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J Biol Chem, Vol. 273, Issue 45, 29489-29496, November 6, 1998
From the Departments of The dermatophyte fungus Trichophyton
exhibits unique immunologic properties by its ability to cause both
immediate and delayed type hypersensitivity. An 83-kDa
Trichophyton tonsurans allergen (Tri t 4) was previously
shown to elicit distinct T lymphocyte cytokine profiles in
vitro. The homologous protein, Tri r 4, was cloned from a
Trichophyton rubrum cDNA library, and the recombinant protein was expressed in Pichia pastoris. This 726-amino
acid protein contained an arrangement of catalytic triad residues
characteristic of the prolyl oligopeptidase family of serine
proteinases (Ser-Asp-His). In addition, a novel
Trichophyton allergen, encoding 412 amino acids, was
identified by its human IgE antibody-binding activity. Sequence
similarity searches showed that this allergen, designated Tri r 2, contained all of the conserved residues characteristic of the class D
subtilase subfamily (41-58% overall sequence identity). Forty-two
percent of subjects with immediate hypersensitivity skin test reactions
to a Trichophyton extract exhibited IgE antibody binding to
a recombinant glutathione S-transferase fusion protein containing the carboxyl-terminal 289 amino acids of Tri r 2. Furthermore, this antigen was capable of inducing delayed type
hypersensitivity skin test reactions. Our results define two distinct
antigens derived from the dermatophyte Trichophyton that
serve as targets for diverse immune responses in humans.
Dermatophyte fungi of the genus Trichophyton colonize
keratinized tissues in humans including nails, hair shafts, and the stratum corneum of the skin. Trichophyton tonsurans,
Trichophyton mentagrophytes, and Trichophyton
rubrum are common causes worldwide of tinea capitis, athlete's
foot, and onychomycosis (infection of the nail beds) (1). An estimated
30-70% of adults are asymptomatic carriers of these pathogens, and
the incidence of symptomatic disease increases with age (2). The immune
response to antigens derived from Trichophyton is unique in
that both immediate hypersensitivity (IH)1 and delayed type
hypersensitivity (DTH) skin test reactions are induced. Studies suggest
that the nature of the underlying immune response to
Trichophyton antigens is related to the severity of dermatophytosis; IH skin tests are associated with chronic recurrent infections characterized by low-grade inflammatory lesions and the
presence of IgE antibodies (Ab) (4-7). In contrast, DTH reactions are
associated with highly inflamed lesions that resolve spontaneously and
a resistance to re-infection (4, 8-13). The implication of these
findings is that cell-mediated immune responses to
Trichophyton are more effective at eradicating infection and
may confer protection. Chronic dermatophytosis has been associated with
allergic disease in the respiratory tract in individuals with immediate
hypersensitivity (14-17). Furthermore, exposure to
Trichophyton proteins may result in bronchial sensitization
and symptomatic asthma that can be controlled with systemic antifungal
therapy (7, 18, 19).
Experimental mouse models support a role for distinct T lymphocyte
helper subsets in fungal infections (20). Furthermore, there is
mounting evidence that a dichotomy in the immune response to a variety
of pathogens, including Trichophyton, exists in humans and
that these responses are regulated by distinct CD4+ T cell
subsets (20-28). Characterization of antigens derived from Trichophyton provides a model system for studying both IgE
antibody- and cell-mediated immune responses in humans; elucidation of
the amino acid sequences of these antigens is relevant to structural analyses of intrinsic antigenic properties governing diverse immune responses and to the identification of antigenic determinants associated with immediate and delayed type hypersensitivity.
Furthermore, elucidation of the biologic function of these unique
antigens may define a role in fungal pathogenicity.
We previously demonstrated that an 83-kDa T. tonsurans
antigen (Tri t 4) elicited IH and DTH skin test reactions in different individuals (27). IH skin tests were associated with IgG, IgE, and IgG4
Ab specific for Tri t 4, whereas DTH reactions were associated with
only low levels of IgG Ab. In addition, short-term T cell lines
specific for Tri t 4 had distinct cytokine profiles characteristic of a
Th1 and Th2/ThO phenotype that correlated with skin test reactivity
in vivo (28). Here we describe the molecular cloning and
expression of the Tri t 4 homologue, Tri r 4, produced by T. rubrum and define its limited sequence identity to the prolyl oligopeptidase family of serine proteinases. In addition, we
characterize a novel T. rubrum allergen (Tri r 2) that has a
high degree of sequence identity to the subtilase enzyme family; this
protein exhibits human IgG and IgE Ab binding properties and the
ability to induce DTH skin test reactions.
cDNA Cloning--
Cultures of T. rubrum, T. mentagrophytes, and T. tonsurans were established in 25 ml of Sabouraud dextrose broth, and culture filtrates were screened
using an assay for Protein IV as described previously (27). The
T. rubrum cultures produced the highest concentration of
Protein IV and were selected for construction of a cDNA library.
Natural Protein IV was previously isolated from T. tonsurans, and this protein is now correctly termed Tri t 4 in
keeping with allergen nomenclature. Thus, the homologous protein
produced by T. rubrum is Tri r 4. Six grams of T. rubrum cells harvested on day 7 were washed in phosphate-buffered
saline and ground with a mortar and pestle pre-cooled at Expression of Recombinant Tri r 4 in Pichia
pastoris--
Recombinant Tri r 4 (r-Tri r 4) was expressed in
P. pastoris as a hexahistidine-tagged protein using the
pPICZ Expression of Tri r 2 in E. coli--
Plasmid DNA containing
T. rubrum clone 9A (~1500 bp) was used as a template to
generate an 867-bp DNA fragment encoding the carboxyl-terminal 289 amino acids corresponding to the putative mature form of Tri r 2. Primers for polymerase chain reaction incorporated EcoRI and
XhoI restriction sites to allow subcloning into the
pGEX-4T-3 expression vector and were as follows:
5'-CCGGAATTCGGGCACTAACCTCACC-3' (sense), containing an EcoRI
restriction site; and 5'-CCGCTCGAGTTTGCCGCTGCCG-3' (antisense),
containing an XhoI restriction site. The 867-bp polymerase chain reaction-amplified DNA fragment was ligated into
EcoRI-XhoI-digested pGEX-4T-3. Expression of the
29-kDa putative mature form of Tri r 2 as a fusion protein with
glutathione S-transferase (GST) was induced in E. coli strain BL21 with 0.2 mM
isopropyl-1-thio- Assays of Enzymatic Activity--
Recombinant GST-Tri r 2 and
Tri r 4 proteins were tested for proteolytic activity using a variety
of substrates. Proteinase K (Sigma) was used as a positive control for
all assays. Negative controls included reactions containing no putative
enzyme. Glutathione S-transferase served as an additional
negative control for assays of GST-Tri r 2 enzymatic activity. All
assays were performed in duplicate. Substrates tested included
azoalbumin, azocasein, azocollagen, and keratin according to methods
previously described (32, 33). Briefly, 10 µg of the sample to be
tested was incubated with 0.1 mg of azoalbumin, 2% (w/v) azocasein, 5 mg of azocollagen, or 5 mg of keratin azure in reaction volumes of 1, 0.2, 0.5, and 0.3 ml, respectively. Reactions were terminated after
incubation for 24 h at 37 °C (azocasein, azocollagen, and
keratin) or after 30 min at 20 °C (azoalbumin). Reaction products
were measured spectrophotometrically. One unit of activity was defined
as an increase in absorbance of 0.01. Recombinant Tri r 2 was also
tested using the anilide substrate
succinyl-(Ala)3-p-nitroanilide (32). Briefly,
reactions contained a final concentration of 10 µg/ml Tri r 2 and
substrate concentrations of 0.1-2.0 mmol/liter; assays of 1-ml volume
were incubated at 20 °C for 1 h, and the absorbance of
liberated nitroaniline was measured spectrophotometrically.
Immunoassays for IgG and IgE Antibodies to Tri r 2 Fusion
Protein--
IgE and IgG Ab to GST-Tri r 2 were measured using an
antigen binding radioimmunoassay according to methods previously
described (27). Serum samples diluted 1:2 and 1:10 (IgE Ab assay) or
1:12.5 and 1:50 (IgG Ab assay) were incubated with
125I-labeled GST-Tri r 2 (~120,000 cpm added) for 4 h at room temperature. IgE myeloma serum (patient P. S.) diluted 1:300
was used as carrier in the IgE binding assay. Immune complexes were
precipitated overnight at 4 °C with 50 µl of sheep anti-human IgE
or 50 µl of sheep anti-human IgG (Binding Site, Inc., San Diego, CA),
and precipitates were counted in a Human Subjects and Skin Testing--
Sera were obtained from 73 subjects previously skin-tested with 0.03 ml of Hollister-Stier
Trichophyton mixture containing T. tonsurans,
T. rubrum, and T. mentagrophytes species (1:200, w/v). Intradermal skin testing was done with 0.03 ml of purified GST-Tri r 2 at 1 and 10 µg/ml diluted in 0.05% human serum albumin in phenol/saline solution. Prick testing was carried out prior to
intradermal testing using a 10-fold higher concentration of protein.
Test sites were examined at 20 min after injection and at 24 and
48 h. Positive delayed reactions were defined as erythema of
Molecular Cloning of Tri r 4--
Screening a T. rubrum
cDNA library with a human serum pool obtained from five individuals
with high titer IgE Ab to n-Tri t 4 failed to identify positive
plaques. However, screening the library with polyclonal serum obtained
from a mouse immunized with n-Tri t 4 resulted in identification of
three clones containing insert sizes of ~1100 bp (clone
tr2), 1600 bp (clone tr1), and 2300 bp (clone
tr3). Nucleotide sequence analysis confirmed that all clones
encoded the same protein. Clone tr3 contained an open reading frame of 2178 nucleotides encoding a 726-amino acid protein (Fig. 1). An estimated molecular mass of
78,193 Da (pK = 2.2) without the signal peptide
sequence was consistent with a non-glycosylated form of n-Tri t 4. Four
potential sites of N-linked glycosylation were identified,
and the presence of a signal sequence with a predicted site of cleavage
between Ala19 and Phe20 was determined. Amino
acid sequences of the amino terminus and six enzymatically generated
internal peptides of n-Tri t 4 (comprising 108 residues) aligned with
the deduced amino acid sequence of clone tr3. This confirmed
that T. rubrum clone tr3 encoded a protein with
high amino acid sequence homology to n-Tri t 4, and we have designated
this recombinant protein Tri r 4.
Amino acid sequence similarity searches identified homology between
r-Tri r 4 and the prolyl oligopeptidase (S9) family of serine
proteinases. These enzymes contain the distinctive Ser-Asp-His arrangement of catalytic triad residues in the carboxyl-terminal portion of the molecule. A short region spanning ~250 residues was
identified within r-Tri r 4 that contained sequence similarity to
prolyl oligopeptidases derived from other eukaryotic sources (20-25%
identity, 231-282-amino acid overlap). A
Gly-X-Ser-X-Gly motif comprising the nucleophile
serine at position 539 was present within this region. The highest
sequence similarity was between r-Tri r 4 and human acylaminoacyl
peptidase (25.6% identity and 57% similarity in a 242-amino acid
overlap) (Fig. 2). Additional homologues
included Saccharomyces dipeptidyl aminopeptidases B and C
and dipeptidyl peptidase IV (DPP4) and dipeptidyl peptidase IV-like
proteins (DPP6) derived from several mammalian species.
Recombinant Tri r 4 was expressed in P. pastoris using the
pPICZ Identification of a Novel T. rubrum Allergen as a Member of the
Subtilase Family of Serine Proteinases--
Since screening the
T. rubrum cDNA library with an initial human serum pool
(Pool 1) failed to identify positive plaques, a second pool was
established (Pool 2) in order to screen for additional putative
T. rubrum allergens. Sera were obtained from four
individuals with high IgE antibody titers and IH skin test reactions to
a Trichophyton extract. All subjects had chronic dermatophytosis, whereas three had asthma and positive bronchial provocation to Trichophyton. Five positive clones were
identified with insert sizes of ~900 bp (clone tr6D), 1000 bp (clone tr6C), 1200 bp (clone tr6B), 1300 bp
(clone tr6A), and 1500 bp (clone tr9A).
Nucleotide sequence analysis confirmed that all five clones encoded the
same protein and that this protein was unrelated to Tri r 4. A
representative clone (tr6B) was screened with sera obtained
from individuals with different skin test reactivity to
Trichophyton. Eight of 10 subjects with IH skin test
reactions displayed IgE antibody binding to this clone, whereas sera
from DTH and negative skin test subjects yielded no positive responders (Fig. 4). Clone 9A contained an open
reading frame encoding 412 amino acids with an estimated molecular mass
of 42,632 Da and a pK value of 1.9 (Fig.
5). The first 20 NH2-terminal
residues contained the conserved features of a signal peptide, and four potential sites of N-linked glycosylation were
identified.
Sequence similarity searches showed a significant homology between the
deduced amino acid sequence of clone 9A and serine proteinases of the
subtilase family (S8) derived from other fungal species (Fig.
6). Conserved amino acid motifs were
identified flanking aspartic acid, histidine, and serine residues,
which form the catalytic triad characteristic of this enzyme family. Over 70 subtilases are currently known, belonging to four subfamilies. The deduced amino acid sequence of clone 9A contained all of the conserved residues characteristic of the class D subfamily, which consists of enzymes found only in yeast, fungi, and Gram-negative bacteria (Fig. 6). The highest degree of amino acid sequence identity was between Tri r 2 and proteinase T produced by the thermophilic fungus Tritirachium album (58.2% identity in a 304-amino
acid overlap). Other enzymes with striking homologies included
proteinases derived from the pathogenic fungus Aspergillus
fumigatus (41.3% identity), the insect-colonizing fungus
Metarhizium anisopliae (42.3%), and the yeast
Schizosaccharomyces pombe (41%) (Fig. 6). Sequence
alignments identified the presence of a putative pro-region in Tri r 2 (residues 21-123) with a predicted cleavage site between asparagine
and glycine residues (positions 123 and 124, respectively) generating a
putative mature product (positions 124-412) with an estimated
molecular mass of 29,171 Da (pK = 1.9) (Fig. 5).
Demonstration of the Immune Response to Tri r 2--
The putative
mature form of Tri r 2 was produced in E. coli using the
pGEX-4T-3 vector. The resulting GST fusion protein purified from
bacterial lysates by glutathione affinity chromatography migrated as a
single 57-kDa band on SDS-PAGE, consistent with the presence of a GST
moiety fused to the 29-kDa putative mature form of Tri r 2. Specific
IgE Ab were measured in 73 sera: the prevalence of IgE Ab was
significantly higher among subjects with IH skin test reactions (43%)
compared with those with DTH or negative skin test reactions (12%)
(p < 0.01) (Fig.
7A). It has been established that GST exhibits IgE Ab binding properties (34); however, absorption of sera with GST did not reduce the prevalence of IgE Ab binding to Tri
r 2. The prevalence of IgG Ab was relatively high in all skin test
groups. However, mean levels of IgG Ab were significantly higher in
subjects with immediate reactions compared with those with delayed or
negative skin test reactions (p < 0.01) (Fig. 7B). Recombinant 125I-GST-Tri r 2 showed strong
reactivity with IgG Ab (up to 46,000 cpm bound) and IgE Ab (up to
10,500 cpm bound), demonstrating that the putative mature form of Tri r
2 retained B cell epitopes. Intradermal skin testing was used to
evaluate the reactivity of recombinant Tri r 2 in vivo. Five
of nine individuals with delayed reactions to the
Trichophyton mixture showed a positive delayed type
hypersensitivity reaction maximal at 24 h; four of these subjects are shown in Table I.
Activity against Protein Substrates--
Given the amino acid
sequence homology to known proteinases, the enzymatic activity of
recombinant Tri r 2 and Tri r 4 was tested using a variety of general
proteolytic substrates including albumin, casein, collagen, and
keratin. No proteolytic activity was observed for GST-Tri r 2 for any
of the substrates tested when compared with a GST control. Furthermore,
this protein exhibited no activity against the anilide substrate
succinyl-(Ala)3-p-nitroanilide. However, r-Tri r
4 exhibited weak activity against keratin (447 units/mg) compared with
proteinase K (7490 units/mg) and showed no activity against the other
substrates tested.
We have reported the amino acid sequences and expression of two
distinct proteins derived from the dermatophyte fungus
Trichophyton. The 83-kDa mannose-rich natural glycoprotein
(n-Tri t 4) was previously shown to elicit IH and DTH skin test
reactions in different individuals (27). To our knowledge, this is the
first reported sequence of a fungal antigen associated with distinct
skin test reactions. The homologous recombinant protein produced by
T. rubrum (r-Tri r 4) is a 726-amino acid protein with
limited amino acid sequence homology to the prolyl oligopeptidase (S9)
family of serine proteinases. Despite the relatively low amino acid
sequence similarity (~20%), several characteristics provide
convincing evidence that r-Tri r 4 belongs to this family of proteins:
the distinctive arrangement of catalytic triad residues and their
localization in the carboxyl-terminal region of the molecule, the
presence of conserved amino acids flanking putative catalytic residues,
its high molecular mass consistent with other members of this family,
and a large variable NH2-terminal portion. In contrast to
subtilases, S9 peptidases do not exist as proenzymes and are
synthesized in an active form (35, 36). These enzymes, which may be
either cytosolic or membrane-bound, exhibit restricted specificities
that may limit degradation of other cell proteins (37). Some family
members have been reported to be involved in a variety of nonenzymatic physiologic processes; for example, the membrane glycoprotein dipeptidyl peptidase IV (CD26) plays a role in cell-matrix adhesion and
transmembrane signaling (38-40). Recombinant Tri r 4 exhibited a low
level of proteolytic activity against keratin. Dermatophyte fungi are
adapted to infect keratinized tissues by virtue of their ability to
utilize keratin as a nutrient source. Whether the natural Tri r 4 and
Tri t 4 proteins are functionally keratinolytic in vivo
remains to be established. If this proves to be the case, the enzymatic
activity of these proteins could facilitate colonization and may
contribute to pathogenicity.
P. pastoris was selected for expression of r-Tri r 4 since a
eukaryotic system is more appropriate for expression of fungal antigens, and high yields of foreign proteins, including some allergens, were previously reported (41, 42). Yields of recombinant protein were very high (~200 mg/liter of culture). SDS-PAGE analysis suggested that r-Tri r 4 was glycosylated to a degree comparable to the
natural antigen (~5% carbohydrate by weight). Despite this, preliminary studies suggest that r-Tri r 4 exhibits partial loss of B
cell epitopes as determined by decreased binding to IgG antibodies compared with natural antigen (data not shown). We hypothesize that
partial loss of conformational epitopes on r-Tri r 4 may result from
incorrect folding owing to its large size, a factor that may also
contribute to its low enzymatic activity. Preliminary results have also
shown that recombinant Tri r 4 failed to elicit DTH skin test responses
in three individuals with DTH responses to natural Tri t 4. These
findings are surprising since only linear antigenic determinants are
required for initiation of T cell responses in vitro. Since
recombinant Tri r 4 is derived from a T. rubrum cDNA
library, and natural Tri t 4 was purified from a T. tonsurans extract, this raises the possibility that antigenic
properties differ between homologous proteins derived from the two
fungal species. Alternatively, it could be hypothesized that
conformational epitopes or post-translational modifications of linear
antigenic determinants required for DTH responses fail to occur in the
recombinant protein. Similar findings have been demonstrated for a
ribosomal protein derived from Brucella melitensis (43).
This antigen typically induces DTH responses in
Brucella-sensitized guinea pigs. However, recombinant
antigen expressed in E. coli produced no skin response. It
was concluded that post-translational acylation of protein is required
for DTH activity. Recombinant Tri r 4 will serve as a valuable tool for
distinguishing the relevance of conformational epitopes or
post-translational modifications in the induction of DTH responses in humans.
The second antigen defined is an allergen with high amino acid sequence
similarity to serine proteinases of the class D subtilase subfamily.
Eight of 10 subjects with IH skin test reactions to Trichophyton displayed IgE antibody binding to this
allergen, five of whom had bronchial reactivity to
Trichophyton. True subtilisins derived from bacteria are
among the best characterized of the subtilase enzyme family. Subtilisin
Carlsberg (Alcalase), a class A subtilase produced by Bacillus
licheniformis, is one of several subtilases used in detergent
formulations. Soon after the initiation of large-scale production of
enzyme-containing detergents, allergic respiratory reactions to the
enzyme components were noted among factory workers (44, 45). Thus, Tri
r 2 is a member of the same enzyme family as an antigen previously
related to asthma.
Bacterial expression of the putative mature form of Tri r 2 in the
absence of a fusion partner resulted in rapid degradation during
purification. One possible explanation is that the predicted site of
cleavage of the pro-region is incorrect and that the presence of
additional NH2-terminal flanking residues is required for
stabilization of the carboxyl-terminal domain containing active-site
residues characteristic of the subtilase family. Alternatively, the
presence of the entire pro-region may be required to serve as a
template for correct folding of this domain, as has been demonstrated
for other subtilase enzymes (46); however, attempts to express Tri r 2 with the putative pro-region were unsuccessful. Production of the
putative mature form of Tri r 2 as a GST fusion protein facilitated
stabilization of this domain. Members of the class D subtilase
subfamily have been shown to exhibit cuticle-degrading and elastase
activities. The class D subtilase ALP (alkaline
proteinase), produced by the pathogenic fungus A. fumigatus, exhibits elastase activity and has been proposed to
contribute to fungal persistence in allergic individuals (47). However,
no enzymatic activity of recombinant Tri r 2 was demonstrated. It is
possible that Tri r 2 is not an enzyme; however, given the high degree
of homology to subtilase enzymes, especially in the putative active
site, it appears more likely that the lack of activity reflects
features intrinsic to the recombinant protein. These may include
suboptimal processing of the recombinant protein owing to the absence
of the putative pro-region, lack of post-translational modification, or
the presence of the NH2-terminal GST moiety. Alternatively, inappropriate substrates may have been selected for study.
Tri r 2 expressed as a GST fusion protein was shown to exhibit IgE and
IgG Ab binding characteristics in addition to mediating DTH skin test
reactions. These findings suggest that expression of the
carboxyl-terminal 289 amino acids containing the putative mature form
of the protein was sufficient for immunologic function. This is
important since the absence of the pro-region or amino-terminal flanking residues could possibly influence immunologic properties. Tri
r 2 is a novel antigen in that it is the first recombinant protein
demonstrated to induce both IgE Ab- and cell-mediated responses in
humans. Furthermore, the high prevalence of IgE antibodies suggests
that this protein is an important allergen among patients with chronic
dermatophyte infection.
Dermatophytosis is an important clinical problem both because of its
chronicity and because current antifungal therapy is only curative in a
small proportion of cases. Identification of the antigenic determinants
associated with protective cell-mediated immune responses would make it
possible to design peptide or recombinant protein vaccines to modify
the natural course of the disease. The 29-kDa antigen Tri r 2 is a good
candidate for the application of overlapping peptide methodology to
define immunodominant epitopes in individuals with either IgE antibody
or DTH reactions. Thus, definition of these proteins will make it
possible to investigate T cell recognition associated with different
responses. Characterization of Trichophyton antigens
provides unique molecular tools not only for the development of
immunotherapeutic strategies related to management of chronic
dermatophytosis and the associated allergic disease, but also for the
analysis of immunologic mechanisms governing diverse immune responses
in humans.
*
This work was supported by National Institutes of Health
Grants AI 30840, AI 34607, and AI 01416.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF082515 (Tri r 2) and AF082514 (Tri r 4).
§
To whom correspondence and reprint requests should be addressed:
Asthma and Allergic Diseases Center, P. O. Box 225, University of
Virginia Health Sciences Center, Charlottesville, VA 22908. Tel.:
804-924-5917; Fax: 804-924-5779; E-mail: jaw4m{at}virginia.edu.
The abbreviations used are:
IH, immediate
hypersensitivity; DTH, delayed type hypersensitivity; Ab, antibody or
antibodies; n-Tri t 4, natural Tri t 4; r-Tri r 4, recombinant Tri r 4; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
Trichophyton Antigens Associated with IgE Antibodies
and Delayed Type Hypersensitivity
SEQUENCE HOMOLOGY TO TWO FAMILIES OF SERINE PROTEINASES*
§,,,
Internal Medicine and
¶ Microbiology, Asthma and Allergic Diseases Center, University of
Virginia, Charlottesville, Virginia 22908
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
70 °C.
Messenger RNA was isolated from 6 g of culture material using a
FastTrack kit (Invitrogen, Carlsbad, CA). A T. rubrum
cDNA library was prepared from 10 µg of mRNA in the UniZAP-XR
phagemid expression vector (Stratagene, La Jolla, CA). cDNA clones
were identified by screening the library with either a 1:5000 dilution
of serum obtained from a mouse immunized with natural Tri t 4 (n-Tri t
4) or a 1:2 dilution of an IgE serum pool from four individuals with
high titer IgE antibodies and IH skin test reactions to a
Trichophyton extract (29). Selected cDNA clones were
screened against individual sera from 10 subjects with IH skin test
reactions to Trichophyton and five individuals with DTH or
negative skin test reactions. DNA sequencing was carried out by
automated sequencing (ABI Prism 377, Applied Biosystems, Inc., Foster
City, CA). Sequences obtained were compared with the National
Biomedical Research Foundation, Swiss-Prot, and GenBankTM
Data Banks using FASTA. Sequence alignments were performed using the
GCG program. The presence of signal peptides was confirmed using the
prediction algorithm developed by Nielsen et al. (30).
A expression vector (Invitrogen). Plasmid DNA (50 ng) encoding
Tri r 4 was used as a template to generate a 2178-bp DNA fragment by
polymerase chain reaction. The following primers for polymerase chain
reaction were synthesized: 5'-CCGGAATTCTTTACCCCAGAGGACTTC-3' (sense),
containing an EcoRI restriction site; and
5'-GCTCTAGAGCGTCGAAGTAAGAGTGAGC-3' (antisense), containing an
XbaI restriction site. The 2178-bp polymerase chain
reaction-amplified DNA fragment was ligated into EcoRI-XbaI-digested pPICZA. Escherichia
coli strain TOP10F' was transformed, and plasmid DNA was purified
from Zeocin-resistant transformants selected on low salt LB medium
containing 25 µg/ml Zeocin. Yeast strain KM71 was transformed by
electroporation (Bio-Rad GenePulser; 1500 V, 25 microfarads, 200 S)
with 5 µg of DNA linearized by digestion with PmeI.
Transformants were selected on yeast extract peptone dextrose agar
containing 100 µg/ml Zeocin after incubation at 30 °C for 3 days.
A single colony was used to inoculate 10 ml of buffered glycerol
complex medium, and cultures were grown at 30 °C in a shaking
incubator (300 rpm) until the culture reached A600 nm = 2.5. The process was repeated using
10 ml of culture to inoculate 1 liter of medium. After reaching
A600 nm = 2.5, cells were harvested by centrifugation
(3000 × g for 5 min) and resuspended in 100 ml of
buffered methanol complex medium containing 0.5% methanol. Expression
of r-Tri r 4 was induced at 30 °C in the presence of methanol for 4 days. The recombinant protein was purified from culture supernatants
using immobilized nickel chelate (Probond resin, Invitrogen). Purity
was assessed by SDS-polyacrylamide gel electrophoresis (PAGE) with
silver staining, and protein yields were measured by the Bradford
assay. Proteins expressed in pPICZA contain an
NH2-terminal factor signal sequence that targets
expressed proteins to the secretory pathway and into the culture
medium. Proteins also contain carboxyl-terminal hexahistidine and Myc
epitope tags. NH2-terminal amino acid sequence analysis of
r-Tri r 4 by Edman degradation confirmed cleavage of the signal sequence and the presence of the first 22 NH2-terminal
residues corresponding to those of n-Tri t 4.
-D-galactopyranoside at 37 °C. The
recombinant protein (GST-Tri r 2) was purified from cell lysates using
glutathione-Sepharose (yield of ~2-5 mg/liter of culture). Amino
acid sequencing of the fusion protein by Edman degradation confirmed
the presence of the first 5 NH2-terminal residues of the
GST moiety. Purified GST-Tri r 2 was dialyzed against
phosphate-buffered saline, and purity was analyzed by silver-stained
SDS-PAGE for the purpose of skin testing. The recombinant protein was
also purified by electroelution from a 12% acrylamide gel for the
purpose of radiolabeling (31).
-counter. Quantitation of IgG Ab
was carried out using a control curve constructed with pooled sera from
patients K. M., J. C., and H. W., assigned to contain 2000 units/ml
IgG antibodies. IgE Ab measurements are expressed as counts bound per
min. Specificity of antibody binding to Tri r 2 was assessed by
comparing values obtained for sera pre-absorbed with GST (5 mg of
GST/ml of cyanogen bromide-activated Sepharose) and non-absorbed sera.
5-mm diameter at 24 h. Subjects were skin-tested with purified GST as a negative control. Skin testing of human subjects using GST-Tri
r 2 was approved by the University of Virginia Human Investigation Committee.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (63K):
[in a new window]
Fig. 1.
Nucleotide and deduced amino acid sequences
of recombinant Tri r 4. The NH2-terminal amino acid
residues (positions 1-19) contain the conserved features of a signal
peptide with a predicted cleavage site between Ala19 and
Phe20. Underlined regions represent amino acid
sequences previously obtained for the NH2 terminus and for
six enzymatically generated peptides of natural Tri t 4. Catalytic
triad residues ( 
) and four potential sites of N-linked
glycosylation (X) are indicated. The stop codon TAG is shown
(*).
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Fig. 2.
Comparison between conserved regions and
catalytic triad residues of recombinant Tri r 4 and serine proteinases
of the prolyl oligopeptidase family. Residues are numbered
according to Tri r 4. Asterisks denote catalytic residues,
and identical residues are depicted in boldface type.
Conservative substitutions of hydrophobic residues present in at least
four family members are indicated ( 
). ACPH, human
acylaminoacyl peptidase; DAP2, Saccharomyces
dipeptidyl aminopeptidase B; DPP6, human dipeptidyl
peptidase IV-like protein; DPP4, human dipeptidyl peptidase
IV; DAP1, Saccharomyces dipeptidyl aminopeptidase
C.
A vector system. SDS-PAGE analysis at 24 h after induction of protein expression revealed the presence of an 85-kDa band in all
recombinants selected consistent with a glycosylated form of r-Tri r 4 containing carboxyl-terminal Myc epitope and hexahistidine tags (Fig.
3A). The recombinant protein
was purified by affinity purification from culture supernatants
harvested on day 4, resulting in a yield of ~200 mg/liter of culture.
The pure protein migrated as a single 85-kDa band on SDS-PAGE (Fig.
3B).
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Fig. 3.
SDS-PAGE analysis of recombinant Tri r 4 expressed in P. pastoris. A,
analysis of culture supernatants 24 h after induction of protein
expression revealed an 85-kDa band in all putative recombinants tested
(Coomassie Blue staining), whereas no protein was visible in a control
recombinant containing vector (Vec.) alone (third
lane). Expression levels were comparable to an albumin
(Alb.) control (second lane). B,
recombinant Tri r 4 was purified from culture supernatants by affinity
purification using immobilized nickel chelate. The recombinant antigen
migrated as a single band on silver-stained SDS-polyacrylamide
gel.
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Fig. 4.
Human IgE antibody binding to T. rubrum
cDNA clone tr6B. Five clones encoding a
single protein were obtained after screening a T. rubrum
cDNA library with pooled IgE antibodies. Individual sera from 10 subjects with IH skin test reactions and from five subjects with DTH or
negative (Neg.) skin test reactions were used to screen each
clone by plaque immunoassay. Results are shown for clone
tr6B. Similar binding patterns were observed for all five
clones. Arrows denote subjects with IgE Ab binding, and
individuals with bronchial reactivity to Trichophyton are
indicated (*).
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Fig. 5.
Nucleotide and deduced amino acid sequences
of T. rubrum cDNA clone 9A encoding Tri r 2. The
NH2-terminal amino acid residues (positions 1-20) contain
the conserved features of a signal peptide.
Double-underlined regions represent conserved amino acid
motifs flanking catalytic triad residues ( ) characteristic of the
subtilase family of serine proteinases. The putative signal peptide
(PRE), the propeptide region (PRO), and the
mature form (MATURE) are shown. Four potential sites of
N-linked glycosylation are indicated (boldface
and underlined). The stop codon TAA (*) is shown, and a
polyadenylation signal sequence (ATAAA) in the 3'-noncoding region is
underlined.
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Fig. 6.
Sequence alignment of the Tri r 2 putative
mature form with catalytic domains of other fungal subtilases. Tri
r 2 showed high sequence identity to proteinase T produced by T. album (GenBankTM P20015), an alkaline proteinase
(ALP) derived from A. fumigatus
(GenBankTM P28296), proteinase Pr1 from M. anisopliae (GenBankTM P29138), and proteinase ISP6
from S. pombe (GenBankTM P40903). Alignments
obtained using the GCG program are shown. Conserved residues of the
class D subfamily of subtilase enzymes (boldface) and
catalytic residues ( ) are indicated. Asterisks denote
identical residues.
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Fig. 7.
IgE and IgG antibody binding to recombinant
Tri r 2. Sera from 73 individuals with different skin test
reactivities to the Trichophyton mixture were analyzed for
IgE and IgG Ab binding to 125I-labeled Tri r 2 fusion
protein. A, the prevalence of IgE Ab binding in IH subjects
and in those subjects with DTH or negative (NEG) skin test
reactions was 43 and 12%, respectively. Arrows denote
individuals with IgE Ab binding as determined by plaque immunoassay.
B, mean IgG Ab levels were significantly higher in subjects
with immediate reactions (p < 0.01). Bars
represent geometric means. Individuals with bronchial reactivity to
Trichophyton are indicated ( ).
Skin test responses and serum antibodies to a GST fusion protein
containing the putative mature form of Tri r 2
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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