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(Received for publication, August 2,
1995; and in revised form, October 27, 1995) From the
The 43,000-Da glycoprotein (gp43) of Paracoccidioides
brasiliensis is an immunodominant antigen for antibody-dependent
and immune cellular responses in patients with paracoccidioidomycosis.
In order to identify the peptide epitopes involved in the immunological
reactivities of the gp43 and to obtain highly specific recombinant
molecules for diagnosis of the infection, genomic and cDNA clones
representing the entire coding region of the antigen were sequenced.
The gp43 open reading frame was found in a 1,329-base pair fragment
with 2 exons interrupted by an intron of 78 nucleotides. The gene is
present in very few copies per genome, as indicated by Southern
blotting and chromosomal megarestriction analysis. A single transcript
of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes
a polypeptide of 416 amino acids (M
Paracoccidioides brasiliensis is a dimorphic fungus
that causes paracoccidioidomycosis, a deep-seated infection, prevalent
in rural workers in several Latin American countries. The yeast phase
is the infective form of the fungus which synthesizes antigenic
heteropolysaccharides, glycoproteins, and glycolipids that may have a
role in pathogenicity and interact with the immune system(1) . The main diagnostic antigen of paracoccidioidomycosis is an
exocellularly secreted glycoprotein of 43,000 Da (gp43) ( In the present work we report on the
characterization of the complete sequence of the gene encoding the gp43
antigen from P. brasiliensis. Determination of the sequence of
peptide fragments derived from the native molecule by enzymatic
proteolysis permitted PCR amplification of a genomic fragment which was
used as a probe to isolate the entire gene from a genomic library of
the fungus. A genomic fragment corresponding to the COOH-terminal
portion of the protein was cloned into pGEX plasmid, and the expression
product reacted in immunoblots with anti-gp43 polyclonal rabbit and
human patient antibodies.
Figure 1:
The gp43
gene cloning strategy. A, the gp43 sequenced peptides are
shown in their assumed positions as inferred from amino acid identities
with S. cerevisiae glucanase. Degenerate, inosine-containing
primers were designed based on the three internal peptides: OLCK53
(forward primer), OLCK59 and OLCK33 (reverse primers). The
oligonucleotide pairs OLCK53/OLCK33 and OLCK53/OLCK59 were used for PCR
amplification of P. brasiliensis genomic DNA and for RT-PCR.
The black box represents the intron. B, ethidium
bromide-stained agarose gel showing the PCR amplified products from the
genomic DNA template of P. brasiliensis using as primers the
OLCK53/33 and OLCK53/59 pairs: fragments of 987 and 570 bp (lanes 2 and 3); and the 492-bp RT-PCR amplified product (lane
4), obtained using as primers the pair OLCK53/59. Lane 1,
molecular weight markers (
Each reaction (25 µl) was carried out in 25 cycles. Optimized
conditions included denaturing at 94 °C (1 min), annealing at 50
°C (1 min), and extension at 72 °C (1.5 min). The PCR fragments
of interest were recovered from agarose gel by using the
Sephaglass(TM) kit (Pharmacia Biotech Inc.). When used as probes,
they were labeled with [
Intact chromosome sized DNA molecules from yeast and mycelial forms
(strain B339) were prepared by lysis and enzymatic proteolysis of cells
embedded in low melting agarose. The chromosomal DNA was digested with
20 units of NotI and SfiI restriction enzymes. The
resulting megarestriction fragments were separated using Saccharomyces cerevisiae pulsed-field gel electrophoresis
conditions. MegaBase I (chromosomal DNA from S. cerevisiae,
Life Technologies, Inc.) was used as the molecular mass standard. The
gels were transferred onto nylon membranes and the chromosomal blots
were hybridized with the above mentioned probe.
Figure 3:
The nucleotide sequence of the gp43 gene
and the deduced amino acid sequence of the 43,000-Da glycoprotein of P. brasiliensis. The TATAAA element, the CAAG motifs, the T
+ C-rich block, the stop codon, and the putative tripartite
consensus sequence for the polyadenylation signal are in bold
type. The amino acid sequence derived from the open reading frame
starts at position nt +1 and ends at position nt +1329. The
intron is written in lowercase letters. The leader
peptide(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) is underlined. The NH
To isolate the entire gp43 gene (GP43G), the 987-bp PCR fragment was
used as a probe in the screening of P. brasiliensis genomic
DNA library. An EcoRI genomic fragment of 3.8 kb carrying the
gp43 gene was isolated and characterized (Fig. 2). This was
subcloned into pUC18 giving rise to pUCGPb16A. Further analysis of
pUCGPb16A with restriction enzymes and Southern blotting probed with
the 987-bp fragment localized the gene in a BglII-XbaI fragment of approximately 2.8 kb.
Figure 2:
Restriction map and sequenced region of
the genomic insert of clone pUCGPb16A. A, showing the relative
positions of the endonucleases restriction sites; E, EcoRI; Bg, BglII; B, BamHI; S, SmaI; H, HindIII; X, XbaI; B, the 1981-kb
sequenced fragment of the genomic insert containing the entire gp43
gene, showing the 5`-upstream region (nts -326 to -1), the
gp43 coding region (nts +1 to +1329), the two exons (1 and 2)
separated by the 78-nt intron (nts +464 to +541) and the
3`-untranslated region (+1330 to
+1655).
The
complete nucleotide sequence of the gp43 gene and its 5`- and
3`-flanking regions is shown in Fig. 3. The deduced amino acid
sequence consisted of 416 residues with an estimated molecular mass of
45,947 Da. The choice of the initiation codon (ATG) was based on the
following evidence: (a) the sequence upstream of the
initiation site contains stop codons in all three reading frames.
Furthermore, as it will be discussed below, this region carries a
putative promoter and other translational initiator consensus
sequences. (b) The predicted protein size without the signal
peptide (42, 281 Da) is comparable to that of the mature gp43 protein. The coding region of the gp43 gene is interrupted by an intron of 78
nt (Fig. 3), with 5` and 3` extremities at positions +464
and +541, respectively. The 5` and 3` extremities of the intron
presented the GT/AG consensus(25) . It is interesting to note
the presence in the intron of a 11-nt motif (TAGAATATCTC) which was
also found perfectly repeated in the 3`-untranslated region of the gp43
gene (positions 1601 to 1612). Several repetitions of the minimotif
(TA) were also detected in the intron. The coding region of the gp43
gene showed A + T content of 48.2%, whereas its 5`- and
3`-flanking regions gave higher A + T contents of 57.5 and 63.8%,
respectively. Analysis of the 5` 326-bp flanking sequence of the
gp43 gene revealed structural feature characteristics of the promoter
regions of eukaryotic genes. A TATA element (TATAAATA) is at position
-80 and a T + C-rich pyrimidine block is found immediately
downstream (nt -71 to -40). The CAAG motif (26) is
found once, 14 nt downstream of the TC block (nt -25), and twice
upstream, at positions -195 and -259. The 3` region
immediately downstream from the gp43 open reading frame contains motifs
thought to be necessary for termination of transcription, processing,
and addition of poly(A) at the 3` terminus. Although a perfect match to
the described eukaryotic polyadenylation consensus
sequence(27) , 5`-AATAAA-3`, is not found, a similar
pentanucleotide (5`-AATAA-3`) is observed at position 1616, 276 nt
downstream from the termination codon. In addition, a tripartite
sequence (TAG . . . TATTT . . . TTT) was identified between nucleotides
1354 and 1388. This sequence presents a high degree of homology to a
tripartite consensus sequence (TAG . . . TATGT . . . TTT) that has been
postulated to be a signal for termination and/or polyadenylation in S. cerevisiae(28, 29) . The size of the gp43
transcript deduced from the nucleotide sequence analysis is in
agreement with the estimated size of transcript (approximately 1.5 kb)
detected in the Northern blot hybridization (see Fig. 7).
Figure 7:
Northern blot hybridization. P.
brasiliensis total RNA (lane 2) and poly(A)
The
gp43 protein has 41.8% nonpolar, 34.5% polar noncharged, 12.8%
positively charged, and 9.7% negatively charged residues. The codon
usage in the gp43 gene showed a relaxed bias toward preferential codons
for the amino acids Ala, Ser, Thr, Val, Ile, Arg, Leu, and Pro. Of the
61 possible codon triplets, all are used in the gp43 gene. From the
calculations described by Bennetzen and Hall (30) we obtained a
codon bias index of 0.476 for the gp43 gene, which for yeasts would
represent a gene of moderate level of expression.
There were two potential N-glycosylation sites at residues
2 and 195 (residues 2-4, NFS, and 195-197, NRT, Fig. 3). Since amino acid 2 is within the signal sequence the
mature gp43 should contain a single N-glycosylation signal.
The net charge of the gp43 gives a pI of 7.43. The polypeptide is
composed of alternating hydrophobic and hydrophilic regions, which is
consistent with the water soluble character of the gp43; there is no
hydrophobic sequence in the mature protein long enough to span the
membrane.
Figure 4:
Immunological reactivity of recombinant
proteins and anti-recombinant protein antiserum. The subfragments of
the internal (residues 110-272) and COOH-terminal (residues
288-411) domains of the gp43 were fused with GST. In A the reaction of an anti-GST rabbit antiserum with the recombinant
proteins (lanes 1 and 2; GST alone, lane 3)
confirms that the products are expressed as fusion proteins; a rabbit
polyclonal monospecific anti-gp43 antiserum (anti-gp43R) and sera
(pool) from patients (anti-gp43H) recognize the recombinant proteins in
immunoblots (lanes 5, 6 and 9, 10, respectively). Lanes 4, 8, and 12, plasmid-less bacterial extracts. Lanes 7 and 11, GST. In B antibodies
generated in rabbits against the recombinant antigen carrying the gp43
COOH-terminal sequence recognized the native gp43 in immunoblots of P. brasiliensis culture filtrate (lane 3) as shown by
Figure 5:
Alignment of the amino acid sequence of
the gp43 with known sequences. Alignment of the gp43 deduced amino acid
sequence with known sequences of cloned fungal exoglucanases: S.
cerevisiae, vegetative (EXG1), and spore specific (SPR1)
exoglucanases, and C. albicans exoglucanase (CAXOG). The
alignment was performed by the J. Hein method. Asterisks and dots indicate identical and similar residues,
respectively.
Figure 6:
Genomic organization of the gp43 gene. A, autoradiogram of a Southern blot of P. brasiliensis genomic DNA digested with restriction enzymes and probed with the
987-bp fragment: 1, EcoRI, a single fragment of 3.8
kb, which corresponds to the size of the insert of the recombinant
The Northern blot hybridization
of the total and poly(A) In this report we describe the cloning and characterization
of genomic and cDNA recombinant clones representing the entire coding
region of the exocellularly secreted glycoprotein of 43,000 Da,
previously described as the main diagnostic antigen of the dimorphic
pathogenic fungus P. brasiliensis(2) . So far, this
is the first gene of the fungus to be cloned, entirely sequenced, and
expressed in bacteria as recombinant fusion proteins. Several lines of
evidence indicate that the sequenced clones encode the gp43 antigen.
First, the deduced amino acid sequence encloses all the previously
characterized partial peptide sequences of the native protein.
Furthermore, it is remarkably consistent with previous analyses of
amino acid composition in different preparations of the native
gp43(7) . Second, different regions of the gp43 protein,
expressed as fusion proteins in E. coli, reacted strongly with
rabbit antiserum against the gp43 native protein and with sera from
patients with paracoccidioidomycosis. On the other hand, the antiserum
elicited against the gp43 recombinant fusion protein specifically
recognized the native gp43 by immunoblotting of the P. brasiliensis culture filtrate. Third, Northern hybridizaton using the cloned
DNA as a probe identified a transcript of 1.5 kb, which is large enough
to encode a protein of the size predicted for native gp43. The
deduced gp43 open reading frame encodes a polypeptide of 416 amino
acids (M Analysis of the sequences
upstream and downstream from the gp43 open reading frame showed several
motifs similar to the consensus elements important for the control of
transcription in eukaryotic organisms. The CAAG motif, closely
associated with the TC block, is correlated with a high level of
expression of certain yeast genes(26, 36) . The
presence of an intron was demonstrated in the GP43G determining two
exons, 1 and 2. The fungal homologous genes of exo-1,3-glucanases of S. cerevisiae and C. albicans have no introns. The
presence of the intron in the P. brasiliensis DNA may
implicate a transcriptional regulation. In fact, P. brasiliensis morphological transition is dependent on temperature, and
therefore adaptation to temperature shifts and environmental stress is
essential for the pathogen to survive in mammalian tissues. It is well
known that heat shock affects RNA metabolism, including RNA processing
as well as mRNA degradation. In dimorphic fungi, those changes require
responses at the gene level and DNA sequences containing introns could
play a vital role in adaptation to the new environment(37) . In spite of the significant identity shared at the amino acid level
with glucanases of the vegetative forms of S. cerevisiae and C. albicans, and with S. cerevisiae spore-specific
glucanase, we could not demonstrate any glucanase activity in the
native gp43 molecule using different substrates. It is also noteworthy
that no immunological cross-reactivity could be detected between gp43
and the glucanases of the other fungi. Although the homologies among
blocks of amino acid residues of the aligned glucanases and the gp43
might suggest functional domains, identification of the amino acid
sequences and conformation of the catalytic and binding domains of the
fungal exo-1,3-glucanases is not possible at present. In many highly
unrelated bacterial 1,4- The two GST-gp43
fusion proteins represent different parts of the whole antigen and were
both recognized by rabbit monospecific polyclonal antibodies and by
human sera from patients with paracoccidioidomycosis, indicating that
the recombinant proteins shared epitopes with native gp43 including
those recognized by antibodies from human patients. Therefore, at least
two different B cell epitopes must be present in the gp43 molecule. The
highest peak of hydrophilicity, computed using an average group length
of 6 amino acids, which might be considered as a third epitope,
corresponds to the sequence GRDAKR (residues 78-83). This
sequence is 1 amino acid shorter than the homologous sequences of C. albicans and S. cerevisiae vegetative glucanases (Fig. 5) which are in turn more hydrophobic. Such variations and
others detected by comparing the hydropathic profiles of the three
amino acid sequences justify the absence of immunological
cross-reactivity of the glucanases with antibodies elicited against the
gp43. Recombinant molecules containing peptide epitopes of the gp43
can, on the other hand, be helpful to increase the specificity of the
diagnostic antigen since all reported cross-reactivities depended on
the N-linked carbohydrate chain of the
glycoprotein(3) . Genomic Southern blot hybridization
indicated that the gp43 is encoded by a gene with very few copies,
similarly to the homologous fungal exo-1,3-glucanase
genes(24, 31, 32) . P. brasiliensis is a multinucleate organism both in the mycelial and yeast
phases(40) . The intensity of the hybridization signals
obtained with the chromosome SfiI megarestriction fragments
was very similar (Fig. 6B) indicating that they may
represent two alleles of the GP43G and therefore, suggesting that both
the yeast and mycelial phases of P. brasiliensis could be at
least diploid. These results are of interest as there is no information
about the fungus ploidy. We have previously described the gp43 from P. brasiliensis as the most specific antigen in the
paracoccidioidomycosis-P. brasiliensis system(2) . It
is presently being used in a variety of serological tests for
diagnostic purposes(41, 42) . The present study opens
the perspective of analyzing the individual peptides and epitopes that
play a role in the interaction of the gp43 with cells of the immune
system, antibodies, and elements of the extracellular matrix.
Recognition of individual epitopes that elicit a favorable
immunological response can contribute for the immunotherapy of this
systemic mycosis. The recombinant molecule and/or the selected epitopes
cloned and amplified will be extremely helpful to define both the
specificities and their functional role in the biology of this
pathogenic fungus. The primary sequence of the gp43 will also permit
evolutionary studies involving related molecules, including the
glucanases of bacteria and fungi.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
U26160[GenBank].
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4553-4560
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
45,947) with a
leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed
similarities of 56-58% with exo-1,3-
-D-glucanases
from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not
cross-react immunologically with the fungal glucanases. Internal and
COOH-terminal gene fragments of the gp43 were expressed as recombinant
fusion proteins, which reacted with antibodies elicited against the
native antigen.
)which reacts with 100% of sera from patients with this
mycosis in double immunodiffusion and immunoprecipitation
reactions(2, 3) . This molecule has been purified by
immunoaffinity chromatography with an anti-gp43 monoclonal antibody (4) . It contains immunodominant peptide epitopes that (a) react with human antibodies and are not affected by N-deglycosylation (5) and (b) elicit T-cell
dependent delayed hypersensitivity reactions(6) . It also
induced the proliferation of T-CD4 lymphocytes in mice primed with the
antigen and of human peripheral lymphoid cells from a sensitized
individual(7) . The role of the gp43 in the pathogenicity of P. brasiliensis was suggested based on some of its properties.
Thus, the gp43 is the main secreted component of the fungus that binds
to murine laminin. It has been shown that laminin-coated yeast forms of
this fungus show a marked increase in their ability to invade and
destroy the infected tissues(8) . As a high-mannose
glycoprotein, detectable in the serum of patients with acute and
chronic paracoccidioidomycosis(9) , the gp43 and other fungal
components binding to concanavalin A may act as metabolic inhibitors or
cause a negative regulation of natural killer lymphocyte
cytotoxicity(10) . Finally, the gp43 has been associated with a
proteolytic activity (4) not necessarily a property of the
glycoprotein itself but that of an aggregated protease of very high
specific activity(11) . Early attempts to clone and express the
gene encoding the gp43 (12) aimed at isolating a recombinant
molecule that could be used in the immunodiagnosis, be sequenced for
peptide epitope identification, and be tested as a virulence factor.
The isolated clone, however, was unstable and could not be used in
subsequent studies.
Purification of the Gp43
Gp43 was purified by
affinity chromatography in columns of Affi-Gel 10 (Bio-Rad) coupled
with rabbit anti-gp43 immunoglobulin G, followed by gel filtration in
Sephacryl S-200, from supernatant fluids of P. brasiliensis yeast cells (strain B339) grown in TOM medium, as described
previously(3, 4, 13) .Amino Acid Sequence Determination
Purified gp43
was dialyzed against Milli-Q grade deionized water and a 829-pmol
sample was used for enzymatic digestion and peptide sequencing at the
Harvard Microchemistry Facility, Harvard University, Cambridge, MA.
Accordingly, peptides obtained by digestion with endoproteinase Lys-C
were fractionated on Hewlett-Packard 1090 HPLC equipped with a 1040
diode array detector, using a Vydac 2.1 
150 mm C-18 column, as
described(14) . Three peaks were selected based on size and
purity, and analyzed by matrix-assisted laser desorption ionization
time of flight performed on a Finnigan Lasermat(TM) mass
spectrometer. Sequencing of the NH
-terminal and of the
internal peptides was carried out by automated Edman degradation on an
ABI 477A protein sequencer with 120A on-line phenylthiohydantoin amino
acid analyzer.Nucleic Acid Isolation, Construction, and Screening of
the Genomic DNA Libraries
Nucleic acids of P. brasiliensis strain B339 were prepared from powdered yeast cells, frozen in
liquid nitrogen, as described previously(12, 15) .
Genomic DNA fragments of 0.5-2.0 and 2.0-7.0 kb obtained by
digestion with EcoRI were inserted into the EcoRI
site of bacteriophage gt11 (12, 15) . The genomic
library in
gt11 was plated with Escherichia coli Y1090
and screened by plaque hybridization (16, 17) using
the gp43 DNA probe described below.
Generation of a Gp43 DNA Probe Using PCR
The yeast
genomic DNA (60 ng) was amplified by PCR using the basic protocol
provided by Perkin Elmer. The oligonucleotide primers used (1
pmol/µl) were designed on the basis of the three peptide sequences
derived from the native gp43. The sequences of the degenerate
oligonucleotide primers corresponding to selected regions of internal
peptides 1 (residues VRIPIGYW), 2 (DTYHNQ), and 3 (QPIWARK), and used
in PCR (Fig. 1) were: (a) OLCK53,
5`-GTIAGAATTCCIATKGGITAYTGG-3` (24-mer); (b) OLCK59,
5`-GGGAATTCYTGRTTRTGRTAIGTRTC-3` (26-mer); (c) OLCK33,
5`-TTAAAGCTTTTTTCTIGCCCARATIGGYTG-3` (30-mer); where I =
inosine; K = G/T; Y = T/C; R = A/G; artificial
sites for restriction enzymes were introduced in the oligonucleotides.
HindIII digested DNA and
174 HaeIII digested replicative form DNA, BRL). C, autoradiogram of the 570- and 492-bp PCR products from the
Southern blot (lanes 2 and 3 stained with ethidium
bromide) hybridized with the [
P]dATP-labeled
987-bp genomic probe. The difference in the size of the hybridized
bands (lanes 4 and 5) corresponds to splicing of the
78-nt intron. Lane 1, M
markers.
-P]dATP using a
random-primer labeling kit (Life Technologies, Inc./BRL). Cloning was
carried out using the Sureclone(TM) kit (Pharmacia) and the
fragments were ligated into dephosphorylated M13 mp10 replicative form
DNA at the SmaI site and sequenced as described(18) .
Southern and Northern Blot Hybridization
Standard
conditions for electrophoresis, Northern and Southern blotting were
used(19) . The PCR product (987 bp) was radiolabeled and used
as a probe. Hybridizations with the labeled probe were carried out at
42 °C, overnight, in 50% formamide, 5 SSC (1 SSC,
0.15 M NaCl, 0.015 M sodium citrate), 5
Denhardt's solution, 50 µg ml yeast tRNA,
100 µg ml
sonicated herring sperm DNA, 10 µg
ml
poly(A), 0.1% SDS, followed by washes in 2
SSC at 28 °C (two, for 30 min), 1 SSC at 56 °C
(one, for 30 min), and 0.1 SSC at 56 °C (one, for 30 min). Nucleotide Sequence Analysis
Nucleotide sequences
were determined by the dideoxynucleotide chain termination reaction
method (16) , using overlapping subclones prepared from either
M13 mp18 and M13 mp19 generated by digestion of the insert with
appropriate restriction endonucleases. Specific oligomers were
synthesized and used as primers to fill in the remaining gaps in the
sequence. Part of the sequence of the entire gene was performed using
as template the recombinant pUCGPb16A and the AutoRead Sequencing kit (Pharmacia). The data were processed by an
Automated Laser Fluorescent DNA sequencer apparatus (Pharmacia/LKB).
Specific fluorescent oligomers were used as primers. The sequences were
analyzed using the PC/GENE software
, A.
Bairoch/University of Geneva, (TM)IntelliGenetics Inc. and Genofit SA,
and the Megalign program of the Lasergene system (DNAstar Inc.).
Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
RT-PCR was used to characterize the intron. Total yeast
RNA (5 µg) was reversely transcribed using the reagents and
conditions provided in the 3` rapid amplification of cDNA ends kit
(Life Technologies, Inc./BRL). The cDNA formed in the reaction was
precipitated with ethanol and after centrifugation the pellet was
resuspended in 50 µl of TE. The cDNA (5 µl) was amplified in a
final volume of 100 µl, for the first PCR reaction using a gene
specific primer (OLCK53, 50 pmol) and the tail sequence of the AP
(Universal Adapter Primer, UAP, 5 pmol), in the following conditions:
94 °C for 5 min, 50 °C for 2 min, and 72 °C for 40 min,
followed by 25 cycles of amplification reaction (20) under the
standard conditions described above for the gp43 gene. The second
amplification step employed the first PCR product (1 µl) and both
the gene specific primers (OLCK53/OLCK59, 50 pmol each). The amplified
cDNA product was digested with EcoRI restriction endonuclease
and ligated into the EcoRI-cut dephosphorylated replicative
form DNA of the M13 mp18 and also in pUC18.Expression and Serum Reactivity of the Gp43 in E.
coli
A HindIII fragment of the 987-bp PCR amplified
product, corresponding to the COOH-terminal coding region for the gp43,
as well as the PCR amplified cDNA fragment were subcloned into pGEX
plasmid, in frame with the glutathione S-transferase (GST)
gene(21) . The fusion proteins were expressed in E. coli DH5, separated by SDS-polyacrylamide gel electrophoresis and
transferred onto nitrocellulose filters(22) . Recombinant gp43
proteins were tested for reactivity with the rabbit anti-gp43
antiserum, pooled sera from human patients, and anti-GST antiserum.
Immunocomplexes were revealed by reaction with goat anti-rabbit and
anti-human IgG-peroxidase conjugates.Preparation and Reactivity of Antiserum Against the
Recombinant Molecule
Antibodies against the recombinant fusion
protein carrying the COOH-terminal domain were elicited in a rabbit by
subcutaneous injections of the partially purified immunogen, using
previously described conditions(21, 23) . The
reactivity with the native antigen of the rabbit antiserum elicited
against the recombinant fusion protein was detected in blots with I-labeled protein A.
Determination of Glucanase Activity
Exoglucanase
activity was investigated in dialyzed culture supernatants using 50
mM acetate buffer, pH 5.5 (250 µl), and laminarin (Sigma), p-nitrophenyl--glucoside (Sigma) and -glucan from P. brasiliensis yeast phase as substrates (0.2% final
concentration) at 37 °C, for 2.5-4 h. Control exoglucanase
activity was obtained from culture supernatant fluids of S.
cerevisiae S288 and Candida albicans ATCCYO1-09
grown in a rotatory shaker at 30 °C for 24 h in YPD medium (1%
yeast extract, 2% casein peptone, and 2% glucose). The reactions with
laminarin and -glucan were terminated by boiling for 10 min and
the liberation of glucose measured by the glucose oxidase method
(Enzymatic Glicemy kit, Wiener Laboratory, Argentina). The reactions
with p-nitrophenyl--glucoside were interrupted by
addition of 2.25 ml of 0.37 M NaCO and the A
measured.
Gp43 Peptide Microsequencing
Peptides obtained
by cleavage of purified gp43 with endoproteinase Lys-C were purified on
high pressure liquid chromatography and subjected to Edman degradation.
The amino acid sequences of the amino terminus (AGSAIYGVNIG) and of
three distinct internal peptides of the gp43 ( Fig. 1and Fig. 3) showed 54-60% identity with sequences present in
the NH- and COOH-terminal domains of the
exo-1,3--D-glucanase of S.
cerevisiae(24) .
-terminal and the internal
peptide sequences obtained from the purified native antigen are
indicated in bold italic type and numbered:
NH-terminal sequence (0), internal peptides (1, 2, and 3). The N-glycosylation site (NRT) is shown underlined and bold.
Cloning and Sequencing of the Gp43 Gene
The
significant degree of homology of all gp43 peptides to the S.
cerevisiae exoglucanase suggested a certain arrangement of the
sequenced peptides in the protein (Fig. 1A). Based on
their assumed positions, degenerate, inosine-containing
oligonucleotides were designed from the peptides 1 (forward primer,
OLCK53), 2 and 3 (reverse primers, OLCK59, OLCK33) and were used for
PCR amplification of the genomic DNA from P. brasiliensis. The
PCR amplified products from the genomic DNA template of P.
brasiliensis using as primers the OLCK53/OLCK33 and the
OLCK53/OLCK59 pairs were of 987 and 570 bp, respectively (Fig. 1B, lanes 2 and 3). The amino acid
sequence deduced from the 987-bp fragment encompassed all three native
internal peptides obtained before, confirming that the PCR product
encoded the expected region of the gene. This was considered as
evidence for the authenticity of the isolated sequence. A stop codon
(TAG) was noticed at position nt 199 of the 987-bp fragment suggesting
the existence of an intron, as we have demonstrated in a later stage.
RNA (lane 3) were isolated from 5-day-old yeast cells
and probed with the radiolabeled 987-bp PCR fragment. Lane 1, Trypanosoma cruzi total RNA (specificity control and M
marker) and lane 4, a RNA ladder. A
single transcript of approximately 1.5 kb was
hybridized.
Protein Structure and Processing
Sequencing of the
NH-terminal segment of the native protein (see above)
indicated that the first residue of the mature protein is the alanine
residue at position 36 (Ala). This gives rise to a mature
peptide of 381 amino acids with a predicted size of 42,281 Da. Thus,
the predicted gp43 gene product has a leader peptide (residues
1-35). The initiator methionine is followed by a block of
hydrophobic amino acid residues typical of signal sequences (residues
1-23, Fig. 3). The deduced amino acid composition of the
gp43, without the leader peptide, is quite similar to that obtained by
conventional chemical methods using the native protein(7) .
Immunological Reactivity of gp43 Recombinant
Proteins
Subfragments of the gp43 gene encoding parts of the
internal and COOH-terminal domains of the protein were inserted in
frame with the GST gene of plasmid pGEX (21) and were expressed
as GST fusion proteins in E. coli. The recombinant fusion
proteins were recognized in immunoblots by a polyclonal rabbit
anti-gp43 antiserum, as well as by sera of patients with
paracoccidioidomycosis (Fig. 4A). The reaction with
rabbit antiserum raised against GST demonstrated that the products were
expressed as fusion proteins (Fig. 4A). Moreover,
antibodies generated in rabbits against the recombinant antigen
carrying the COOH-terminal domain of the gp43 recognized the native
antigen in immunoblots of P. brasiliensis culture filtrate (Fig. 4B).
I-protein A binding; lanes 1 and 2,
reactions with rabbit preimmune serum and with the monospecific rabbit
antiserum (anti-gp43R), respectively.
Comparison of the Amino Acid Sequence of gp43 with Known
Sequences
Previous data base search had shown that peptide
sequences (Fig. 3) shared homology with regions of the S.
cerevisiae exo-1,3--D-glucanase (EXG1)(24) .
Comparison of the gp43 deduced amino acid sequence with those of known
cloned fungal exoglucanases from S. cerevisiae (vegetative,
EXG1, and spore specific, SPR1) (24, 31) and C. albicans (CAXOG) (32) showed that they
shared, respectively, 49.8, 48.3, and 50.7% identity at the amino acid
level, and if conservative substitutions were included, 56.3, 56.0, and
57.9% similarity. Fig. 5shows the multiple alignment of the
amino acid sequences encoded by the GP43G and those of S.
cerevisiae and C. albicans exoglucanases. The entire gp43
sequence can be divided into parts of high and low homology, that could
suggest functional domains. There are several blocks of 2 to 9 amino
acids of 100% identity among the known glucanases separated by
divergent regions. Some bacterial glucanase and/or cellulase sequences
of the GenBank or EMBL data bases showed minor homology to the gp43
deduced amino acid sequence. The comparison of the secondary structures
of the four aligned proteins (Fig. 5) showed a similar pattern
of folding, especially at those regions indicated as extended in each
individual molecule.
Exoglucanase Activity
Culture supernatant fluids
(50 µl) of S. cerevisiae and C. albicans dialyzed
against 50 mM acetate buffer, pH 5.5, showed exoglucanase
activity when either laminarin or p-nitrophenyl--glucoside were used as substrates, whereas
purified gp43 at 840 µg/ml or P. brasiliensis culture
filtrates were negative. No hydrolysis of P. brasiliensis -glucan was detectable with purified gp43 in the same
conditions.Genomic Organization and Transcription of
GP43G
Southern blot of P. brasiliensis genomic DNA
digested with several restriction enzymes was probed with the 987-bp
fragment derived from the coding region of the GP43G. The Southern blot
autoradiogram showed a single hybridized band of 3.8 kb with the EcoRI-digested genomic DNA, which corresponds to the size of
the gt11 purified inserts. The hybridization profile of the other
fragments was quite simple suggesting that the GP43G should occur as a
single copy or have very few copies in the genome (Fig. 6A). The chromosomal blot hybridization of NotI and SfiI megarestriction fragments showed a
210-kb NotI fragment and two, 300- and 440-kb SfiI
fragments (Fig. 6B).
gt11 clone; 2, BamHI; 3, HindIII; 4, SmaI; 5, EcoRI
and BamHI, double digestion; 6, EcoRI and HindIII; 7, EcoRI and SmaI; 8, BamHI and HindIII; 9, BamHI and SmaI; 10, HindIII and SmaI; 11, EcoRI, BamHI, and HindIII, triple digestion; 12, EcoRI, BamHI, and SmaI. B, an ethidium bromide
stained pulsed field gel of chromosomal megarestriction fragments from P. brasiliensis mycelium (M) (lanes 2 and 4) and yeast phase (Y) (lanes 3 and 5). Digestions employed the rare cutting enzymes NotI (lanes 2 and 3) and SfiI (lanes 4 and 5). S. cerevisiae intact chromosomes were
used as molecular weight markers (lane 1). The corresponding
autoradiogram shows that the NotI digested M and Y chromosomes hybridized with a single 210-kb fragment (lanes 7 and 8).The M and Y SfiI
digests hybridized with two fragments of approximately 330 and 440 kb (lanes 9 and 10). Lane 6, S. cerevisiae DNA.
RNA using the same probe
showed, on the other hand, a single transcript, a well defined band of
approximately 1.5 kb which is large enough to encode a protein of
45,947 Da (Fig. 7).
45,947), with a leader peptide of 35
amino acids which was defined by peptide sequencing of the NH terminus of the mature protein. The leader peptide has a
hydrophobic sequence and a putative signal peptide cleavage site after
residue 23
(Ala-Ser
)(33, 34) . If the
signal peptidase cleavage observes these rules there could be a second
post-translational processing of residues 24-35, which are not
present in the secreted antigen. The specificity of the second
proteolysis step would differ from that of the Kex2-like proteinases
which process the homologous fungal exoglucanases at a cleavage site
consisting of basic amino acid residues,
KR(24, 32, 35) . This basic pair is absent in
the gp43 leader peptide (Fig. 3).
-endoglucanases and in two fungal
endoglucanases, however, the amino acid sequence NEP flanked by
hydrophobic amino acid sequences is a conserved structure. Another
similar sequence, LEP, was noted in bacterial and fungal glucanases.
The NEP sequence is essential for the catalytic activity as
demonstrated by site-directed mutagenesis of this conserved motif in
two highly unrelated bacterial endo--glucanases (38) . It
has been suggested that the E residue of the NEP sequence could be the
proton donor in the hydrolysis process. In fact, the NEP sequence is
similar to the MNEP sequence that exists in human lysosomal
-glucosidase and human and rabbit isomaltase and sucrase and
reacts with the sugar unit of the substrate in the pyranose
configuration(38, 39) . The LEP and NEP sequences are
conserved in C. albicans (residues 64-66 and
229-231, respectively) and S. cerevisiae EXG1 (residues
64-66 and 231-233) and SPR1 (residues 65-67 and
232-234)
exo-1,3-glucanases(24, 31, 32) . The gp43 has
the LEP sequence (residues 51-53), but the NEP is altered to NKP
(residues 207-209). Such difference in the amino acid sequence at
the catalytic site with the introduction of a basic amino acid
replacing an acidic one can itself account for the absence of glucanase
activity in the gp43 protein. With the present evidence, one can
suggest that the fungal glucosidases as well as the gp43 may have had a
common ancestral gene and that a divergent evolutionary processing of
these molecules has occurred. In the absence of a functional
glucosidase activity, the gp43 may have been conserved mainly as an
immunomodulating antigen and a virulence factor.
)
We thank the Department of Biophysics of the Federal
University of São Paulo (UNIFESP) for the
oligonucleotide syntheses, and Susana N. Diniz for the technical
support.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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