ARTICLE
Auteur(s) : Ana María ABRÉU-VÉLEZ1, Pablo
JAVIER PATIÑO2, Fernando MONTOYA2, Wendy B.
BOLLAG1
1 Institute for Molecular Medicine and Genetics,
Medical College of Georgia, CB 2803, 1120 15th Street,
Augusta, GA, 30912-2630, USA.
2 Group of Primary immunodeficience, University of
Antioquia, Medellin, Colombia, SA
Reprints: A. M. Abréu-Vélez Fax: (+ 1) 706 721-7915
E-mail: aavelezmail.mcg.edu
Article accepted on 7/5/2003
Abbreviations: Dsg1, Desmoglein 1; EEF, epidermal
envelope fractions; El Bagre EPF, endemic pemphigus foliaceus from
El Bagre, Colombia; FS, fogo selvagem; ICS, intercellular staining;
PF, pemphigus foliaceus; PV, pemphigus vulgaris; PVDF,
polyvinylidene difluoride; SDS-PAGE, sodium
dodecylsulfate-polyacrylamide gel electrophoresis; TBS,
Tris-buffered saline.
The search for autoantigens recognized by sera from patients with
different types and sub-variants of pemphigus has played an
important role in the attempt to understand these diseases [1]. The
importance of autoantibodies in pemphigus disease was reinforced by
the demonstration that injection of autoantibodies from sera of
patients with pemphigus vulgaris (PV), pemphigus foliaceus (PF),
and fogo selvagem (FS) into rabbit, monkey and neonatal mouse
models resulted in the development of intercellular
immunoreactivity and temporary lesions resembling those occurring
in pemphigus [2]. A significant contribution to our understanding
of these diseases was made by Stanley and colleagues, who were the
first to show by immunoblotting (IB) that one third of the FS and
PF sera bind desmoglein 1 (Dsg1) [3-5]. Important contributions
were made subsequently by Labib, Diaz and colleagues in their
investigations to elucidate antigenic PF and FS moieties [6-14].
These investigators obtained three pools of PF and FS antigens: the
first pool was obtained from a highly-insoluble trypsin-resistant
fraction bound to the epidermal cell-envelope fraction that was
only further released by repeated sonication and papain treatment
to produce a 50 kDa antigen that was immunoprecipitated by
15 FS sera [7]. Using a similar technique and antigen source
(the highly-insoluble trypsin-resistant fraction bound to the
epidermal cell-envelope-fraction), another set of antigenic
moieties of 80, 62 and 45 kDa were prepared by repeated
sonication and trypsinization [8]. Using 5 FS sera that
specifically immunoprecipitated the 80 kDa PF antigen, these
investigators identified this 80 kDa fragment as the
N-terminal part of the mature form of bovine Dsg1 [9]. However,
this same group of investigators suggested the existence of two
pools of PF and FS antigens in addition to the trypsin-resistant
fragments isolated from the cell-envelope fraction [8]. The
recognition of these additional pools of PF antigen as proposed by
Labib et al., was an important step in explaining variations
in the solubility of the PF antigen reported by various
investigators [8, 10-12]. One of these pools is intracellular,
soluble in non-ionic detergents and recognized by some PF and FS
sera. A second consists of a cell-surface-exposed PF and FS antigen
which is immunoprecipitated by all sera from patients with
clinically active superficial pemphigus and by half of those with
PV. This last pool contains a 45 kDa proteolytic tryptic
fragment (from the cell-surface-exposed and trypsin-sensitive pool
associated with desmosomal cores) which is the most common epitope
recognized by these sera [10] using immunoprecipitation (IP)
[10-14]. The nature of this 45 kDa antigen is not yet known
despite multiple attempts at identification, including those by
Labib and collegues [10-14] and by Calvanico and Swartz [14]. These
last authors reported the isolation of a 45 kDa proteolytic
tryptic fragment (from the cell-surface-exposed and
trypsin-sensitive pool associated with desmosomal cores (PF
antigen)) exhibiting the following N-terminal sequences: DLEKDFQNIH
and NYIPFAKTYDS. These peptides did not correspond to any protein
sequence identified to date in any data base [14]. Finally, after
nearly a decade, we report here the nature of the 45 kDa
tryptic fragment derived from the cell-surface-exposed and trypsin-
sensitive pool associated with desmosomal cores.
Materials and methods
Pemphigus and control sera
All the subjects of the study participated willingly and signed or
agreed to a consent form approved by the Institutional Review Board
(IRB) and Human Assurance Committee (HAC) in accordance with the
Scientific and Ethics committees of the institutions. We tested
50 sera from patients with PV, 50 sera from patients with
PF and 50 sera from patients suffering a new variant of EPF
that resembles Senear-Usher syndrome (pemphigus and lupus) but
occurs endemically in El Bagre, Colombia [16-18]. This disease
occurs in 4.7 % of middle aged and older men or
post-menopausal women from these rural areas and differs from
previously described forms of endemic pemphigus [16-18]. It shares
some heterogeneous immunoreactivity similar to paraneoplastic
pemphigus but with no known association with malignant tumors
[16-18]. Controls included 50 sera from El Bagre EPF patients’
relatives living in the same endemic area in El Bagre, and
50 normal controls from outside of the endemic area. The
diagnosis of PF or PV was based on the results of clinical and
histopathological results, including direct immunofluorescence
(DIF) analysis of skin biopsies and indirect immunofluorescence
(IIF) against monkey esophagus, as well as immunoblotting (IB) as
previously described [1, 3, 4, 6]. 80 % of the PF, El Bagre
EPF, and PV patients from which sera were obtained were receiving
systemic corticosteroids (between 10 and 40 mg/day)
according to their clinical condition, and/or topical
corticosteroids, sun screen and H1 and H2 anti-histamine therapy.
Adsorption of autoantibodies by bovine epidermal antigens
The bovine epidermal antigens used in the immunoprecipitation and
the immunoadsorption protocols were obtained from
keratome-separated epidermis from fresh cow snouts using procedures
reported in previous publications [10-11] and fractions were stored
at – 70 °C until use. Briefly, the epidermis was
digested with trypsin and the soluble extract partially purified by
chromatography on a Concanavalin A (Con A) column. Fractions were
then tested for their ability to preabsorb autoantibodies, as
previously reported [10-12, 18]. Briefly, different amounts of the
Con-A eluent (10, 25, 50 μl and the flow through) were
incubated at room temperature for 30 minutes with a fixed
volume (50 μl) of a 1:8 dilution of positive PF and El
Bagre EPF sera [15-17]. Each aliquot of serum was then evaluated
for the presence of immunoreactive molecules by IIF in comparison
with similar aliquots of unabsorbed serum and by preadsorbing the
serum with the same volume of a control antigen (bovine serum
albumin) in TBS/Ca++ . An additional two slides
were also incubated with a normal human serum as a negative control
and with Con-A beads to eliminate possible inhibitory effects of
the beads themselves on the reaction of pemphigus sera [18]. Con-A
fraction A was found to preabsorb immunoreactive autoantibodies.
This fraction was also radiolabeled using the chloramine T method
as described previously [19]. Aliquots of this radiolabeled
fraction A were used for IP, as a tracer for electrophoresis, and
for testing the efficiency of coupling of immunoglobulins from PF
serum to a protein-A sepharose CL-immunoaffinity chromatography
column (see below). Immunoprecipitation was performed using the PF
and El Bagre EPF sera as previously reported [10, 11]. Under these
experimental conditions, the antigen of interest was specifically
precipitated along with the IgG but without nonspecific
radiolabeled bands. The immunoprecipitate was re-dissolved in
0.1 % SDS in TBS buffer without Ca++ and
analyzed by SDS-PAGE. The proteins immunoprecipitated by each
individual serum were separated by 10 % SDS-PAGE under
reducing conditions as described previously [10-13]. The gels were
stained with 0.2 % Coomassie blue and dried before exposure to
X-ray film. Radiolabeled bands were analyzed by autoradiography.
The apparent molecular weights of the radiolabeled and the
Coomassie blue-stained proteins were determined by comparison with
broad molecular weight standards (Bio Rad).
Coupling of protein-A sepharose and the immunoglobulins from a
PF serum
The serum from a PF patient was incubated with protein-A sepharose
CL-4B gel, which was resuspended in 0.2 M triethanolamine, pH
8.2, as described elsewhere [9, 21]. Cross-linking was initiated by
the addition of dimethyl pirimedilate (6.6 mg/ml) (Aldrich,
Milwaukee,USA). After washing with 50 mM sodium borate buffer,
the remaining binding sites of the gel were blocked by addition of
0.1 M ethanolamine, pH 8.2. Finally, the
immunoglobulin-coupled protein-A Sepharose was washed with 1 M
NaCl in TBS/Ca++ . Leakage of immunoglobulin from
the column was determined by testing aliquots of eluent before and
after cross-linking using 12 % SDS-PAGE. This pre -and
post-linking determination of the heavy and light immunoglobulin
chains in the eluent was visualized by staining with Coomassie
blue.
Purification of the antigenic peptide by affinity
chromatography
A portion of non-radiolabeled Con-A fraction A was incubated with
the immunoglobulin-coupled protein-A sepharose and after extensive
washing, the bound fraction was eluted in one ml fractions with
0.2 M glycine, pH 2.8, containing 5 mM EDTA [9, 21]. Each
eluted fraction was adjusted to pH 7.4 with 0.1M NaOH and the
OD280 monitored. A small aliquot of radiolabeled Con-A
fraction A was added to each aliquot in order to provide a
co-migrating tracer. After mixing with an equal volume of 2-times
concentrated sample buffer [20], half of each sample was run
separately on two 10 % SDS-PAGE gels. One gel was stained with
Coomassie brilliant blue, and the other gel was transferred to a
polyvinylidene difluoride (PVDF) membrane and silver stained. The
regions of the gel demonstrating a protein band that co-migrated
with components of the radiolabeled fraction were collected, and
the procedure was repeated several times to obtain sufficient
quantities for amino acid sequencing. The final product containing
the protein was concentrated 3,000 times in an Amicon PM-30,
dialyzed first against 0.1 % TBS/Ca++ , and
subsequently against H2O, and lyophilized. 125I-Labeled
bovine epidermal antigen extract (approximately 1,500 cpm) was
added to samples as a tracer prior to analysis by one dimensional
10 % SDS-PAGE. Each sample was run in triplicate; two were
used for Coomassie brilliant blue staining and silver staining,
respectively, to verify the molecular weight of the peptide of
interest, and the other was blotted onto PVDF membrane (Immobilon
P, Millipore) for amido black staining and sequencing.
Autoradiography was performed to verify comigration of the
125I- labeled 45 kDa PF antigen and the stained
bands.
Amino acid sequence analysis
The electroblotted 45 kDa PF antigen, stained with amido-black
and shown to colocalize with the 125I-labeled
45 kDa PF antigen, was excised and subjected to sequence
analysis after acid hydrolysis using a Porton/Beckman gas phase
sequencer LF3000 (Palo Alto, CA, USA). PVDF strips with the protein
band were incubated in 5.7 N HCl-buffer containing 0.02 %
β-mercaptoethanol [22]. Tubes were evacuated and sealed under
N2, and hydrolysis was allowed to proceed for
20 hours at 110 °C. The samples were then dried,
re-dissolved in sodium citrate buffer, pH 2.4, and run on the amino
acid analyzer. Cysteine and tryptophan could not be detected by
this procedure because previous carboxymethylation was not
performed [21].
Structural analysis of the 45 kDa peptide
Analysis of Dsg1 was performed based on theoretical and
mathematical models to determine hydrophobicity (Kyte-Doolittle),
surface probability (Emini), chain flexibility (Karplus Schulz),
secondary structure (Garnier-Osguthorpe-Robson) and antigenicity
index (Jameson-Wolf). In addition, the PeptideStructure program was
used to predict secondary structure such as alpha helices, beta
sheets, coils and turns, as well as antigenicity, flexibility,
hydrophobicity, and surface probability. PlotStructure displays the
predictions graphically. All analyses were performed using the
Sequence Analysis Software Package of the Genetics Computer Group
(GCG), Madison, Wisconsin, U.S.A. Sequences were compared to the
Swiss Protein data bank and Genbank.
Results
Extraction and partial purification of an El Bagre EPF
antigen
In order to confirm the presence in the Con A column eluent of a
soluble peptide that maintains conformation-dependent epitopes
recognized by El Bagre EPF sera, four fractions (A, B, C and D), as
well as the flow-through, were concentrated to 3 mg/ml
(approximately 1.358 OD reading) and were tested for their
ability to immunoadsorb autoantibodies from the PF and El Bagre EPF
sera. Thus, 5 PF and 5 El Bagre EPF sera were
immunoadsorbed with these fractions before incubation with normal
human skin and visualization with fluorescein-conjugated secondary
antibodies recognizing total IgG as well as IgG1, 2, 3 and
4 subclasses. With the PF sera a complete block of the
intercellular staining (ICS) of foreskin keratinocytes was observed
with fraction A using all volumes (10, 25 and 50 μl) and
secondary antibodies to total IgG and all subclasses (Fig. 1). Similar results
were reported by Labib et al. [12]. However, when using the
5 El Bagre EPF sera, the ICS staining was completely inhibited
by preadsorption with the Con-A fraction A when visualized using
secondary antibodies to total IgG and IgG1, 2, and 4 for all
5 sera; however, with anti-IgG3 intracytoplasmic staining was
still observed in all cases, in contrast to the results with the PF
sera. Note that IgG3 does not bind to Protein A; therefore, this
subclass of IgG will not be coupled to the affinity column (Fig. 1).
Immunoprecipitation of antigen(s) in the extract by El Bagre
EPF sera
An IP was performed using patient sera to demonstrate a peptide
with conformation-dependent epitopes in this Con A fraction.
SDS-PAGE of the immunoprecipitated proteins revealed a 45 kDa
band, which was recognized by 47/50 sera from patients with El
Bagre EPF and also by 48/50 sera from PF patients and by
24/50 sera from patients with PV. No control sera from outside
the endemic area of El Bagre EPF recognized the 45 kDa band;
however 13/50 controls from within the endemic El Bagre area,
9/13 of which were genetically related to El Bagre EPF
patients, immunoprecipitated the 45 kDa band (Fig. 2). Other radioactive
bands of approximately 21, 36, 62, 80, 117, 120 kDa and
300 kDa were also immunoprecipitated, mainly by the sera from
people affected by El Bagre EPF.
Autoantibodies in the serum of a PF patient were cross-linked to
sepharose as described for immunoaffinity purification of the
antigen. Leakage of the immunoglobulin from the sepharose-coupled
column was determined by testing aliquots of eluent before and
after cross-linking using 12 % SDS-PAGE. This pre-and
post-linking determination of the heavy and light immunoglobulin
chains in the eluent was visualized by staining with Coomasie blue
(Fig. 3). In
order to determine if the clused product of the immuno-affinity
column maintained conformational epitopes capable of blocking the
ICS of PF and El Bagre EPF sera in normal human skin and/or of
being immunoprecipitated by these sera, experiments were performed
as described for the Con-A eluent. Neither block of the ICS nor
specific IP was observed, likely indicating loss of conformational
antigenicity, possibly as a result of the use of acid for elution
of the antigen from the immunoaffinity column.
From 8 cow snouts to 9 pg of the Dsg1 ectodomain
From 8 cow snouts, representing 49 grams of epidermal
tissue with some dermal remnants, we obtained 185 ml of
typsinized soluble extract. Six Con-A columns were run and
13 ml of eluent (fraction A) with a protein concentration of
0.328 mg/ml were obtained. Next, the Con-A fraction A was run
through six immuno-affinity columns, and the product was
concentrated to a value of 1.82 mg protein/ml. This product
was separated by SDS-PAGE and the 45 kDa band excised for
sequencing. Finally, 9 pg of the protein target were sequenced
(Fig.
3).
Amino acid sequence analysis of the 45 kDa protein
The eluent from the immuno-affinity column was transferred onto a
PVDF membrane and stained with amido black. The 45 kDa band
was excised and subjected to N-terminal protein sequence analysis
and the sequence “EXIKFAAAXREGED” obtained. A computer search
showed a 100 % homology of the determined amino acids with
both human and bovine Dsg1 (Fig. 4). The amino acid
corresponding to position 2 is tryptophan and position
9 in bovine Dsg1 is cysteine. These amino acids are sensitive
to oxidation and are easily lost during immunoblotting. Based on
previous sequencing experience, it was thought that these positions
in the 45 kDa protein were also tryptophan or cysteine. Our
sequence also showed a 64 % identity with the N-terminal
domain of mature human Dsg3. Submission of the amino acid
percentages for this 45 kDa protein (see Table I) to AACompident at the Expasy-Prosite web
site (URL http://www.expasy.org/tools) resulted in retrieval of
Dsg1 as a possible match, providing further support that this
protein represents a fragment of Dsg1.
Table I. Amino Acid
Composition of the 45-kDa PF Antigen in Comparison with Desmoglein
1 (Amino acids 50-473)
|
|
Mole
Percentage
Observed |
Mole
Percentage Expected
(for Bovine Dsg 1, amino
acids 50-473*) |
|
Ala
|
7.6 |
5.9 |
|
Arg
|
3.9 |
5.2 |
|
Asx
|
10.7 |
15.1 |
|
Glx
|
14.2 |
11.1 |
|
Gly
|
18.0 |
5.9 |
|
His
|
1.2 |
0.5 |
|
Ile
|
6.0 |
9.2 |
|
Leu
|
8.0 |
7.3 |
|
Lys
|
2.6 |
4.2 |
|
Met
|
0.3 |
3.1 |
|
Phe
|
3.8 |
4.0 |
|
Pro
|
5.3 |
3.8 |
|
Ser
|
8.2 |
7.1 |
|
Thr
|
4.1 |
5.4 |
|
Tyr
|
0.7 |
3.1 |
|
Val
|
5.4 |
7.1 |
|
Cys
|
n.d.** |
1.2 |
|
Trp
|
n.d.** |
0.9 |
* Amino acids 50-473 yield a protein fragment with a
predicted molecular weight of ~ 47.6 kDa, the actual
value estimated for the “45 kDa antigen” by SDS-PAGE using
molecular weight standards.
** n.d. = not detected.
Structural characteristics and comparison of the peptide
isolated with the theoretical expected from the mature form of
Dsg1
An apparent molecular weight of the PF antigen was determined by a
typical calibration curve using standard proteins separated by
SDS-PAGE. The full length of the bovine Dsg1 ectodomain consists of
498 amino acids with a predicted molecular weight very close
to 55 kDa (Swiss-Prot, GCG analysis). However, additional
tryptic cleavage sites abound, the utilization of which are
predicted to yield proteins fragments of 45-48 kDa. Table I illustrates the amino acid
composition of the 45-kDa PF antigen in comparison with Dsg 1
(amino acids 50-473). Using an antigenicity-determining program we
also detected five putative antigenic sites, one with higher
probability than the others, located in the four-barrel structure
of the ectodomain of Dsg1.
Discussion
In addition to autoantibodies to Dsg1 and Dsg3, autoantibodies to
other autoantigens have been reported in the sera of patients
suffering pemphigus and pemphigoid diseases. The discovery of “new
pemphigus antigens” may be related to the improvement of molecular
techniques, solubilization methods, and enzymatic cleavage
conditions. Some of the identified autoantigens include keratin
59 [22, 23], a ubiquitin carrier protein [24], desmocollins
[25], envoplakin [26, 27], periplakin [26, 27], and acetylcholine
receptors [28] as well as a double band of about 190 and
185 kDa localized to desmosomal plaques and the basement
membrane zone; however, the nature of this antigen remains unknown
[29, 30].
Calvanico and colleges reported that the antigen(s)
immunoprecipitated by sera from FS patients separates as a
260 kDa protein following 5 % SDS-PAGE of unboiled
samples [8]. However, this 260 kDa complex becomes
undetectable upon boiling or by lowering the pH to 2.8 with
glycine HCl [8]. Under these conditions, three lower weight bands
become prominent; two intense bands of 80 and 62 kDa and
a third one weakly visualized at 45 kDa [8]. The 80 and
the 62 kDa bands chromatographed as a solitary peak after IgG
on Bio Gel A-1.5 m chromatography [8]. Olague-Marchan et
al. [9] isolated the 80 kDa band, and found it to
represent the N-terminal domain of Dsg1. Since the 45 kDa
protein described here has also been identified as an N-terminal
antigen recognized by autoantibodies from patients with PF, FS,
Senear-Usher syndrome, and El Bagre EPF, this procedure represents
a simple and reproducible method for preparation of a soluble EPF
antigen(s). On the other hand, one of the unique features of El
Bagre EPF was the presence of IgG3 immunoreactivity against
intracellular autoantigens, the nature of which remains unknown. In
addition, El Bagre EPF patients demonstrate reactivity towards
desmoplakin 1, periplakin and envoplakin in addition to Dsg1
[31].
Other studies have also shown the importance of the ectodomain of
Dsg1. In one study, this fragment was placed into
baculovirus-secreted domain-swapped Dsg1 molecules, expressing
different parts of this domain, to demonstrate the epitopes
recognized by pemphigus sera [32]. The swapped molecules were
portions of the N-extracellular domain of Dsg1 (amino acids 1-496)
and the binding of the autoantibodies to these engineered molecules
was assessed by competitive ELISAs [33]. The domain-swapped
molecules containing the N-terminal 161 residues of Dsg1 were
found to exhibit 50 % competition in 30/43 (68 %) PF sera
[33]. Moreover, other reports suggest that mutations located in the
ectodomain of Dsg1 can play a role in autoimmunity, since
polymorphism has been reported in this molecule and may contribute
to disease susceptibility in PF [34]. On the other hand, other
portions of Dsg1 also seem to be important in terms of the complex
antigenic sites recognized by pemphigus sera. As an example,
Dmochowski and colleagues [35] showed that the extracellular amino
terminal domain of bovine Dsg1 was recognized only by certain PF
sera, whereas its intracellular domain was recognized by both PV
and PF sera [35]. Similar results were reported by Ohata et
al. [36]. Additional studies implicate Dsg1 also as a potential
part of the pathogenic mechanisms involved in spreading of
Staphylococcus aureus in bullous impetigo as well as in
staphylococcal scalded skin syndrome (SSSS), and also as one of the
antigens for IgA pemphigus [37, 38]. Dsg1 is not only
immunologically but also biologically important. Thus, a spectrum
of dominant mutations in Dsg1 causes striate palmoplantar
keratoderma, in which affected individuals have marked
hyperkeratotic bands without blisters on their soles or palms [40].
These mutations also affect the N-terminal domain of Dsg1 [39].
In our study, we confirmed that sera from patients with active PF,
FS, and PV disease, as well as those from patients with this new
variant of EPF in El Bagre, recognize a 45 kDa tryptic
fragment prepared from viable bovine skin [39, 40]. This antigen
was identified as the ectodomain of Dsg1; our tryptic fragment
sequence was similar to the theoretical expected fragments of a
tryptic digestion of the ectodomain of Dsg1 (GCG software). The use
of trypsin on viable cells results in cleavage of only the
ectodomain [41, 42], although the ectodomain of Dsg1, in fact
possesses multiple potential tryptic cleavage sites resulting in
various-size fragments predicted to result from proteolysis.
Indeed, it is possible that the 45 kDa band excised for
analysis of amino acid sequence and composition may represent a
mixture of related peptides, thus possibly explaining in part the
lack of complete identity between the predicted bovine Dsg1 amino
acid composition and the observed amino acid composition.
Alternatively, glycine remaining from the electrophoretic
separation and/or another protein may contaminate the excised
band.
Our study confirms the biological importance of the ectodomain of
Dsg1, and shows that this molecule constitutes an immunodominant
moiety for pemphigus diseases [43], including the novel variant of
EPF found in El Bagre, Colombia. Every day new molecules
potentially involved in the pathogenesis of pemphigus and
pemphigoid diseases are discovered [44, 45]. Our experiments show
the continued importance of the 45 kDa conformational epitope
of Dsg1 as an autoantigen involved in the development of these
autoimmune diseases.
Acknowledgements.
Our special thanks to Professor David Garrod (University of
Manchester) for his valuable scientific comments on this
manuscript. We also want to thank Drs. Luis A. Diaz, Monica
Olague-Marchan and Liane M. Mende-Mueller as well as Ms. Argelia
Lopez-Swidersky for their scientific support. The immunochemical
characterization of this fragment was performed at the Dermatology
Department at the Medical College of Wisconsin, Milwaukee, WI,
U.S.A. This work was performed as part of the doctoral (Ph.D.)
thesis of Ana María Abréu Vélez M.D. at the University of
Antioquia. Dr. Abreu was the recipient of a scholarship from
Colciencias, Colombia. <
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