
XhoI to release the
-agglutinin/GFP fragment, and the
7.0-kb vector fragment containing the p423GAL1 backbone and
invertase secretion signal was gel purified. pRL10 was also digested
with EcoRI and BglII, and the
1.3-kb product containing the
ALS5 Ig-T fragment was gel purified. pRL10' was
digested with BglII and XhoI to release the
2.0-kb
ALS5 C-terminal fragment and gel purified. The three fragments
were then ligated together, and subsequent cloning steps followed the
procedures described above. A similar procedure was followed to create
plasmid pRL08, with a V5 epitope added immediately after the signal
sequence.
Plasmids pDG100 (containing two tandem repeats) and
pDG102 (containing four tandem repeats) were derived from pGK114
through homologous recombination between ALS5 tandem repeats
in S. cerevisiae
(23). pGK114 was digested
with BtgI at a unique restriction site located in the second tandem
repeat of ALS5. The linearized plasmid was transformed into
S. cerevisiae, and the yeast cells were plated onto synthetic
complete-Trp plates and grown at 28°C for 2 days. Only colonies
with recircularized recombinant plasmids grow under these conditions.
Colonies were scraped using a sterile cell scraper and suspended in 400
µl sterile H2O. Total DNA was isolated by glass
beads and phenol-chloroform
(31) and transformed into
E. coli, and transformants were selected on ampicillin plates.
Plasmids isolated from the E. coli transformants were analyzed
by restriction digestion with ClaI and SphI. The wild-type
ALS5 gives a DNA fragment of approximately 1,200 bp, and
mutant plasmids with four or two tandem repeats have approximately
1,000- or 800-bp fragments, respectively. The TR regions of two
plasmids with 1,000-bp fragments and two plasmids with 800-bp fragments
had the expected sequences and were transformed into S.
cerevisiae. The members of each pair of similar plasmids resulted
in similar phenotypes, and one of each pair was used in subsequent
experiments.
Protein expression and purification of Als5p1-664 (Ig-T-TR6) and Als5p1-431 (Ig-T) fragments.
The growth of S. cerevisiae
cells containing the appropriate plasmid in defined medium without
uracil with galactose as a carbon source resulted in expression of
soluble proteins. Similarly, surface-displayed proteins were expressed
through growth in defined medium without tryptophan (pGK114, pDG100,
and pDG102) or histidine (pRL08 and pRL10). Protein excretion into the
culture supernatant was verified by dot blot analysis as described
below. Als5p cell culture supernatants (1 to 4 liters) were harvested,
and phenylmethylsulfonyl fluoride was added to a final
concentration of 10 µM. The supernatant was concentrated to
approximately 150 ml through a Millipore filtration apparatus fitted
with a 30-kDa molecular mass cutoff membrane. The concentrated
supernatant was adjusted to pH 7.0 using 1 M Tris and chromatographed
on a nickel-nitrilotriacetic acid column (QIAGEN) preequilibrated with
20 mM imidazole, 300 mM NaCl, and 50 mM sodium phosphate
buffer, pH 7.00 (buffer A). The column was washed with
buffer A to remove nonspecific proteins. Als5p fragments were eluted
with 500 mM imidazole, 300 mM NaCl, and 50 mM sodium phosphate buffer,
pH 7.00 (buffer B). The presence of Als5p fragments was determined by
dot blot analysis. Fractions containing Als5p fragments were pooled and
dialyzed exhaustively into 20 mM sodium phosphate and
0.01%NaN3, pH 7.00 buffer (buffer C), using a 3,500-kDa
molecular mass cutoff membrane. The sample was concentrated by further
dialysis against buffer C supplemented with 10% polyethylene glycol
35000 (molecular mass). Protein concentration was determined as
A280. Several milligrams of Als5p1-664
and Als5p1-431 were
purified.
Polyacrylamide gel electrophoresis.
Als5p
samples were electrophoresed in sodium dodecyl sulfate (SDS)
polyacrylamide gels (4 to 20% precast gels; Bio-Rad Laboratories) and
stained with Coomassie blue dye. Immunoblot analyses were performed by
standard procedures
(14).
Dot blot analyses of Als5p.
Als5p samples were immobilized on
nitrocellulose membranes and allowed to dry. Membranes were blocked for
1 h using phosphate-buffered saline (PBS) with 5.0% (wt/vol)
dry milk (PBSTM) (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4,
0.1% Tween 20, pH 7.0) and washed with PBS-Tween 20 (PBST)buffer three times. Next, the membrane was probed for 1 h
using anti-His6 or anti-V5 peroxidase-conjugated antibody (Invitrogen)
at a working concentration of 1:5,000 and washed. Proteins were
detected by incubating the membranes with equal parts of SuperSignal
West Pico stable peroxide and SuperSignal West Pico luminol/enhancer
solutions (Pierce). The membrane was exposed to film and developed
using an X-OMAT developing
apparatus.
Indirect immunofluorescence assays.
Immunofluorescence assays
were performed as previously described with a polyclonal antibody
raised against Als1p
(30).
Fibronectin binding assays.
Adherence
assays were performed as described previously
(9). Briefly, S.
cerevisiae cells containing plasmids encoding wall-bound adhesins
were grown with shaking at 30°C in YPGal medium (10 g of yeast
extract per liter, 20 g of peptone per liter, and
20 g of galactose per liter) to stationary phase for Als5p
expression. Cells were harvested and washed with Tris-EDTA (TE) buffer
(pH 7.0) three times and then resuspended in TE buffer. In a 13- by
100-mm glass tube, Als5p-expressing cells were mixed with fibronectin
(FN)-coated magnetic beads at a cell-to-bead ratio of 100:1 in TE
buffer, briefly vortexed, and incubated at room temperature with gentle
shaking for 30 to 45 min. Each tube was vortexed briefly and
immediately placed into a magnetic separator (Dynal). Adherent and
aggregated cells were gently washed three times with TE buffer while
the tube remained within the magnet. The cells were resuspended in TE
buffer, and a sample was placed onto a microscope slide for
examination. Adherent cells were quantified by hemacytometer counting
after dissociation with 0.1 M NaOH
(9). Experiments were
performed at least three independent times.
Cells were viewed
with a Nikon Optiphot-2 microscope equipped with a Sony DK-5000 camera.
For assays in which the effects of additives on adhesion were
investigated, additives were added to the cell-bead mixture at the
onset of the adherence assays.
Analysis of protein glycosylation.
Dot blot analyses were performed as
described above with the exception that blots were probed with 0.5
µg/ml concanavalin A (ConA) in PBSTM buffer supplemented with
10 µM each of CaCl2 and MnSO4. Hexose was
quantified by the phenol-sulfuric acid method
(5).
CD spectroscopy.
Far UV
spectra of Als5p1-664 (Ig-T-TR6) and
Als5p1-431 (Ig-T) were recorded using AVIV model 215
spectropolarimeters, using quartz, thermo-regulated cuvettes (Hellma)
with a 0.1-cm path length. Protein solutions (0.4 mg/ml) in 20 mM
sodium phosphate buffer, pH 7.0, were multiply scanned, averaged,
smoothed, and corrected by subtraction of the 20 mM
NaH2PO4 buffer baseline spectra. Molar
ellipticities were calculated based on the amino acid composition and
molar extinction coefficient for each fragment
(12). CD data were
analyzed by the self-consistent method with CONTINLL, using data
between 205 and 250 nm
(35).
Fibronectin binding assay for soluble Als5p fragments.
In a polystyrene 96-well plate, a
1-mg/ml solution of fibronectin (Sigma) was serially diluted across the
row (1 mg/ml to 10 ng/ml in 10-fold decrements). All samples were
analyzed in duplicate. Each well contained a volume of 50 µl,
and the plate was incubated for 2 h at room temperature or
overnight at 4°C. Next, the wells were washed three times with
PBST buffer, blocked for 1 h with 200 µl of PBST
buffer supplemented with fresh native bovine serum albumin (BSA) at 1
mg/ml, and washed. A total of 15 µg of the designated Als5p
fragment was added to the wells, incubated for 2 h at room
temperature, and washed. The wells were then incubated for 2
h with primary anti-V5 antibodies at a working concentration of
1:5,000, washed, and incubated with secondary anti-mouse alkaline
phosphatase-conjugated antibodies at a working concentration of
1:10,000 for 1 h. Hydrolysis of p-nitrophenol was
monitored as A420 in a microplate reader after 30
min or more of color
development.
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RESULTS
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Construction and purification of the Als5p Ig-T and Ig-T-TR6 domains.
We constructed
S. cerevisiae plasmids that encoded and expressed both cell
surface-bound and soluble forms of Als5p. ALS5 gene fragments
were amplified by PCR and subcloned into the pYES shuttle vector to
create plasmids pRL02 and pRL09, respectively (Fig.
1). The proteins produced
by the various plasmids were named for the amino acid residues encoded.
For example, Als5p1-431-W has Als5p residues 1 to 431 and
650 to 1419 ("-W") but lacks residues 432 to 649 in the
TR region.
Als5p1-431 (Ig-T) and Als5p1-664
(Ig-T-TR6) fragments, each without the wall-anchoring
residues but with C-terminal V5 and His6 tags, were
harvested from yeast culture supernatants, and dot blot s analysis
verified secretion (Fig.
2). The proteins were purified by Ni-nitrilotriacetic acid column
chromatography, and SDS-polyacrylamide gels showed that they were the
major stainable proteins present (Fig.
3). Immunoblotting showed the presence of the V5 epitope in the isolated
protein (data not shown).

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FIG. 2. Expression
and secretion of soluble forms of Als5p. Equal aliquots of His-TRAP
elution fractions were spotted on nitrocellulose and probed with
peroxidase-conjugated anti-His6. White vertical arrows
denote antigen-negative fractions. Horizontal arrowheads denote
positive controls (black arrowhead, purified Als5p1-664 or
His6-labeled -agglutinin
[36]) and negative
controls (white arrowhead,
BSA).
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FIG. 3. Purified
soluble proteins. Purified Als5p1-431 (lane 1) and
Als5p1-664 (lane 4) were electrophoresed on a 4 to 20% gel
and stained with Coomassie blue. The apparent molecular masses for
standard proteins (lane 3) are on the left. Lane 2 is
empty.
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The Als5p T region is critical for protein secretion.
Both the Als5p1-431 (Ig-T)
and Als5p1-664 (Ig-T-TR6) fragments contain the T
region of the protein. In contrast, Als5p1-329 (Ig) without
the T region was not secreted into the culture supernatant (Fig.
2). Dot blot analysis
demonstrated that the protein was translated and was present in the
lysate of Als5p1-329-expressing yeast cells. Extracts of
control cells without plasmid showed no immunoreactivity. In addition,
Als5p1-329-W, a deletion mutant of Als5p that lacked the T
and TR regions, did not localize the protein on the cell wall (Fig.
1). When the T region was
reinserted (Als5p1-431-W), the protein was successfully
localized on the cell wall (Fig.
1 and see below).
Therefore, the T region was required for proper secretion of Als5p
N-terminal fragments in S.
cerevisiae.
Structural characterization of the Als5p N-terminal and central domains.
CD spectroscopy studies have reported
that Als5p1-329 (Ig regions) and Als1p1-432 (Ig-T
regions) fragments have far UV CD spectra with a single minimum close
to 212 nm, near the characteristic negative peak for proteins with
mostly antiparallel ß-sheet and very little
-helical
content (17,
32). There was a similar
spectrum for Als5p1-431, the Als5p fragment containing the
Ig and T regions (Fig.
4). CD spectra were also obtained for Als5p1-664, which contains
six repeats in the TR region as well. These spectra showed substantive
differences, including more-negative ellipticity around 200 nm, less
negativity at the 212-nm peak, and additional negative shoulders near
227 nm and in the region >235 nm (Fig.
4). These shoulders are
also present in the Als5p1-431 spectrum, but overlap with
the greater negative ellipticity of the 212-nm band makes them less
distinct.

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FIG. 4. Far
UV circular dichroism spectra for Als5p1-431 and
Als5p1-664. Multiple spectra were averaged, smoothed, and
baseline corrected for 0.4-mg/ml solutions of each protein, as
described in Materials and Methods. The buffer was 20 mM sodium
phosphate, pH
7.0.
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Despite the differences, CONTINLL analyses of the
spectra showed that the Ig-T-TR6 and Ig-T proteins had
similar fractions of each secondary structure, that is, the TR region
contributed to the ß-sheet, turn, and aperiodic conformations
but had few, if any, residues in helical conformation (Table
2). The peaks at 227 to 235 nm are characteristic of CD contributions from
aromatic amino acid side chains in an asymmetric environment
(20).
SDS-polyacrylamide
gel electrophoresis showed that Als5p1-431 had an apparent
molecular mass of 66 kDa, about 12 kDa greater than the calculated size
of 53.6 kDa. This lower mobility is common in proteins with a high
content of hydroxy amino acids
(29).
Als5p1-664 had a much greater discrepancy: its apparent
molecular mass was 125 kDa, 52% greater than the expected size of 81.7
kDa (Fig. 3).Since no N-glycosylation sites exist within Als5p1-664, we
tested whether the protein was O glycosylated. Dot blot analysis using
ConA-horseradish peroxidase as a probe showed a positive reaction for
Als5p1-664, an indication of the presence of
-linked mannose or glucose (Fig.
5). Glycosylation was specifically localized to the TR region,
because ConA blots with Als5p1-431 were negative,
in agreement with published results
(17). Quantification of
the hexoses associated with Als5p1-664 demonstrated
approximately 90 mol of hexose per mole of protein, or
1.5
molecules of hexose per Ser or Thr residue in the TR6
region.

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FIG. 5. Dot
blots with horseradish peroxidase-conjugated concanavalin A. Aliquots
of His-TRAP elution fractions were spotted on nitrocellulose and probed
with peroxidase-conjugated concanavalin A. White vertical arrows denote
antibody-negative fractions. Horizontal arrowheads denote positive
controls (black arrowhead, purified His6-labeled
-agglutinin
[36]) and negative
controls (white arrowhead,
BSA).
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Effect of TR on Als5p Ig-T fragment binding to fibronectin.
The differences between
Als5p1-431 (Ig-T) and Als5p1-664
(Ig-T-TR6) implied that there might be differences in
function as well. Therefore, we tested binding ability in a
semiquantitative assay. The two Als5p fragments were bound to FN-coated
microtiter plate wells, and the amount bound was determined by an
enzyme-linked immunosorbent assay using antibody to V5. Both Als5p
fragments bound to the FN at levels above those of binding to wells
coated only with native BSA (Fig.
6). Negative controls omitting primary antibodies, FN, or Als5p showed
absorbance values that were not statistically different from baseline
values from wells in which the secondary antibody or
p-nitrophenol had been omitted. Als5p1-664 bound
with much greater affinity than Als5p1-431 (Fig.
6). The difference in
apparent affinities was not due to differences in antibody binding,
because both Als5p fragments bound similar amounts of antibody at
similar concentrations in assays which measured antibody binding to
immobilized Als5p (data not shown). Estimation of the FN concentration
at half-maximal binding (Fig.
6) implied that
Als5p1-664 had a dissociation constant in the nanomole
range.

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FIG. 6. Binding
of Als5p1-431 and Als5p1-664 to FN-coated
microtiter wells. Wells were coated at the indicated
concentration of FN (determined as protein added), and Als5p binding
was assayed as described in Materials and Methods. The results for
three independent experiments with different preparations of Als5p are
shown. Error bars are ranges for duplicate samples in each experiment.
(Inset) Expanded scale showing binding for Als5p1-431; the
filled diamond indicates the binding in wells coated with BSA
alone.
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To determine whether the two forms of Als5p had similar
amounts of active protein, we assayed various concentrations of Als5p
binding to excess immobilized FN. In these assays, there were similar
ratios of bound to free protein for Als5p1-431 and
Als5p1-664 (data not shown). Therefore, similar amounts of
active protein were bound at each concentration of Als5p, implying
equivalent molar fractions of active protein. The results confirmed the
affinity differences shown in Fig.
6, because 200-fold-higher
concentrations of FN were required for binding of the
Als5p1-431 Ig-T fragment than for the Als5p1-664
Ig-T-TR6 fragment. Taken together, these results show that
the Als5p Ig-T-TR6 regions bound fibronectin better than the
Ig-T regions alone and that the TR region modifies Ig domain
function.
The TR region enhances cell-to-cell aggregation.
In order to further
understand the role of the Als5p domains in mediating adhesion and
cell-cell aggregation, we expressed a cell surface construct containing
a deletion of the TR region (pRL10/Als5p1-431-W) (Fig.
1). After ALS5
expression was induced by growth in YPGal, immunofluorescence
microscopy of intact cells and aggregates revealed uniform fluorescence
(Fig.
1 and
7F).

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FIG. 7. Adhesion
to FN-coated beads and yeast cell aggregation. S. cerevisiae
cells expressing Als5p (A and B), Als5p1-431-W (C to F),
Als5p1-17,432-664-W (G), or no Als (H) were
incubated with FN-coated magnetic beads (orange-brown), and the
aggregates were separated and photographed
(9). Panels B and F show
immunofluorescence with anti-Als1p to demonstrate surface expression of
Als5p in the aggregates. For scale comparison, the magnetic beads have
a diameter of 4.5
µm.
|
|
Adhesion
assays demonstrated the adhesion and aggregation activity of cells
expressing Als5p1-431-W (Ig-T-W). Als5p1-431-W
cells adhered to FN-coated beads similarly to Als5p cells. However,
cell-to-cell binding was drastically reduced for
Als5p1-431-W cells compared to Als5p (wild-type) cells (Fig.
7, compare panel A with
panels C to E). Posttreatment of cellular aggregates with anti-Als1p
antibodies (28,
30) revealed similar
levels of protein on the cell surface (Fig.
7B and F). Therefore, the
TR region contributed significantly to Als5p-mediated cellular
aggregation.
The contribution of the TR region was confirmed in
adhesion/aggregation assays with surface-displayed Als5p with zero,
two, four, or six copies of the repeats. We quantified adhesion to
beads and cell-to-cell aggregation for Als5p1-431-W
(Ig-T-W), Als5p1-505-W (Ig-T-TR2-W),
Als5p1-577-W (Ig-T-TR4-W), and complete Als5p
(Ig-T-TR6-W) (Fig.
8). The intensity of cell surface immunofluorescence with anti-Als1p
antibody was similar for each of these proteins (Fig.
1), implying similar cell
surface concentrations. Although cells expressing any of these versions
of Als5p adhered well to FN-coated beads, the number of cell-to-cell
interactions, shown in Fig.
8 as cell-to-bead ratios,
increased with each increase in the number of
repeats.

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|
FIG. 8. Adhesion
to FN-coated beads and aggregation of S. cerevisiae cells
expressing Als5p with different numbers of tandem repeats. Binding in
the bead aggregation assay was quantified for Als5p containing six
repeats (Als5p), four repeats (Als5p1-575-W), two repeats
(Als5p1-503-W), or no tandem repeats
(Als5p1-431-W). Error bars show standard deviations for
quadruplicate samples. Surface expression levels were similar for all
constructs (Fig.
1).
|
|
Adherence properties of the Als5p Thr-rich repeat region.
Plasmid pRL08 encodes
Als5p1-17,432-664-W, a surface-bound V5 epitope-tagged
version of Als5p without the Ig-like region (Fig.
1). YPGal cultures of
cells carrying this plasmid showed variable expression of detectable
surface-bound antigen. Positive cultures, such as that shown in Fig.
1, were tested in the
adherence assay (Fig. 7G).
These cells showed poor binding to beads but had more cell-to-cell
binding than similar cells grown in glucose or than cells without
plasmid grown in YPGal (Fig.
7H). Thus, the TR and
stalk regions alone can mediate cell-cell interactions. There was also
limited binding to FN-coated beads (not
shown).
Substrate specificity of surface-bound Als5p.
The
surface display model was used to test substrate specificity of Als5p
binding. When Als5p-expressing cells were incubated in microtiter wells
coated with various proteins, the cells bound to wells coated with FN
or with Als5p1-664 (Ig-T-TR6). Cells bound poorly
to uncoated wells or to wells coated with native BSA (Table
3). Cells not displaying Als5p did not bind to wells coated with any tested
substrate. Thus, Als5p1-664 was a ligand for
surface-displayed Als5p. Therefore surface-displayed Als5p mediated
binding to protein ligands but not to uncoated
polystyrene.
 |
DISCUSSION
|
|---|
The
TR region of Als5p consists of six copies of a 36-mer
repeat that is rich in Thr and other ß-branched amino acids
(8). Such repeats are
common to all Als proteins and have been implicated in activity; those
Als proteins with more repeats are often associated with a stronger
adherence phenotype (16,
32). However, much of the
adherence activity resides in the Ig-T region, because deletions in
this region abrogated >90% of the binding activity in
surface-displayed Als1p, and the adhesion characteristics of
surface-displayed Als5p/Als6p chimeras were determined by which Ig-T
region was expressed (28,
32). These results and
sequence analyses have led to suggestions that the TR region was either
a spacer region or a site for cross-linking to the wall matrix
(16,
19,
28,
32). In contrast, we find
that the TR region has a defined secondary structure and contributes
positively to the activity of the Als5p Ig-T region in vitro. In the
surface display model, the repeats have adherence activity and, in
addition, they augment the binding affinity and aggregation activity of
surface-displayed Als5p.
Glycosylation in the tandem repeats.
In
Als5p1-664 (Ig-T-TR6) produced in S.
cerevisiae, there was substantial glycosylation in the
fragment with the TR repeats but not in the Als5p1-431
fragment that lacks the repeats. This glycosylation was reflected in
the reduced mobility on gel electrophoresis, the reactivity with ConA,
and the hexose content of the purified protein fragment. The
glycosylation must be O linked because there are no
N-glycosylation sites in the Ig, T, or TR region of Als5p
(although some are present in the TR regions of other Als proteins). It
is likely that this O glycosylation is important for structural and
functional properties of this region. The O glycosylations in C.
albicans and S. cerevisiae are similar, except that the
C. albicans glycans lack an
-1,3-mannosyl residue
present at the nonreducing end in S. cerevisiae
(1,
15).
The
lack of glycosylation in Als5p1-431 implies that the T
region is not O glycosylated, despite its high Thr content (35%). This
finding is consistent with its inclusion in models of Ig-like domains
of Als5p (32). The
occurrence of O glycosylations in only some domains is common with the
homologous adhesin
-agglutinin of S. cerevisiae. In
this protein, Ser and Thr residues N terminal to Ser282 are
unglycosylated, but all tested hydroxy amino acids C terminal to
position 282 are mannosylated
(3). To our knowledge,
such mapping has not been done for C. albicans mannoproteins,
but the similarities in binding abilities of C.
albicans and Als5p-expressing S.cerevisiae imply that glycosylation differences do not
greatly affect the function of Als5p
(7,
22,
28,
30).
The T region of Als5p is important for protein secretion.
The initial isolation and primary
sequence profiling of ALS5 identified the region with high
ß-sheet potential in between the Ig domain and TR region as the
conserved threonine region
(8). Models of Als
proteins include the T region as part of the Ig domain despite the fact
that this region does not display structural or sequence similarity to
immunoglobulin domains
(32). We find that this
region is essential for the secretion of soluble forms of the
N-terminal regions of Als5p and for secretion and cell wall anchorage
in the surface display model. Because proper folding is essential for
secretion, it is likely that the T region forms a conserved part of the
compact Ig-like region of Als
proteins.
The TR region contributes to the secondary structure of the Als5p Ig-T region.
CD spectroscopy shows that Als Ig and
Ig-T regions have ß-sheet-rich structures, as expected for
tandem Ig-like domains
(17,
32). The CONTINLL
analyses of CD spectra for Als5p1-664 (Ig-T-TR6)
also show a ß-sheet-rich structure (Table
2). Therefore, the
structure of the TR fragment has the same average conformation as the
Ig-T fragment alone. This result is in accord with secondary structure
predictions for this sequence, which is rich in ß-branched
amino acids with high ß-sheet potential
(4).
Because the TR
region changed the CD spectrum somewhat, it must have a structure
distinct from that of the Ig-T region. This difference is not due to
differences in secondary structure content, so there must be
differences in tertiary structure and glycosylation. Also,
Als5p1-664 has different conformational states at different
temperatures (data not shown), a characteristic not shared by the
Als5p1-431 fragment.
The Als5p TR region significantly enhanced binding to fibronectin.
The versions of Als5p that included the
TR showed greater activity than those with TR deleted. Specifically,
the relative affinity in vitro of the Als5p soluble fragments showed
that the TR region modifies binding activity independent of any role in
extension from the wall
(16) or cell wall
anchorage (32). The FN
titration assays with a constant Als5p concentration showed that
Als5p1-664 (Ig-T-TR6) had affinity that was
several orders of magnitude higher than that of Als5p1-431
(Ig-T) (Fig. 6).
Titrations with increasing Als5p concentrations at a constant FN
concentration confirmed the difference in affinity and also showed that
Als5p1-664 binding was complex and not saturable at excess
Als5p (data not shown). This nonsaturability was consistent with the
finding that Als5p can act as its own ligand, as illustrated in Table
2 and Fig.
7, which shows
TR-dependent cell-to-cell binding. The overall conclusion is that the
TR region of Als5p alters the structure of the binding regions and
increases affinity for FN and for binding to itself as well. In the
surface display model, the TR region itself had adherence
activity.
Like the T region, the Als TR region is rich in Thr and
other ß-branched residues. This composition is found in
adhesins of several pathogenic organisms, mucin proteins, and S.
cerevisiae flocculins
(26,
27). There may be
analogous functional consequences to the Thr-rich regions in these
proteins.
Cellular consequences of Als structure.
Our results modify
the idea that Als binding activity is localized to the Ig region alone
and demonstrate a positive role for the TR in Als5p activity. This role
could well have been missed in previous studies because CFU counting
assays used to assess adherence are insensitive to aggregation of the
fungal cells: an adherent single cell or a large aggregate will give
rise to a single colony. Also, there must be some adherence activity in
regions other than the Ig-T region, because ALS1 strains with
deletions in Ig-T show residual ability to bind endothelial cells
(28). We have
specifically documented that the TR repeats in Als5p have a defined
secondary structure and are highly O glycosylated.
The repeats themselves have limited cell-to-cell bindingactivity, and they greatly increase cell-to-cell binding when
coexpressed with the Ig-T regions. Versions of Als5p that included
TR6 also have increased affinity for FN in vitro. Thus, the
activity of Als5p is described by a model in which the Ig-T region is
necessary for most binding, with the T region being essential for
folding and secretion. The TR region positively modulates adherence,
increasing avidity for FN and facilitating the cell-to-cell
binding that is a characteristic of C. albicans
(2,
33).
 |
ACKNOWLEDGMENTS
|
|---|
We thank Dixie Goss and
Yu-Jia Xu for use of the CD spectrometers.
This work was
supported by NIH NIGMS SCORE Program grant S06 60654 (P.N.L.) and a VA
Merit Review grant (N.K.G.). J.M.R. was a recipient of the NIH Ruth
Kirschstein predoctoral fellowship F31 GM070122. Research
infrastructure at Hunter College is supported by NIH RCMI grant
RR030307.
 |
FOOTNOTES
|
|---|
* Corresponding author. Present address: Dept. of Biology, Brooklyn College, 2900
Bedford Ave., Brooklyn, NY 11210. Phone: (718) 951-5000, ext. 1949.
Fax: (718) 951-4659. E-mail: lipke{at}genectr.hunter.cuny.edu. 
Published ahead of print on 25 August 2006. 
Present
address: Dept. of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032. 
 |
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Eukaryotic Cell, October 2006, p. 1664-1673, Vol. 5, No. 10
1535-9778/06/$08.00+0 doi:10.1128/EC.00120-06
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