From the Department of Biological Sciences and the
Institute for Biomolecular Structure and Function, Hunter College of
the City University of New York, New York 10021 and the
¶ Department of Microbiology and Molecular Genetics, University of
Vermont, Burlington, Vermont 05405
Received for publication, November 16, 2000, and in revised form, February 5, 2001
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ABSTRACT |
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a-Agglutinin from Saccharomyces
cerevisiae is a cell adhesion glycoprotein expressed on the
surface of cells of a mating type and consists of an anchorage subunit
Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of
Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C.,
Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and
had low molar specific activity relative to its receptor
The Saccharomyces cerevisiae agglutinins are cell
surface mannoproteins that mediate cell-cell adhesion of haploid cells
during mating. The a- and a-Agglutinin is the product of the genes AGA1 and
AGA2. The 725-amino acid Aga1p precursor includes
hydrophobic N and C termini implicated in transfer into the secretory
pathway and GPI anchor attachment, respectively. The remainder of Aga1p
has 55% serine and threonine residues, consistent with the high level
of O-glycosylation reported in analogs from other yeasts (1,
14). The ten cysteine residues in mature Aga1p are clustered in two
Cys-rich repeats containing five cysteine residues each (Fig. 1).
aga1 mutations allow secretion of Aga2p into the growth
medium, consistent with the role of Aga1p in cell wall attachment of
Aga2p (15, 16). Aga1p is expressed by both MATa
and MAT Aga2p is a small glycoprotein expressed only by
MATa cells. It consists of 69 residues after
cleavage of an 18-residue signal sequence, and at least 10 of the 21 serine and threonine residues are O-glycosylated (19). Cys
residues at positions 7 and 50 of mature Aga2p (corresponding to
positions 25 and 68 of the open reading frame) anchor it to the cell
surface through Aga1p; both cysteine residues must be mutated to lose
cellular agglutinability (20). Aga2p is necessary and sufficient for binding to We purified and characterized Aga2p alone and in complexes with
fragments of Aga1p to characterize its activity and structure. We have
also characterized the effects of a number of mutations in
AGA2. These studies have identified regions and specific
residues in each subunit important in subunit interactions. The results have also clarified the role of specific Aga2p residues in interaction with the receptor Strains--
Escherichia coli strains TG1 and DH5
The a-agglutinin constructs were expressed in S. cerevisiae strains isogenic to W303-1A (MATa
ade2-1 can1-100 trp1-1 ura3-1 his3-11,15 leu2-3,112). The
biochemical analysis of a-agglutinin utilized the W303
MATa/MAT Plasmids--
Agglutinin subunits were expressed in S. cerevisiae from YEpPGK, a URA3-containing
expression vector that contains the PGK promoter and
terminator sequences (22); pRS316, a URA3-containing centromeric plasmid (23); or pRS424, a TRP1-containing YEp
plasmid (24). All primer sequences and construction details are
available upon request from the corresponding author.
N- and C-terminal Histidine-tagged versions of Aga2p were constructed
in YEpPGK using pPGK-AGA2 as template with
standard PCR and restriction techniques (16). These constructs were
sequenced to confirm the correct insertion. An AGA2 clone
containing its homologous promoter was created by PCR amplification of
about 1200 bp of the AGA2 upstream sequences from W303-1A
genomic DNA. The product was subcloned into pRS316 to make
pRS316-AGA2-us. An intact AGA2 gene was
reconstructed by subcloning the EcoRI-BamHI AGA2 fragment from YEpPGK-AGA2 into
pRS316-AGA2-us. The EcoRI-BamHI fragments from various AGA2 mutants were also subcloned into
pRS316-AGA2-us to construct mutant genes expressed from the
AGA2 promoter on a CEN plasmid.
AGA1 DNA was amplified from genomic DNA by PCR (Expand Long Template
PCR system, Roche Molecular Biochemicals, Basel, Switzerland). The
product was cloned into pGEM-T to make pGEM-AGA1 (Promega, Madison, WI) and was subsequently used as template for polymerase chain
reactions in the construction of other plasmids. pRS424-AGA1 (containing the entire open reading frame of AGA1) and
pRS424-AGA1-(1-664) (which lacks the GPI anchor
sequence) were constructed following amplification of
pGEM-AGA1. To construct
YEpPGK-AGA1-(1-149), which encodes the
22-residue signal sequence and the first 127 amino acid residues of the
mature Aga1p, insert DNA was amplified using pGEM-AGA1 as
template and subcloned into YEpPGK. The
YEpPGK-AGA1-(1-149) XhoI-SalI fragment, which contains
AGA1-(1-149) and the PGK promoter and
terminator, was subcloned into pRS424 to create
pRS424-AGA1-(1-149). All other truncation and deletion
plasmids shown in Fig. 1B, as well as a C-terminal HA-tagged
version of pRS424-AGA1-(1-149) were constructed by PCR
using pGEM-AGA1 as template with appropriate primers and
subcloning the product into pRS-424-AGA1-(1-149).
Random Mutagenesis of AGA2--
Random mutagenesis
followed by gap repair recombination was performed as described in
Muhlrad et al. (25). Random PCR mutagenesis was carried out
on YEpPGK-AGA2, varying ratios of MgCl:MnCl and limiting
amounts of each nucleotide (25). The PCR products were ethanol-precipitated and isolated from an agarose gel with the QIAquick
gel extraction kit (Qiagen Inc., Valencia, CA).
Site-directed Mutagenesis--
C-terminal AGA2
mutants were generated using YEpPGK-AGA2 DNA as template in
polymerase chain reactions (PCR) using an upstream oligonucleotide
containing an EcoRI site and downstream oligonucleotides with the desired mutation and a BamHI site. The resulting
mutant AGA2 EcoRI-BamHI fragments were subcloned
into YEpPGK. Additional mutants were made by the QuikChange
site-directed mutagenesis method (Stratagene, La Jolla, CA). The mutant
aga2 genes were sequenced to confirm that no extraneous
mutations were present.
Internal regions of AGA2 for which mutations obtained in the
random mutagenesis affected agglutination were subjected to further mutagenesis using a double-PCR method (26). A primary PCR reaction with
pPGK-AGA2 as a template used appropriate primers to create a
fragment of ~200 bp. This fragment was purified and used as an
upstream mega-primer in a second PCR reaction; the products were
cloned into YEpPGK and sequenced.
Production and Purification of a-Agglutinin
Polypeptides
Aga2p expressed from pPGK-AGA2 was dialyzed
against 20 mM sodium acetate, pH 5.5, 1 mM
EDTA, 0.01% NaN3. The suspension was lyophilized,
resuspended in the same buffer with 10% (v/v) glycerol, clarified by
centrifugation, and applied to a Bio-Gel P-60 column. The eluted
fractions were examined by gel electrophoresis, and stained with
Coomassie Blue or periodic acid Schiff reagent (PAS) (27). (Coomassie
Blue-stained Aga2p was not stably fixed in the gels, and bands
disappeared after 24 h at room temperature). Extended storage or
lyophilization of purified Aga2p generated in a ladder of bands of
apparent molecular size 30 kDa, 60 kDa, and larger upon SDS-gel
electrophoresis. These apparent multimers were reduced to 30 kDa
following treatment with dithiothreitol.
For His-tagged versions of a-agglutinin, the concentrated
culture supernatant was dialyzed overnight against 20 mM sodium phosphate buffer, pH 7.45, 500 mM NaCl (buffer A),
then centrifuged to clarify the suspension. The supernatant was applied to a His-Trap column (Amersham Pharmacia Biotech and Upjohn, Inc., Downers Grove, IL), washed in buffer A, and eluted with buffer A
containing 500 mM imidazole. The eluate was dialyzed
against buffer B (10 mM sodium phosphate, pH 6.7, 0.01%
NaN3). In some cases, the material was further purified by
gel filtration on Bio-Gel P-60.
Protein Analysis--
Polyacrylamide gel electrophoresis of
proteins was according to the method of Laemmli (28). Protein
concentrations were determined by A280, using an
extinction coefficient of 1 per mg/ml for impure solutions, and for
pure polypeptides, the extinction coefficients were calculated from the
amino acid composition (29). Pyroglutaminase was used according to
manufacturer's directions (Roche Molecular Biochemicals). Partial
deglycosylation was effected with jack bean
For immunoblots and dot blots, the proteins were transferred to
polyvinylidene difluoride or nitrocellulose paper under standard conditions. The primary antibodies were anti-HA (Roche Molecular Biochemicals), anti-His-Tag (Amersham Pharmacia Biotech), and polyclonal antibodies to Aga2p (provided by W. Tanner (30) or produced
for this study); these polyclonals were multiply adsorbed with W303-1B
and X2180-1B cells. An anti-rabbit antibody conjugated to horseradish
peroxidase (Sigma) was used as a secondary antibody, developed with
4-chloro-1-naphthol (Sigma) or ECL reagent (Amersham Pharmacia Biotech,
Arlington Heights, IL).
Agglutinin Activity Assays--
Agglutinins were assayed by
published procedures (14, 31). Wild type and mutant aga2
genes expressed from the AGA2 promoter on CEN
plasmids were also tested for the ability to complement the
aga2::LEU2 agglutination defect by
cellular agglutination assays (14).
Our results demonstrate that Aga2p is unstable and most of the
purified subunit is inactive. Aga2p can be stabilized in an active form
by a small fragment of Aga1p, and the two subunits interact through a
pair of disulfide bonds and at least one other region of Aga2p.
Biochemical and molecular analyses imply that the C-terminal-most
residues of Aga2p are critical for binding to its receptor
Biochemical Properties of Aga2p--
Aga2p expressed from plasmids
had properties similar to that released from cell surfaces (14, 19,
20). For both Aga2p and Aga2p with His6 immediately
following the secretion sequence (His6-Aga2p), the yield
averaged 1-2 × 104 units/liter of culture, and the
purified protein showed a single diffuse band on gel electrophoresis
when stained with Coomassie Blue or the glycoconjugate stain PAS. The
M
The specific activity of purified Aga2p or His6-Aga2p
ranged up to 1.4 × 1011 units/mol, comparable to most
previous reports (14, 19, 20). However, we found that the activity of
both Aga2p and His6-Aga2p decreased during storage except
at Co-expression of Aga2p with Aga1p--
Disulfide linkage of Aga2p
to Aga1p involves two disulfide bonds (19). The ten Cys residues in
Aga1p are present in two repeats with five Cys residues each (Fig.
1A). We constructed short
versions of Aga1p containing a single Cys repeat to determine whether
such fragments might support secretion of higher levels of Aga2p
activity. Aga1p-(1-149) contains the signal sequence, a Ser/Thr-rich
region, and the first Cys-rich region. Strains expressing this form of
Aga1p alone did not secrete measurable a-agglutinin
activity. However, when this construct was co-expressed with
His6-Aga2p, secreted activity was 10-fold higher than when
His6-Aga2p was expressed alone. Nickel-affinity
chromatography of the supernatant yielded a complex of
His6-Aga2p with a larger component, presumably
Aga1p-(1-149), as well as His6-Aga2p monomer and dimer
(Fig. 2B, lane 5),
which were removed by gel filtration (lanes 6-10). The
purified complex stained with PAS, indicating glycosylation (Fig.
2C), and upon reduction with DTT yielded two bands, one with
the size of His6-Aga2p near 30 kDa, and a band of apparent
size 105-110 kDa. The upper band was not stained with Coomassie Blue (Fig. 2C). The 30-kDa subunit was also
stained by antibody to His6 or to Aga2p (data not shown).
When His6-Aga2p was co-expressed with Aga1p-(1-149)-HA,
the larger subunit was stained with antibody to the HA epitope (data
not shown). Therefore, co-expression of His6-Aga2p with
Aga1p-(1-149) resulted in secretion of an active complex of these two
proteins.
Properties of the His6-Aga2p·Aga1p-(1-149)
Complex--
The specific activity of the
His6-Aga2p·Aga1p-(1-149) complex was (6 ± 1) × 1012 units/mol (S.E., six independent
determinations on two preparations), which was 43-fold higher than the
activity of Aga2p or His6-Aga2p alone and similar to the
specific activity of purified
The His6-Aga2p component of the complex had smaller
apparent size than the corresponding subunit when expressed alone (Fig. 2A, lane 5 versus lane 3).
This result implied that the subunit in the complex might contain less
carbohydrate than when expressed alone. When His6-Aga2p or
the His6-Aga2p·Aga1p-(1-149) complex was digested with
jack bean Role of Aga1p in Stabilization of a-Agglutinin--
In
addition to high specific activity and measurable secondary structure,
the purified His6-Aga2p·Aga1p-(1-149) complex was stable
for several months at Characterization of the Stabilizing Region of Aga1p--
To
determine the minimal region of Aga1p needed to stabilize Aga2p, we
constructed deletions within the AGA1 gene and co-expressed the various forms of Aga1p with His6-Aga2p. Aga1p-(1-136)
terminates after the intact Cys-rich region and allowed secretion of
activity equivalent to Aga1p-(1-149) (Fig. 1B). In
contrast, Aga1p-(1-132) lacks two Cys residues and supported activity
only at a level equivalent to that of Aga2p alone. Therefore, it is
likely that Cys133, Cys136, or both are
important for maintenance of activity of Aga2p.
To determine the N terminus of the domain required for stabilization of
Aga2p, we constructed internal deletions that fused Aga1p residues
1-29, including the signal sequence, in-frame to various positions.
Expression of Aga1p-(1-29,71-136), which consists only of the signal
sequence and the intact cysteine-rich region, stabilized Aga2p
activity, as did Aga1p-(1-29,106-136), which lacks the first Cys and
34 other residues but retains four Cys residues. More extensive
deletions, to give Aga1p-(1-29,111-136) and Aga1p-(1-29,112-136),
which retain three and two cysteine residues, respectively, still
stabilized activity at levels about 25% of the constructs with four
Cys residues (Fig. 1B). In conclusion, activity of
a-agglutinin was maximal when the binding subunit Aga2p was
coexpressed with fragments of Aga1p that contained the last four Cys
residues of the first Cys-rich repeat. The last two Cys residues, at
positions 133 and 136 are particularly important.
The complex containing Aga1p-(1-29,111-136) with
His6-Aga2p was purified to yield material with specific
activity of 1.2 × 1012 units/mol, 8.6-fold greater
than Aga2p alone and about 5-fold less that the complex with
Aga1p-(1-149). The far-UV CD spectrum was consistent with a structure
containing ~7% A Region of Aga2p Promotes Interaction with Aga1p--
Random
mutagenesis of AGA2 produced three mutations, F56S, Y58C,
and S61P, with an unexpected phenotype: Cells expressing these mutant
genes showed increased agglutinin activity in the cell culture
supernatants (Table I). Serial dilution
dot blots (not shown) demonstrated proportional increases in the amount of protein compared with the wild type, implying that these mutations produced Aga2p with specific activity similar to wild type. To study
these effects further, additional mutations of this region were
constructed. Two- to 4-fold increases in secreted activity were
observed with many of the single amino acid substitution mutations in
this region. Overexpression of Aga1p-(1-149) in two of these mutants,
Y58C and S61P, had no effect on secreted activity. Moreover, in
cellular agglutination assays, mutant S61P did not complement the
aga2::LEU2 agglutination defect, and
mutant Y58C showed a significant reduction in activity relative to wild
type. When residues 56-60 were deleted or when three Ala residues were inserted between Tyr58 and Tyr59, the secreted
agglutinin had activity similar to wild type Aga2p. However, these
mutations were unable to complement
aga2::LEU2 cells, even though expressed
from high copy plasmids. These results could be explained by a defect
in the interaction with Aga1p. Such an effect would reduce or eliminate
cell surface localization of Aga2p, increase the amounts of secreted
Aga2p, and prevent complementation of
aga2::LEU2.
Role of Cys Residues in Aga2p--
Cappellaro et al.
(20) showed that both Cys residues of Aga2p needed to be mutated to
lose the ability to complement an aga2 mutation, indicating
that only a single disulfide bond was sufficient for cellular
agglutination activity (20). However, such cellular agglutination
assays are less sensitive in determining loss of activity than the
assays of secreted Aga2p (see below). To test whether disulfide bonding
to Aga1p through one or both Cys residues is important for maintaining
the active conformation of Aga2p, we tested single and double
Cys-to-Ser mutations of Aga2p for stabilization of activity by
co-expression of Aga1p-(1-149). Dot blots with anti-Aga2p indicated
that all supernatants contained similar amounts of Aga2p. With Aga2p
expressed alone, the single mutations (C25S and C68S) did not affect
activity, and the double mutant showed a mild decrease in activity
(Fig. 5). In this experiment co-expression of Aga1p-(1-149) with wild type Aga2p resulted in a
4-fold increase in activity. The Aga2p C25S,C68S double mutant should
not be disulfide-linked to Aga1p, and as expected, co-expression of
Aga1p-(1-149) did not affect activity. Interestingly, co-expression of
Aga1p-(1-149) also did not increase the activity of either Aga2p
single mutant. These results suggest that both disulfide bonds are
necessary for the Aga1p interaction to stabilize Aga2p in its active
conformation.
Mutational Analysis of Aga2p Determinants for
The affinity of agglutinins or other adhesion proteins can be estimated
from assays using different concentrations of the proteins. Although
protein concentrations in bulk solution can be easily estimated, the
actual cell surface concentrations are significantly higher, so
cellular assays will show lower affinity interactions observable at
higher agglutinin concentrations (34). We can estimate the agglutinin
concentration within 0.5 µm of the cell surface, corresponding to a
spherical shell with 1.7-µm inner radius (the cell diameter) and
2.2-µm outer radius. For a spherical cell of radius 1.7 µm, the
volume is 23 × 10
Thus intact cells present interacting agglutinins at extremely
high concentration relative to assays of nanomolar to micromolar soluble agglutinins. Consequently, many mutants showed decreases in
activity when assayed as soluble agglutinins, but were
indistinguishable from wild type in cellular agglutinability assays for
complementation of the aga2::LEU2 defect.
Peptides containing the C-terminal ten amino acids of Aga2p bind to
Single mutations in this region were generated by site-specific and
random mutagenesis. These mutants showed a similar trend; mutations in
residues closer to the C terminus had greater defects (Table II).
Mutations that resulted in secretion of inactive Aga2p altered the two
most C-terminal residues (F87A, F87L, and V86G). The severity of the
defect varied with the particular amino acid substitution, with F87Y,
V86A, and V86I showing 20- to 50-fold decreases in activity when
expressed alone, but each showed activity close to wild type when
coexpressed with Aga1p-(1-149). In addition, the Y85A mutant showed a
20-fold decrease in activity, and was stabilized significantly by
coexpression with Aga1p-(1-149). Other mutations near the C terminus,
Q84A, T83A, I81A, and P80A, also reduced activity about 2- to 4-fold,
and had close to wild type activity when coexpressed with
Aga1p-(1-149). Again, all mutant proteins were secreted at levels at
least 25% that of wild type. No single mutation inactivated Aga2p
completely; all mutants complemented the
aga2::LEU2 mutant under conditions that
measured binding affinities in the millimolar range. Only the
C-terminal F87A mutant showed a detectable reduction in complementation
of aga2::LEU2. These results indicate
that the most C-terminal residues of Aga2p are critical for its binding
activity. In accordance, a His tag at the C terminus of Aga2p
eliminated activity, indicating that activity is dependent on a free C
terminus (data not shown).
Other Regions of Aga2p May Contribute to Ligand Binding--
Many
protein-protein interactions involve multiple noncontiguous regions of
protein (12). AGA2 was therefore randomly mutagenized by
PCR, and the products were co-transformed with a gapped AGA2 plasmid to obtain intact genes by homologous recombination. Those plasmids mediating altered a-agglutinin activity were
isolated, retested to confirm the phenotype, and sequenced to identify
the mutation(s).
Of the missense mutants, N48I showed almost a 3-fold reduction in
secreted Aga2p activity that was only partially complemented by
Aga1p-(1-149). Alanine scanning mutagenesis of the surrounding residues identified several mutants that also showed mild decreases in
secreted Aga2p activity (Table III), but
all of these mutants could complement the cellular agglutination defect
in aga2::LEU2 cells (not shown). The
reduced activity of some of these mutants suggests that this domain may
contribute to S. cerevisiae Roles of the Anchorage Subunit Aga1p in Aga2p
Activity--
Purification of Aga2p alone led to an unstable protein
with low specific activity. Co-expression of fragments of Aga1p along with Aga2p potentiated a-agglutinin activity, increasing secreted activity 10- to 20-fold (Fig. 1B), and increasing
molar specific activity 43-fold. In addition, disruption of the
Aga1p·Aga2p complex decreased a-agglutinin activity (Fig.
4). CD spectroscopy identified well-defined secondary structure for
Aga2p·Aga1p complexes, whereas isolated Aga2p did not show
well-defined structure. This Aga1p-mediated secondary structure
derived, at least in part, from the Aga2p component, because the total
The Aga2p·Aga1p interaction involves disulfide linkages to the two
Cys residues in mature Aga2p, Cys25 and Cys68.
Cappellaro et al. (20) showed that both Cys residues must be
mutated to lose cellular agglutinability, indicating that both are
involved in the disulfide linkage. There have been no previous reports
of which of the ten Aga1p Cys residues are involved. These residues are
grouped into two repeats with five Cys residues each. Only a small part
of Aga1p, including the N-terminal Cys repeat was necessary to
stabilize a-agglutinin, and the Aga1p-(1-149)·Aga2p complex had the highest specific activity so far reported for any form
of the agglutinin. Deletion of sequences from the N-terminal through
the second Cys residue (Cys106) resulted in Aga1p fragments
that supported high activity. However, the C terminus of the repeat,
including the fourth and/or fifth Cys residues, was essential for
increased activity. Even a short fragment with the third, fourth, and
fifth Cys residues (amino acids 111-136 fused to the signal sequence)
supported increased secretion of a-agglutinin activity and
an 8-fold increase in specific activity of the purified complex
relative to Aga2p alone.
The a-agglutinin analogs from Pichia amethionina
and Hansenula wingei contain multiple binding
subunits disulfide-linked to a single anchorage subunit (1). Therefore,
the Aga1p homologs from these yeasts may contain multiple Cys-rich
clusters, with each cluster involved in disulfide linkage to a single
Aga2p homolog. Similar clusters are found on many fungal cell wall
proteins and are common in mammalian extracellular matrix proteins
(36-38).
Regions of Aga2p--
Cappellaro et al. (19) have
reported that a C-terminal decapeptide of Aga2p has
a-agglutinin activity about 20% that of intact Aga2p. Our
results further define the binding region: Mutations of this C-terminal
region reduced Aga2p activity, with the last two residues,
Val86 and Phe87, showing the most important
role. No other region of Aga2p that is critical for activity was
identified in the random mutagenesis, but mutations in a central region
(residues 48-50) decreased activity, suggesting that this region may
also participate in binding.
Although Aga2p alone and the C-terminal decapeptide can bind
A complementary but unexpected phenotype was observed with mutations of
residues between 56 and 61. These mutations increased secretion of
fully active Aga2p, but the mutated proteins were not stabilized by
co-expression of the Aga1p fragment. In addition, these mutations
reduced the ability to complement an aga2 mutation in the
cellular agglutinability assay. These results suggest that this region
may be important for interaction of Aga2p with Aga1p; mutations in this
region would therefore prevent cell surface anchorage of Aga2p.
The central region of Aga2p (residues 45-72) is 37% identical and
81% similar to sequences in several Nod genes from soy
(Fig. 6) (39, 40). Also within this
region of Aga2p, a short sequence motif shows similarity to von
Willebrand Factor sequence (41). This region of Aga2p includes residues
48-50 in which mutations resulted in mild defects in the
In summary, Aga2p contains all the determinants for binding to
-agglutinin. Aga2p co-expressed with a 149-residue fragment of Aga1p
formed a disulfide-linked complex with specific activity 43-fold higher
than Aga2p expressed alone. Circular dichroism of the complex revealed
a mixed
/
structure, whereas Aga2p alone had no periodic
secondary structure. A 30-residue Cys-rich Aga1p fragment was partially
active in stabilization of Aga2p activity. Mutation of either or both
Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the
roles of Aga1p include both cell wall anchorage and
cysteine-dependent conformational restriction of the
binding subunit Aga2p. Mutagenesis of AGA2 identified only
C-terminal residues of Aga2p as being essential for binding activity.
Aga2p residues 45-72 are similar to sequences in soybean
Nod genes, and include residues implicated in
interactions with both Aga1p (including Cys68) and
-agglutinin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-agglutinins, expressed by
MATa and MAT
cells, respectively,
interact directly with 1:1 stoichiometry. Exposure to the pheromone
expressed by the opposite mating type induces agglutinin expression and
thus facilitates the agglutination reaction (1).
-Agglutinin is a single polypeptide that contains both N-
and O-linked carbohydrate. The
-agglutinin gene
AG
1 (2, 3) encodes a 650-residue precursor
with an N-terminal signal sequence that directs
-agglutinin to the
secretory pathway and a C-terminal hydrophobic sequence that results in
attachment to a glycosyl phosphatidylinositol
(GPI)1 anchor (4, 5).
Attachment of many cell wall proteins, including the agglutinins,
involves transfer from a plasma membrane-linked GPI anchor to cell wall
glycan by a transglycosylation reaction (5, 6). The N-terminal half of
Ag
1p contains the binding site for a-agglutinin and is a
globular region rich in antiparallel
-sheets. Homology models
predict that there are three immunoglobulin-like folds within this
region (7-11). Residues involved in ligand binding were identified
through site-directed mutagenesis of the third Ig domain (12). Homologs
of
-agglutinin mediate adhesion of the pathogenic yeast
Candida albicans to epithelia (13).
cells in a pheromone-responsive manner (15-17).
It is required for efficient mating in liquid cultures and in cells
with a deletion of the homolog FIG2. The deletion can be
complemented by overexpression of related genes FIG2,
FLO11, or FLO10 (18).
-agglutinin, and binding is tight and practically irreversible over the time course of a mating reaction (21, 44).
Synthetic or proteolytic peptides containing the ten C-terminal residues of Aga2p are sufficient to bind
-agglutinin, but at concentrations 4- to 5-fold higher than the full-length peptide (20),
indicating that this region has a major binding determinant and that
carbohydrate or structural features of the full-length protein
contribute to binding.
-agglutinin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were used for plasmid analysis and subcloning. Supercompetent E. coli strain XL2 Blue (Stratagene), was used to clone inverse PCR products.
diploid or the isogenic
MAT
ag
1::LEU2 haploid. Assays
of mutated AGA2 alleles were done in the isogenic
MATa strain W2-UL, which was created by replacing
the aga2::URA3 allele in strain W2 (16) with an aga2::LEU2 derived from the
NdeI-DraIII
ura3::LEU2 fragment from
pRS306
U-LEU (provided by S. Cunningham).
a-Agglutinin polypeptides expressed from
PGK plasmids were produced in W303 diploid or W303-1B
ag
1::LEU2 haploid cells.
Cells were grown to density of 2--
2.5 × 107/ml in
synthetic medium before harvest and collection of the excreted protein
in the culture supernatants. The supernatants were buffered to pH 5.8 with sodium acetate and concentrated ~10-fold through a Millipore
filter with a 30,000-Da cutoff.
-mannosidase (100 µg/ml, used according to manufacturer's directions; Sigma Chemical
Co., St. Louis, MO). Peptide sequences were generated on an ABI gas
phase sequencer in the Hunter College Sequence and Synthesis Facility.
CD spectra were obtained on a JASCO J710 spectropolarimeter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-agglutinin
70 °C. Activity was not restored or preserved by glycerol,
dithiothreitol, EDTA, divalent metal ions, or peroxide oxidation of Cys
residues. CD spectroscopy of the purified Aga2p or
His6-Aga2p yielded spectra typical of denatured peptides, with only aperiodic structures (data not shown). These results implied that much of the isolated Aga2p was in an inactive and
denatured form.
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Fig. 1.
Features of a-agglutinin. A,
diagram of a-agglutinin subunits. The hydrophobic N-terminal
signal sequences and C-terminal GPI anchor signal are solid
black. Cys residues are marked as vertical lines, and
Cys-rich regions are cross-hatched. B, features and activity
of AGA1 constructs. Internal deletions in AGA1
are indicated as lines. All constructs were coexpressed
under the PGK promoter along with His6-Aga2p,
and the activity of culture supernatants was assayed in at least three
independent experiments. The criteria for activity levels were: ±,
mean activity < 25 units/ml, maximum activity < 50 units/ml; +, 70 units/ml < mean activity < 100 units/ml;
++, mean activity> 150 units/ml.
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Fig. 2.
SDS-gel electrophoresis of
His6-Aga2p and the His6-Aga2p·Aga1p-(1-149)
complex. A, Coomassie Blue-stained
His6-Aga2p (lane 3) and
His6-Aga2p·Aga1p-(1-149) complex (lane 6).
Fractions were treated as marked (see text for details). B,
purification of the His6-Aga2p and
His6-Aga2p·Aga1p-(1-149) complex: all lanes are stained
with Coomassie Blue: 1, molecular size markers;
2, culture supernatant; 3, material not binding
to His-TRAP column; 4, wash from His-TRAP column;
5, imidazole eluant from His-TRAP column; 6-10,
successive fractions from BioGel P-30 column. C,
left, glycoconjugates stained with PAS reagent: lane
1, native complex; lane 2, after treatment with DTT
before electrophoresis; right, similar samples stained with
Coomassie Blue.
-agglutinin. The far-UV CD spectrum of
the His6-Aga2p·Aga1p-(1-149) complex showed a positive
rise centered at 204 nm and negative peaks at 208, 212, and 217 nm
(Fig. 3). A slight negative shoulder is
visible at 222 nm. These positions and intensities resulted in
an estimate based on the program SELCON that the structure
included 13%
-helix, 27%
-sheet, 24% turn, and 35% aperiodic
structures (32, 33).
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Fig. 3.
Circular dichroism spectra of
His6-Aga2p·Aga1p complexes. Ten spectra were summed
and background was subtracted for each spectrum: solid line,
His6-Aga2p·Aga1p-(1-149) complex, mean residue mass
105.77 Da; dotted line,
His6-Aga2p·Aga1p-(1-29,111-136) complex, mean residue
mass 107.51 Da.
-mannosidase, the apparent size was reduced (Fig.
2A). Aga2p expressed alone was reduced to about 19 kDa
(lane 2), whereas Aga2p released from the complex generated heterogeneous material with apparent size ranging from the dye front up
to 25 kDa (lane 4). Within the smear there were visible bands at 15 kDa (poorly visible in the photo), 23 kDa, and 25 kDa.
Deglycosylated Aga2p isolated alone or in complex with Aga1p-(1-149) remained stainable by PAS reagent (data not shown).
20 °C. To determine whether disulfide bonding of the subunits stabilized an active form of
a-agglutinin, the complex was treated with DTT for various
times, diluted, and assayed (Fig. 4). The
activity decreased with a half-time of about 20 min; the unreduced
complex was much more stable under these conditions.
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Fig. 4.
Effects of dithiothreitol on
a-agglutinin activity. His6-Aga2p·Aga1p-(1-149)
complex was incubated at 25 °C without ( ) or with (
) DTT (5 mM, pH 6.9) for the indicated time before assay.
-helix, 31%
-sheet, and 15%
-turn (Fig. 3)
(32, 33). Thus, a construct expressing only 31 residues of Aga1p,
including five post-signal residues and the last three Cys of the first
Cys-rich region, was sufficient to maintain secondary structure of the
complex of 108 amino acid residues (75 from His6-Aga2p and
31 from Aga1p).
Mutations that increased activity of secreted Aga2p
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Fig. 5.
Effect of Aga2p Cys Ser mutations on a-agglutinin activity. Culture supernatants
from cells expressing the labeled form of Aga2p with (solid
bar) or without (open bar) Aga1p-(1-149) co-expressed
were assayed in quantitative agglutination assays. Values shown are
grand mean and standard error from duplicate assays in each of two
independent experiments.
-Agglutinin
Binding--
We used mutagenesis to identify regions of Aga2p
important for activity. The mutants were tested using several assays.
First, assays of culture supernatants from cells overexpressing Aga2p were used to determine gross activity. Second, assays of supernatants from cells coexpressing Aga2p mutants and Aga1p-(1-149) were used to
determine whether the mutated forms of Aga2p were stabilized by Aga1p.
Finally, we tested the ability of mutant forms of aga2 genes
expressed on CEN or high copy plasmids from the
AGA2 promoter for the ability to complement the
aga2::LEU2 mutant using cellular agglutination assays.
18 liters. For the
2.2-µm sphere the volume is 45 × 10
18
liters. On average there are 1-5 × 104 molecules of
agglutinin within this spherical shell (19, 21, 35); therefore the
concentration is as follows: ~104 molecules/(4.5 × 10
19
2.1 × 10
19 liters) = ~104 molecules/2 × 10
19 liters = 5 × 1022 molecules/per liter = 0.08 M.
-agglutinin (Ref. 20 and data not shown), indicating that this
region comprises part or all of the
-agglutinin binding site.
Therefore this region of Aga2p was explored by site-specific mutagenesis to identify residues important for activity (Table II). In the first series of mutants,
pairs of residues were mutated to alanine. The most C-terminal double
mutations, VF[86-87]AA and QY[84-85]AA showed undetectable Aga2p
activity when assayed alone or when coexpressed with Aga1p-(1-149).
The agglutination defects became less severe for more N-terminal
mutations, with GS[78-79]AA showing wild type activity. When these
mutants were re-assayed in the more sensitive cellular agglutination
assays for complementation of
aga2::LEU2, most showed some activity. The exception was VF[86-87]AA, which did not show detectable
activity. Immunoblotting of the supernatants showed that all mutant
forms were secreted at levels of at least 25% of wild type level.
Mutant analysis of the C-terminal region of Aga2p
-agglutinin binding.
Internal alanine scan of Aga2p
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-agglutinin has been extensively
characterized and shares features with mammalian adhesion proteins,
including immunoglobulin fold domains. In contrast, structure and
function of the complementary adhesion protein a-agglutinin
remains poorly understood. It is composed of two subunits with
well-defined functions: one for
-agglutinin binding and one for cell
surface anchorage (1). Although C. albicans homologs of
-agglutinin are known (13), no homologs of the
a-agglutinin binding subunit Aga2p have been reported. The
results of this paper define molecular roles for individual residues
and regions in the binding subunit and show a previously unrecognized
role for the anchorage subunit in maintenance of the active
conformation of the binding subunit. These findings are summarized
in Fig. 7.
-helical,
-sheet, and
-turn content (53% of the residues) was
greater than the fraction of residues contributed by Aga1p to the
complex (30%) for the His6-Aga2p·Aga1p-(1-29,111-136)
complex. This result was consistent with the high fraction of
structured residues in the larger
His6-Aga2p·Aga1p-(1-149) complex as well, with the
difference in secondary structure content implying that the extra
residues in Aga1p contributed additional
-helix and
-turn
regions. These results indicate that Aga1p stabilizes the structure of
the binding determinants in Aga2p.
-agglutinin, a highly glycosylated small peptide such as Aga2p may
not have enough hydrophobic character to form a stable structure on its
own. The ability of the Aga1p Cys-rich domain to stabilize Aga2p in an
active structure suggests the interaction between the two proteins
constrains Aga2p to a functional conformation. Because mutations of
either Aga2p Cys residue involved in disulfide linkage eliminated this
stabilization, both disulfide bonds appear to be critical for this constraint.
-agglutinin interaction (residues 48-50) and residues 56-61, which
were implicated in the Aga1p interaction. These latter residues show
strong propensity to adopt a
-strand-turn structure with the Cys
residue in the turn. Thus this sequence motif in Aga2p has properties
consistent with a bridging or "adapter" function between two other
proteins,
-agglutinin and Aga1p.
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Fig. 6.
Alignment of residues 45 to 72 of Aga2p with
similar sequences. Above the Aga2p sequence the phenotype is
summarized: , no effect of tested mutations;
, affects interaction
with
-agglutinin; 1, may affect interaction with Aga1p;
there is no mark above residues that were not mutated. The alignment
shows identities to Aga2p as boldface capital letters and
similarities as unbolded capital letters. Shown on the
right are the percent identity and similarity to Aga2p. The
GenBankTM accession numbers are: g416593, g81796, g128411, g340360.
The last row shows GOR strong predictions for secondary
structure:
, beta strand; T, loop or turn
(43).
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Fig. 7.
Features of a-agglutinin. The N and C
termini of the subunits are marked. A few of the glycosylation sites
are shown in glycosylation-rich regions (1, 18). The
a-agglutinin regions contributing to subunit interactions
are marked "a." In Aga2p the region similar to soy Nod
proteins is boxed, and the regions implicated as binding
determinants for interaction with -agglutinin are marked "
."
Note that disulfide bonds have not been mapped, but our results are
consistent with bonding of the two Aga2p Cys residues to the third,
fourth, and/or fifth Cys residues of Aga1p, with the last two being
most likely (Fig. 1).
-agglutinin, with the C terminus playing the most critical role.
However, the highly active conformation of the protein is dependent on
several factors. Previous results point to the importance of
O-glycosylation in maintaining activity of Aga2p (19, 42). Our studies indicate that in addition to its role in cell surface anchorage, the interaction of Aga1p with Aga2p through disulfide linkage is critical for maintaining Aga2p in its highly active form for
the interaction with
-agglutinin.
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ACKNOWLEDGEMENTS |
---|
We thank Yu Sheng Wu, Leonel Edwards, and Eren Hock for technical assistance. We thank Dr. Widmar Tanner for the kind gift of anti-Aga2p, Dr. Dixie Goss for use of the CD Spectrometer, and Dr. Y. K. Yip for peptide sequencing and syntheses.
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FOOTNOTES |
---|
* Supported by the Grants 1R01-GM47176 and 2SO6-GM60654 from NIGMS, National Institute of Health (NIH) and by Grant RR-03037 from the Research Centers in Minority Institutions Program of NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
Present address: Anesthesia Research Laboratories, Brigham and
Women's Hospital, Harvard Medical School, Boston, MA 02115.
** Prestent address: Genencor International, P. O. Box 218, 2300 AE, Leiden, Netherlands.
To whom correspondence should be addressed: Dept. of
Biological Sciences, Hunter College, 695 Park Ave., New York, NY 10021. Tel.: 212-772-5235; Fax: 212-772-5227; E-mail:
lipke@genectr.hunter.cuny.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010421200
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ABBREVIATIONS |
---|
The abbreviations used are: GPI, glycosyl phosphatidylinositol; bp, base pair(s); CD, circular dichroism; DTT, dithiothreitol; PCR, polymerase chain reaction; PAS, periodic acid Schiff reagent.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Lipke, P. N., and Kurjan, J. (1992) Microbiol. Rev. 56, 180-194[Abstract] |
2. | Lipke, P. N., Wojciechowicz, D., and Kurjan, J. (1989) Mol. Cell. Biol. 9, 3155-3165[Medline] [Order article via Infotrieve] |
3. | Hauser, K., and Tanner, W. (1989) FEBS Lett. 255, 290-294[CrossRef][Medline] [Order article via Infotrieve] |
4. | Lu, C. F., Kurjan, J., and Lipke, P. N. (1994) Mol. Cell. Biol. 14, 4825-4833[Abstract] |
5. | Lu, C. F., Montijn, R. C., Brown, J. L., Klis, F., Kurjan, J., Bussey, H., and Lipke, P. N. (1995) J. Cell Biol. 128, 333-340[Abstract] |
6. |
Hamada, K.,
Terashima, H.,
Arisawa, M.,
Yabuki, N.,
and Kitada, K.
(1999)
J. Bacteriol.
181,
3886-3889 |
7. | Wojciechowicz, D., Lu, C. F., Kurjan, J., and Lipke, P. N. (1993) Mol. Cell. Biol. 13, 2554-2563[Abstract] |
8. | Williams, A. F., and Barclay, A. N. (1988) Annu. Rev. Immunol. 6, 381-405[CrossRef][Medline] [Order article via Infotrieve] |
9. | Vaughn, D. E., and Bjorkman, P. J. (1996) Neuron 16, 261-273[Medline] [Order article via Infotrieve] |
10. |
Lipke, P. N.,
Chen, M. H.,
de Nobel, H.,
Kurjan, J.,
and Kahn, P. C.
(1995)
Protein Sci.
4,
2168-2178 |
11. | Grigorescu, A., Chen, M.-H., Zhao, H., Kahn, P. C., and Lipke, P. N. (2000) IUBMB Life 50, 105-113[CrossRef][Medline] [Order article via Infotrieve] |
12. | de Nobel, H., Lipke, P. N., and Kurjan, J. (1996) Mol. Biol. Cell 7, 143-153[Abstract] |
13. | Hoyer, L. L., Payne, T. L., Bell, M., Myers, A. M., and Scherer, S. (1998) Curr. Genet. 33, 451-459[CrossRef][Medline] [Order article via Infotrieve] |
14. | Sijmons, P. C., Nederbragt, A. J., Klis, F. M., and Van den Ende, H. (1987) Arch. Microbiol. 148, 208-212[Medline] [Order article via Infotrieve] |
15. | Roy, A., Lu, C. F., Marykwas, D. L., Lipke, P. N., and Kurjan, J. (1991) Mol. Cell. Biol. 11, 4196-4206[Medline] [Order article via Infotrieve] |
16. | de Nobel, H., Pike, J., Lipke, P. N., and Kurjan, J. (1995) Mol. Gen. Genet. 247, 409-415[Medline] [Order article via Infotrieve] |
17. |
Roberts, C. J.,
Nelson, B.,
Marton, M. J.,
Stoughton, R.,
Meyer, M. R.,
Bennett, H. A.,
He, Y. D.,
Dai, H.,
Walker, W. L.,
Hughes, T. R.,
Tyers, M.,
Boone, C.,
and Friend, S. H.
(2000)
Science
287,
873-880 |
18. |
Guo, B.,
Styles, C. A.,
Feng, Q.,
and Fink, G. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12158-12163 |
19. | Cappellaro, C., Hauser, K., Mrsa, V., Watzele, M., Watzele, G., Gruber, C., and Tanner, W. (1991) EMBO J. 10, 4081-4088[Abstract] |
20. | Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744[Abstract] |
21. | Lipke, P. N., Terrance, K., and Wu, Y. S. (1987) J. Bacteriol. 169, 483-488[Medline] [Order article via Infotrieve] |
22. | Kang, Y. S., Kane, J., Kurjan, J., Stadel, J. M., and Tipper, D. J. (1990) Mol. Cell. Biol. 10, 2582-2590[Medline] [Order article via Infotrieve] |
23. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
24. | Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H., and Hieter, P. (1992) Gene 110, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
25. | Muhlrad, D., Hunter, R., and Parker, R. (1992) Yeast 8, 79-82[Medline] [Order article via Infotrieve] |
26. | Kallal, L., and Kurjan, J. (1997) Mol. Cell. Biol. 17, 2897-2907[Abstract] |
27. | Gander, J. E. (1984) Methods Enzymol. 104, 447-451[Medline] [Order article via Infotrieve] |
28. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
29. | Gill, S. C., and Hippel, P. H. v. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve] |
30. | Watzele, M., Klis, F., and Tanner, W. (1988) EMBO J. 7, 1483-1488[Abstract] |
31. | Terrance, K., and Lipke, P. N. (1981) J. Bacteriol. 148, 889-896[Medline] [Order article via Infotrieve] |
32. | Woody, R. W. (1995) Methods Enzymol. 246, 34-71[Medline] [Order article via Infotrieve] |
33. | Woody, R. W., and Dunker, K. (1996) in Circular Dichroism and the Conformational Analysis of Macromolecules (Fasman, G. D., ed) , pp. 109-157, Plenum Press, New York |
34. | Creighton, T. E. (1993) Proteins , W. H. Freeman, New York |
35. | Wojciechowicz, D., and Lipke, P. N. (1989) Biochem. Biophys. Res. Commun. 161, 46-51[Medline] [Order article via Infotrieve] |
36. |
Verna, J.,
Lodder, A.,
Lee, K.,
Vagts, A.,
and Ballester, R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13804-13809 |
37. | Bhargava, A. K., Woitach, J. T., Davidson, E. A., and Bhavanandan, V. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6798-6802[Abstract] |
38. | Caro, L. H., Tettelin, H., Vossen, J. H., Ram, A. F., van den Ende, H., and Klis, F. M. (1997) Yeast 13, 1477-1489[CrossRef][Medline] [Order article via Infotrieve] |
39. | Sandal, N. N., Bojsen, K., and Marcker, K. A. (1987) Nucleic Acids Res. 15, 1507-1519[Abstract] |
40. | Jacobs, F. A., Zhang, M., Fortin, M. G., and Verma, D. P. (1987) Nucleic Acids Res. 15, 1271-1280[Abstract] |
41. | Sadler, J. E., Shelton-Inloes, B. B., Sorace, J. M., Harlan, J. M., Titani, K., and Davie, E. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6394-6398[Abstract] |
42. | Yen, P. H., and Ballou, C. E. (1974) Biochemistry 13, 2428-2437[Medline] [Order article via Infotrieve] |
43. | Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120[Medline] [Order article via Infotrieve] |
44. |
Zhao, H.,
Shen, Z.-M.,
Kahn, P. C.,
and Lipke, P. N.
(2001)
J. Bacteriol.
183,
2874-2880 |