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The Journal of Immunology, 2006, 176: 1968-1977.
Copyright © 2006 by The American Association of Immunologists

Identification of an Epitope within Human Integrin {alpha}6 Subunit for the Binding of Autoantibody and Its Role in Basement Membrane Separation in Oral Pemphigoid1

Khwaja Aftab Rashid*, Joel N. H. Stern* and A. Razzaque Ahmed2,{dagger}

* Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA 02115; and {dagger} Center for Blistering Diseases, Department of Medicine, New England Baptist Hospital, Boston, MA 02120


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin {alpha}6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin {alpha}6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin {alpha}6. Control sera did not bind to the full-length integrin {alpha}6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin {alpha}6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mucous membrane pemphigoid (MMP)3 is an autoimmune mucocutaneous blistering disease characterized by autoantibodies to several target Ags present in the basement membrane zone (BMZ) (1). Oral pemphigoid (OP) is a subset of MMP characterized by vesiculobullous lesions, limited only to the oral mucosa (2). Patients with OP do not have involvement of other mucosae or the skin. A linear deposition of Ig, complement, or both is observed along the BMZ in perilesional tissues both in MMP and OP. Recent data from biochemical and in vitro studies with human tissue suggest that an integrin {alpha}6 subunit is a putative autoantigen for OP (3). The integrin subunits {alpha}6 and beta4 are present as a heterodimer (4) in the BMZ (5, 6).

In OP patients, a disruption of {alpha}6beta4 occurs along the BMZ and, in some cases, a complete loss of the integrin {alpha}6 subunit is seen in the region in which the bullae are observed (7). Mice with knockout integrin {alpha}6 or beta4 gene die shortly after birth. These mice have an extensive blistering of the skin and other stratified squamous epithelium, and lack hemidesmisome formation (8, 9, 10). In patients with pyloric atresia and junctional epidermolysis bullosa, which manifest as cutaneous blistering, mutations in the integrin {alpha}6 or beta4 gene are present, and the severity of the disease is related to the nature of the mutation (11). Hence, it is believed that a primary function of the integrin {alpha}6beta4 heterodimer is to anchor the basal epithelial cells to the BMZ.

The integrins make up a large family of heterodimeric cell surface receptors that mediate several functions. Some of these are cell adhesion to extracellular matrix and to other cells, signal transduction (12, 13, 14), and hemidesmosomal assembly (15, 16). The hemidesmosomes facilitate and provide adhesion of the basal keratinocytes to the basement membrane (17, 18). To date, 24 {alpha} and 9 beta subunits have been identified (19).

The integrin {alpha}6 subunit is a ~120-kDa transmembrane protein that contains 1073 aa. The large extracellular domain has 1011 aa, the transmembrane domain has 26 aa, and the intracellular domain has 36 aa (20). The two variants, A and B, of the integrin {alpha}6 can dimerize with either beta1 or beta4 subunits (21). The extracellular domain forms a receptor for the protein laminin, while the short intracellular domain links the integrins to the cytoskeletal proteins (22, 23, 24).

The integrin {alpha}6beta4 is believed to play a complex role in epithelial carcinogenesis (25). The focal loss of the integrin {alpha}6beta4 at tumor margins frequently corresponds with the loss of attachment of tumor cells to the underlying basement membrane (26). The overexpression of integrin {alpha}6beta4 may promote tumor metastasis by stimulating cell motility (27, 28).

The integrins are involved in wound healing and skin inflammation, and may regulate the balance between proliferation and differentiation (29, 30). Changes in the integrin expression, mutation, or dissociation of the subunits lead to several pathological conditions (31, 32).

Identification of binding sites for pathogenic autoantibodies of OP within the integrin {alpha}6 subunit is important for understanding the pathophysiology of OP and the molecular mechanisms for the disruption of the cell adhesion to basement membranes.

In this study using sera from OP patients, the autoantibody-binding epitope within integrin {alpha}6 subunit is identified and characterized. Using an organ culture model, we demonstrate that Abs to this epitope play a role in the BMZ separation of normal human oral mucosa.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
This section confirms adherence to the Declaration of Helsinki.

Patient sera

Sera used in this study were obtained from 20 untreated patients with active OP. These patients had the pemphigoid disease process limited to the oral cavity. These patients did not have pemphigoid disease involving any other mucosal tissue or the skin, during a long-term follow-up, mean 5.2 years (range 3.8–9.6). At the time of diagnosis and during follow-up, involvement of other sites was excluded by detailed history; physical exam; ears, nose, and throat exam; eye exam; and endoscopic examinations. The clinical diagnosis of OP was established by routine histology and confirmed by direct immunofluorescence. The presence of IgG or complement was detected in the BMZ of oral mucosa. On the salt split skin (SSS), the sera of OP patients bound to the epidermal side of the split. Control sera were obtained from 10 healthy individuals, 10 patients with pemphigus vulgaris (PV), 10 with pemphigus foliacious (PF), 10 with MMP, 10 with bullous pemphigoid (BP), and 10 with ocular cicatricial pemphigoid (OCP). Blood samples were collected after informed consent, and the study was approved by the Institutional Review Board.

Antibodies

The custom Abs services of Sigma Genosys were used to produce polyclonal Abs in rabbits. New Zealand rabbits were immunized s.c. with 100 µg of purified fragments of the integrin {alpha}6 subunit. Pre- and postimmunization sera were collected from respective rabbits.

The mouse anti-human mAb BQ16 (Ancell), the mouse anti-rat mAb GoH3 (R&D Systems) against the integrin {alpha}6 subunit, mouse anti-human mAb UMA9 (Ancell) against the integrin beta4, mouse anti-human GB3 (Harlan Bioproducts for Science) against laminin 5, and mouse mAb to His-Tag (EMD Biosciences) were purchased.

Analysis of antigenic determinants

The protein sequence of the integrin {alpha}6 was analyzed for antigenicity, flexibility, and beta turn with PCGENE software. Peak values were assigned for each criterion. Overlapping regions of the three criteria and peak values greater than 1.0 were selected.

Cloning of fragments representing the extracellular and intracellular domains of the integrin {alpha}6 subunit

Fragments representing different parts of the sequence of the human integrin {alpha}6 subunit were generated by PCR amplification from the full-length molecule. The full-length human integrin {alpha}6 subunit clone was a generous gift by Prof. Arnoud Sonnenberg (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Fragments representing the extracellular and intracellular domains were amplified and sequenced by the di-deoxy chain termination method. For accuracy purposes, both of the strands were sequenced. The sequences of the fragments were compared with the sequence of the human integrin {alpha}6 molecule, accession AF166335.1.

The fragments representing the extracellular domain are designated as A (23–462 aa) and B (463–1011 aa). The fragments A1 (23–131 aa) and A2 (217–462 aa) are the subfragments of the extracellular fragment A. The fragment C (857–1073 aa) represents part of the extracellular portion and the complete transmembrane and intracellular portion, and fragment D (1012–1073 aa) represents the complete intracellular portion of the integrin {alpha}6 molecule. The fragments we used in this study are represented schematically in Fig. 1. Fragments B, C, and C, D overlap each other.


Figure 1
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  		FIGURE 1. Binding specificity of OP patient serum and rabbit Abs against fragments A, B, C, and D of integrin {alpha}6 subunit. Immunoblot analysis of the binding of the serum from OP patient and rabbit Abs against fragments of integrin {alpha}6 subunit. Lysate of DU145 cell line was resolved on 4–20% SDS-PAGE, and proteins were transferred to nitrocellulose membrane and reacted with NHS, OCP serum, PV serum, PF serum, OP serum, and rabbit antisera against fragments A, B, C, and D and mAb GoH3.

 
The PCR products were separated on 1% agarose gel, purified by means of a QIAquick purification kit (Qiagen), digested with the restriction endonucleases, and ligated into a Gateway entry vector pENTR IA (Invitrogen Life Technologies). The restriction sites for SalI and EcoRV were created in the 5' and 3' primers, respectively, to facilitate the subcloning procedure. The ligated products were transformed into Escherichia coli DH5{alpha}, and the positive clones were identified by restriction endonuclease analysis. The plasmid isolated and purified from the positive clones was fused with Gateway destination vector pDEST14 (Invitrogen Life Technologies) by LR recombination reaction with clonase enzyme (Invitrogen Life Technologies). The correctness of the sequence was verified by sequencing the fragments after cloning them in gateway entry and destination vectors.

Expression and purification of the fragments of the integrin {alpha}6 subunit

Various domains of the integrin {alpha}6 subunit were expressed in BL21{lambda}DE3plysS strain of E. coli. The pDEST14 vector (Invitrogen Life Technologies) allows expression by means of the bacteriophage T7 promoter. To facilitate protein purification, a His-Tag sequence was fused to the N terminus of the cloned fragments. Cells of the resultant transformants were grown to an OD600 of ~0.4 at 37°C in liquid broth containing ampicillin (50 µg/ml), induced by the addition of 1 mM isopropyl-beta-D-thiogalactopyranoside. After a 3-h incubation under induction conditions, the cells were harvested by centrifugation at 3000 x g for 20 min at 4°C. The cell pellets were resuspended and solubilized in bug-buster reagent (EMD Biosciences) with protease inhibitor mixture (Roche) and 1 mM PMSF (Sigma-Aldrich). The inclusion bodies were prepared and solubilized in 8 M urea, 20 mM phosphate buffer (pH 7.8), and 500 mM NaCl. The solubilized inclusion bodies were passed through Ni-NTA column equilibrated in the same buffer. Contaminating proteins were washed twice with 4 vol of 8 M urea, 20 mM phosphate buffer (pH 6.0), and 500 mM NaCl. The column was then washed twice with 5 vol of 50 mM phosphate buffer (pH 8.0), 500 mM NaCl, and 20 mM imidazole. Proteins containing His-Tag were finally eluted in 0.5-ml fractions with 50 mM phosphate buffer (pH 8.0), 500 mM NaCl, and 250 mM imidazole.

Characterization of OP sera and Abs against cloned fragments of integrin {alpha}6 subunit by immunoblot using DU145 cell lysates

The DU145 cell line was obtained from American Type Culture Collection. The cells were lysed in TXSWB (1% Triton X-100, 100 mM Tris-Cl (pH 8.0), 100 mM NaCl, and 10 mM EDTA) with 1 mM PMSF and protease inhibitor mixture (Roche) and incubated on ice for 20 min. The lysate was cleared of debris by centrifugation at 10,000 x g for 15 min at 4°C. The immunoblot was conducted by separating the protein on 4–20% acrylamide gels at 150 V for ~1 h with Tris-glycine-SDS running buffer, under reducing conditions. The resolved proteins were transferred onto a nitrocellulose membrane at 20 V, 250 mA for 1 h with a semidry blotting system. The membrane was blocked by 1% alkali-soluble casein (Boston Bioproducts) in TBS (0.5 M NaCl and 20 mM Tris-HCl (pH 7.4)). Patient sera or primary Abs with appropriate dilution in 1% alkali-soluble casein were added to the membrane and incubated for 1 h with gentle shaking. The membrane was then washed three times with TBSTT (0.5 M NaCl, 20 mM Tris-HCl, 0.2% v/v Triton X-100, and 0.05% v/v Tween 20 (pH 7.5)) and twice with TBS, 10 min each. After washing, appropriate secondary Abs conjugated with HRP were added and incubated for 1 h. The membrane was then washed, as above. The substrate solution was added to the membrane for 1 min, and the membrane was then exposed to x-ray film and developed.

Characterization of OP sera; Abs against the cloned fragments of integrin {alpha}6 by immunoprecipitation

OP sera and Abs against cloned fragments of integrin {alpha}6 were further characterized by immunoprecipitation. OP patient serum and Abs to cloned fragments A, B, C, and D were incubated with the cleared lysates of DU145 cells for 1 h at 4°C. The protein A-agarose beads were washed and equilibrated in TXSWB and added to the Ag-Ab complexes. The tubes were rotated overnight at 4°C. Subsequently, the protein A-agarose beads were washed three times with TXSWB and twice with TBS. The immunoprecipitated proteins were released by boiling for 10 min in SDS loading buffer containing 0.5 M DTT and analyzed by Western blot, using GoH3 mAb.

Characterization of the integrin {alpha}6 fragments and their binding with OP patient serum by Western blot analysis

Characterization of the purified integrin {alpha}6 fragments was done by standard Western blot analysis. The fragments of the integrin {alpha}6 were separated using semipreparative, 4–20% acrylamide gels and transferred to nitrocellulose membrane under the conditions described above. After the transfer of the proteins, the membranes were cut into 5-mm-wide strips. The membrane strips were blocked with 1% alkali-soluble casein for 1 h and incubated with OP patient sera at 1/100 dilution. Then they were probed with HRP-conjugated goat anti-human IgG.

Peptide synthesis

To delineate the approximate amino acid sequences that bind to OP Abs, small peptides were synthesized at GL Biochem. Based on the PCGENE analysis, a sequence of 14-aa residues (range 292–305 aa) within the fragment A2, considered to be antigenic, was synthesized as peptide A2.1 (LKRDMKSAHLLPEH) and conjugated with BSA with amide bonds. Another peptide (A2.2) of 11-aa residues (range 320–330 aa), within fragment A2 was synthesized (VAVVDLNKDGW) and conjugated with BSA by amide bonds, to serve as a control peptide. Analysis by HPLC showed 96.5% purity.

Blocking of OP Ab epitope in fragment A2

The specificity of the OP autoantibodies for fragment A2 of the integrin {alpha}6 subunit was further tested by blocking the epitope on fragment A2 with rabbit antisera to fragment A2. Fragment A2 was transferred onto a nitrocellulose membrane. The membrane was blocked with 1% alkali-soluble casein and incubated with rabbit Abs to fragment A2 (1:500). The membrane was washed and treated with OP patient serum (1:100). Then it was reacted with HRP-conjugated goat anti-human IgG. As a control, another blot of fragment A2 was reacted with OP serum and rabbit Abs to fragment A2; the blot was probed by HRP-conjugated goat anti-human and goat anti-rabbit IgG, respectively.

Characterization of Ag binding site (epitope) on fragment A

To purify and to test the reactivity of OP autoantibodies and to test the presence of additional epitopes in fragment A of the integrin {alpha}6 subunit, OP patient serum was passed through a column of the integrin {alpha}6 fragment A2.1 coupled to CNBr-activated Sepharose 4B. The beads were swollen for 30 min at room temperature (RT) and washed with 1 mM HCl. The slurry was then washed three times with 5 vol of distilled water and then equilibrated with 0.1 M NaHCO3 and 0.5 M NaCl (pH 8.3) (binding solution). The purified fragment A2.1 of the integrin {alpha}6 was equilibrated in the binding solution by dialysis and coupled to the beads in separate tubes by mixing at RT for 2 h. The unbound protein was removed, and beads were washed with the binding solution several times. The unreacted groups on the beads were blocked by 0.2 M glycine (pH 8.3) for 2 h at RT. The beads were then packed into columns and equilibrated with binding solution. The OP serum was passed through these columns, and bound proteins were washed with binding solution. The Abs were eluted with acetate buffer (pH 4.5) and neutralized. The unbound proteins (flow through) and eluted Abs were tested for their reactivity with fragments A2 and A2.1 by Western blot.

Characterization of binding of Abs to cloned fragments of the integrin {alpha}6 subunit in normal human oral BMZ

Sections each of 4 µm thickness of normal human oral mucosa were incubated with: 1) OP sera; 2) immunoaffinity-purified OP sera; 3) Abs to cloned fragments of integrin {alpha}6 subunit; and 4) mAb GoH3 and BQ16. The sections were stained by immunoperoxidase reaction and viewed under a microscope. Normal human sera were the negative control.

Effect of OP Abs on oral mucosa in organ culture

To determine the ability of test Abs to cause BMZ separation in human oral mucosa, we used an in vitro culture model with normal human oral mucosa, as previously described (2). Patient serum, immunoaffinity-purified OP serum, Abs for fragments of integrin {alpha}6 subunit, and mAb BQ16 and GoH3 were tested. Normal human mucosa was obtained by biopsy from volunteers, after informed consent. Pieces of this normal oral mucosa were incubated in a 24-well tissue culture plate in complete RPMI 1640 medium supplemented with different test reagents. Next, 30% v/v of sera from patients with OP, immunoaffinity-purified OP sera, preimmune rabbit sera, and rabbit Abs to recombinant fragments of integrin {alpha}6 subunit were added to the wells and incubated with 5% CO2 for 48 h at 37°C. Control test sera consisted of sera from untreated patient with generalized active PV. Earlier in vitro organ culture experiments had demonstrated that optimal BMZ separation was observed after 48 h of incubation (3). After incubation, tissue samples were processed and observed by routine H&E stain.

Binding of OP autoantibody to SSS

We used 1 M NaCl to produce SSS substrate to determine binding of the integrin {alpha}6 subunit. The SSS slides were incubated with sera from an untreated patient, with active OP, GoH3, BQ16, and rabbit Abs to fragments of integrin {alpha}6 subunit. Appropriate fluorescent-conjugated Abs were added, washed, and viewed under fluorescent microscope, as described (33, 34). Controls for this experiment were sera of a patient with BP and sera of a patient with epidermolysis bullosa acquisita.

Confocal microscopy

The 4-µm-thick sections of oral mucosa were simultaneously incubated with sera of OP patients, rabbit Abs to cloned fragments A and A2 of the integrin {alpha}6 subunit, and mAb GB3. Alexa 488 and 647 fluorochromes were added. Sections were incubated, washed, and excited at 488 nm 25 mW and 633 nm 10 mW laser, respectively. We observed the binding by confocal microscope, as described (35).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Binding of OP autoantibody with full-length native integrin {alpha}6 molecules

Using an immunoblot and immunoprecipitation assays with DU145 cell lysate as substrate, we demonstrate that Abs in sera of OP patients bind to ~120-kDa protein. Similar binding is observed with antiserum to recombinant fragments of integrin {alpha}6 subunit. The positive control GoH3 Abs also bound to a ~120-kDa protein, while no binding was seen with normal human serum (NHS) nor serum of OCP, PV, and PF patients (Fig. 1).

Expression and purification of extracellular and intracellular fragments of the integrin {alpha}6 subunit

The full-length human integrin {alpha}6A gene cloned in pRc/CMV vector (21) was used to generate fragments representing the extracellular and intracellular domains of the integrin {alpha}6 (Fig. 2A). The approximate size of the fragments were: A, 1370 bp; A1, 327 bp; A2, 738 bp; B, 1647 bp; C, 651 bp; and D, 84 bp. Fragments isolated and purified from agarose gel were subcloned in Gateway entry vector pENTR1A and then fused with destination vector pDEST14. The sequence analysis of the cloned fragments matched the integrin {alpha}6 sequence in the National Library of Medicine PubMed database sequence. The fragments were expressed in E. coli.


Figure 2
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  		FIGURE 2. A, Schematic representation of the cloned fragments of the full-length human integrin {alpha}6 subunit. The fragments, A (23–462 aa) and B (463–1011 aa), represent the extracellular domains. Fragment C (857–1073 aa) represents part of extracellular domain and full transmembrane and intracellular domains. Fragment D (1012–1073 aa) represents full intracellular domain. Fragments A1 (23–131 aa) and A2 (217–462 aa) are the subfragments of fragment A. Fragments A2.1 (292–305 aa) and A2.2 (320–330 aa) are the synthetic peptides within the fragment A2. EC, extracellular; TM, transmembrane; IC, intracellular. B, Biochemical characterization of the cloned fragments of the human integrin {alpha}6 subunit. Immunoblot analysis of the various fragments representing the different domains of the human integrin {alpha}6 subunit. The protein fragments expressed and purified were separated by SDS-PAGE, blotted onto nitrocellulose membrane, and probed with anti-His-Tag mAbs. Lanes A–D, Represent fragments A (~80 kDa), A1 (~33 kDa), A2 (~40.8 kDa), B (~82.5 kDa), C (~40.8 kDa), and D (~7 kDa), respectively.

 
Protein fragments were isolated in the form of inclusion bodies and purified on an Ni-NTA column, first with denaturing buffer and then with nondenaturing buffer. The fusion proteins were resolved on a 4–20% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with mAb to His-Tag. The protein fragments were of the following sizes: A, ~80 kDa; A1, ~33 kDa; A2, ~40.8 kDa; B, ~82.5 kDa; C, ~40.8 kDa; and D, ~7 kDa (Fig. 2B). The mAb GoH3 detected fragments A and A2 by immunoprecipitation (Fig. 3I) and immunoblotting (Fig. 3II), while mAb BQ16 detected fragment B only by immunoblotting (Fig. 3III).


Figure 3
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  		FIGURE 3. Binding characteristics of rat mAb GoH3 and murine mAb BQ16 to cloned fragments of integrin {alpha}6 subunit. I, Immunoblot analysis showing binding of mAb GoH3 to fragments A and A2. II, Represents immunoprecipitation of all the fragments of {alpha}6 subunit with mAb GoH3. Note that GoH3 immunoprecipitates only fragments A and A2. III, Demonstrates immunoblot analysis of binding of BQ16 to cloned fragments of {alpha}6 subunit. Note that mAb BQ16 binds only to fragment B.

 
Binding of OP sera to fragments of the integrin {alpha}6 subunit

These experiments were done to test the binding of OP patient sera with various fragments of the integrin {alpha}6 subunit by Western blotting. The binding pattern of OP sera to fragments A, B, C, and D is presented in Fig. 4A. For representation, the binding pattern from one patient serum sample is shown. All of the OP sera (n = 20) demonstrated binding to subfragment A2 of fragment A (Fig. 4B). None of the OP sera showed binding with the fragments A1, B, C, and D. Normal human sera (n = 10) samples and sera from 10 patients with OCP, 10 with MMP, 10 with BP, 10 PV, and 10 PF did not demonstrate any detectable level of binding to any of the cloned fragments of the integrin {alpha}6 molecule.


Figure 4
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  		FIGURE 4. A, Binding specificity of OP sera to cloned fragments of the integrin {alpha}6 subunit. Immunoblot analysis of the binding of the serum from one OP patient, with cloned fragments A, B, C, and D of the human integrin {alpha}6 subunit. Note binding of OP serum is observed with fragment A only. No binding is seen with fragments B, C, and D. Similar binding was observed with all 20 test OP sera. B, Binding specificity of OP sera to subfragments of A of the integrin {alpha}6 subunit. Immunoblot analysis of the binding of the serum from one OP patient, with cloned subfragments A1 and A2 of the fragment A of human integrin {alpha}6 subunit. Note binding of OP serum is observed with fragment A2 only. No binding is seen with fragment A1. Similar binding was observed with all 20 test OP sera. C, Binding specificity of OP sera to synthetic peptide of the integrin {alpha}6 subunit. Immunoblot analysis of the binding of the serum from one OP patient with the synthetic peptide of the human integrin {alpha}6 subunit. Note binding of OP serum is observed with peptide A2.1 only. No binding is seen with peptide A2.2. The peptide was conjugated with BSA; therefore, its observed molecular mass was ~65 kDa.

 
The PCGENE software analysis showed four potential antigenic regions on the full-length integrin {alpha}6 subunit molecule. The two domains lie within fragment A between aa 105–118 (present in subfragment A1) and 292–306 (present in subfragment A2). The other two domains are in fragment B between aa 520–533 and 890–903. These fragments were not studied further because OP sera did not bind to fragments B, C, and D. Based on our earlier observations of OP serum binding with subfragment A2 only, we synthesized peptide A2.1 (292–305 aa) for further studies and peptide A2.2 (320–330 aa) was used as a negative control. The peptides A2.1 and A2.2 conjugated with BSA demonstrate a molecular mass of ~65 kDa on 4–20% SDS-PAGE. All OP sera (n = 20) demonstrated binding with peptide A2.1 only, while none of the sera bound to the control peptide A2.2. The binding pattern from one representative patient with OP is shown in Fig. 4C.

Determining the autoantibody binding site(s) within cloned fragment A2

The purpose of this experiment was to determine whether multiple binding sites exist for the OP autoantibodies within the fragment A2. The peptide A2.1 was immobilized on a CNBr-activated Sepharose 4B column, and OP sera passed through the column. The unbound OP serum was tested for reactivity with the fragments A2 and A2.1. The unbound serum fraction did not show any reactivity with the fragments A2 and A2.1. Thereafter, Ab that had bound to the column was eluted and reacted with the fragments A2 and A2.1. Binding was observed with fragments A2 and A2.1 (Fig. 5). These observations would suggest that the sera available to us and tested in these experiments recognized only one epitope A2.1 in the {alpha}6 subunits of integrin.


Figure 5
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  		FIGURE 5. Determining the OP autoantibody binding site(s) within fragment A2. OP serum was passed through a column of CNBr-activated Sepharose 4B beads conjugated with peptide A2.1. Absorbed OP sera and Abs eluted from column were tested for reactivity with fragments A2 and A2.1 by immunoblot. The absorbed OP serum did not react with fragments A2 and A2.1. The eluted Abs bound to ~40.8-kDa fragment A2 and ~65-kDa fragment A2.1.

 
Determining the binding specificity of the epitope by blocking experiments

The purpose of this experiment was to determine whether serum from OP patient and rabbit antiserum bind to the same epitope(s) in fragment A2. Fragment A2, transferred onto nitrocellulose membrane, was incubated with rabbit Abs to fragment A2 and then reacted with OP serum. Binding of OP serum was not observed (Fig. 6B). This indicated that rabbit Ab to fragment A2 blocked binding of OP serum to a portion in fragment A2. Rabbit Abs to fragment A2 and OP serum when incubated with fragment A2 demonstrated appropriate binding is seen in Fig. 6, A and C, respectively, and served as controls for this experiment. Similarly, when a 4-µm-thick cryostat section of normal human oral mucosa is first reacted with rabbit Abs to fragment A2 and then with OP serum, no binding is observed. In reverse experiment, when the sections were first incubated with OP sera and then reacted with rabbit Abs to A2, no binding was observed (data not shown).


Figure 6
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  		FIGURE 6. Determination of the binding specificity of the epitope by blocking experiment. Fragment A2 transferred onto nitrocellulose membrane was incubated with OP serum (A), rabbit antiserum to fragment A2 and then OP serum (B), rabbit antiserum to fragment A2 (C). Nitrocellulose membranes A and B were probed by secondary Abs, goat anti-human conjugated with HRP, and membrane C was probed by secondary Abs and goat anti-rabbit conjugated with HRP. Clear bands are seen in membranes A and C. Fragment A2 blocked by rabbit Abs did not react with OP serum.

 
Absorption studies to determine binding specificity of multiple OP sera

The purpose of this experiment was to verify the earlier observation that all 20 sera bind to fragment A2. First, the serum of OP patients was passed through a column of CNBr-activated Sepharose 4B with immobilized fragment A2. The absorbed sera were subsequently incubated with nitrocellulose membrane with DU145 cell lysate. On the immunoblot, the absorbed sera did not demonstrate binding to integrin {alpha}6 subunit. Unabsorbed sera, GoH3 and BQ16, demonstrated binding to a ~120-kDa protein. These results demonstrate that sera of 20 different patients recognize the same epitope within integrin {alpha}6 subunit (data not shown).

Immunoperoxidase examination of normal human oral mucosa

Smooth linear homogenous BMZ staining of normal human oral mucosa was observed when reacted with OP sera, immunoaffinity-purified OP autoantibodies, and rabbit Abs to the cloned fragments (A, A1, A2, B, C, and D) of integrin {alpha}6 subunit, mAb GoH3, and BQ16 (Fig. 7). No binding was seen with NHS samples (data not shown).


Figure 7
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  		FIGURE 7. Immunoperoxidase staining of normal human mucosa with test reagents. Sections of normal human oral mucosa were incubated with: 1, OP serum; 2, affinity-purified OP Abs; 3, rabbit antiserum to fragment A; 4, rabbit antiserum to fragment B; 5, rabbit antiserum to fragment C; 6, rabbit antiserum to fragment D; 7, rabbit antiserum to fragment A1; 8, rabbit antiserum to fragment A2; 9, GoH3; and 10, BQ16. Binding to BMZ by rabbit Abs to cloned fragments of integrin {alpha}6 subunit, GoH3, and BQ16 was similar to that observed by OP sera and affinity-purified OP sera.

 
SSS analysis

The sera of OP patients, mAb GoH3, mAb BQ16, and rabbit Ab to the cloned fragments (A, B, C, and D) bound to the epidermal side of SSS. No binding was seen at the base of the blister with any of these Abs. Control BP sera bound to the epidermal side of the split, while sera from patients with epidermolysis bullosa acquisita bound to the dermal side of the split (data not shown).

Confocal microscopy

Simultaneous staining with immunoaffinity-purified OP sera and GoH3 demonstrated binding to the epidermal side, while Abs to laminin bound to the dermal side of the vesicle, in a biopsy of an oral lesion, from an OP patient (Fig. 8, A and B).


Figure 8
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  		FIGURE 8. Confocal microscopy of vesicle from oral mucosa of a patient with OP. Confocal microscopy was used to determine in vivo presence of molecules in the roof and floor of the vesicle. A, Immunoaffinity-purified OP Abs bind to the roof of the vesicle. B, Immunoaffinity-purified OP Abs bind to the roof, and Abs to laminin 5 (GB3) bind to the base of the vesicle.

 
In vitro organ culture model

Sections of normal human buccal mucosa, obtained from biopsies of oral cavity, were incubated with a panel of Abs, OP patient serum (A), immunoaffinity-purified OP patient serum (B), NHS (C), rabbit antiserum to fragment A (D), rabbit antiserum to fragment A2 (E), PV patient serum (F), rabbit antiserum to fragment B (G), mAb GoH3 (H), and mAb BQ16 (I). BMZ separation in the normal human oral mucosa is observed with OP serum, immunoaffinity-purified OP patient serum, rabbit antiserum to fragment A and A2, and mAb GoH3. PV serum caused typical acantholysis of epithelial cells. NHS, rabbit antiserum to fragment B, and mAb BQ16 did not cause BMZ separation (Fig. 9). These experiments indicate that Abs to epitope within the fragment A2 region of integrin {alpha}6 are capable of producing BMZ separation in normal human oral mucosa in organ culture.


Figure 9
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  		FIGURE 9. In vitro organ culture model to study BMZ separation in normal human oral mucosa. Normal human oral mucosa was incubated with different test reagents: A, OP patient serum; B, immunoaffinity-purified OP patient serum; C, NHS; D, rabbit antiserum to fragment A; E, rabbit antiserum to fragment A2; F, PV patient serum; G, rabbit antiserum to fragment B; H, mAb GoH3; and I, mAb BQ16. Note that rabbit antiserum to fragment A (D), A2 (E), and mAb GoH3 (H) produces BMZ separation, which is histologically similar to that produced by OP serum (A) and immunoaffinity-purified OP patient serum (B). NHS (C), rabbit antiserum against fragment B (G), and mAb BQ16 (I) did not cause BMZ separation. PV sera demonstrate acantholysis of epithelial cells (triangular arrow) in oral mucosa (F). Magnification, x20.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have identified the epitope in the human integrin {alpha}6 subunit to which OP Ab in sera of patients with OP bind. Our previous studies showed that autoantibodies in the serum of OP patients bound to a ~120-kDa protein in oral mucosa, identified as the integrin {alpha}6 subunit (2). The titers of autoantibodies to the human integrin {alpha}6 subunit correlate with disease extent and severity (36).

Sera of 20 untreated patients with active OP bound to native full-length integrin {alpha}6 subunit in DU145 cell lysate. The same sera bound to cloned fragment A of integrin {alpha}6. No binding was observed with any other recombinant protein fragments of integrin {alpha}6. Fragment A was subdivided into A1 and A2. Further immunoblot analysis demonstrated that binding to fragment A was entirely due to a peptide in fragment A2. No binding was observed with peptides in fragment A1. Immunoblot analysis demonstrated that all 20 sera samples bound to a 14-aa synthetic peptide, A2.1 (292–305 aa), within fragment A2. Absorption experiments further confirmed this binding.

The validity of the presence of the epitope to which OP Abs bind is confirmed by absorption of OP Abs by fragment A2.1 when immobilized on a CNBr-activated Sepharose 4B bead column. Experiments demonstrate that the sera tested bind to a single epitope within fragment A2 of integrin {alpha}6 subunit. However, the authors realize that there may be additional epitope(s) present either within fragment A or other fragments of integrin {alpha}6 subunit. The possible reason for detecting only one epitope is that we studied only 20 sera. If a larger sample of sera were available, then other epitope(s) may have been detected. Commercially available mAb GoH3 and BQ16 bind to two different epitopes, indicating that Abs binding to integrin {alpha}6 subunit may be pathogenic based on their epitope specificity. mAb GoH3 causes subepithelial separation in organ culture, while BQ16 does not. It is possible that epitope-binding specificity of OP autoantibody may be MHC or other gene restricted. All of the patients in this study were Caucasian living in New England. When samples of sera from different regions of U.S. and the world are tested, it is possible that other epitope(s) may be detected.

In vitro organ culture experiments, using normal human oral mucosa, demonstrated that rabbit Ab to fragments A, A2, and A2.1 cause BMZ separation, similar to that produced by OP serum and immunoaffinity-purified OP sera. Rabbit Ab to other fragments did not produce BMZ separation in organ culture. Hence, we concluded that at least one binding site for the OP autoantibodies resides in the fragment A2. Sera depleted of Ab to fragments A, A2, and A2.1 did not produce BMZ separation in organ culture.

A logical experiment to follow these studies would be to inject the pathogenic sera into neonatal BALB/c mice. Such an in vivo assay has been used to demonstrate pathogenicity of PV, PF, BP, and antiepiligrin cicatricial pemphigoid (AECP) autoantibodies (37, 38, 39). This experiment was not included in this study because the authors prefer to use an animal model in which oral tissues would be accessible and large enough to reveal blisters superficially. The i.p. injection of anti-{alpha}6 Abs in neonatal mice may cause blistering in the skin; however, that would not validate their activity to cause blister in the oral cavity, and thus have a limited direct application to pathogenesis of OP. Furthermore, studies in in vivo BALB/c mice were preceded by human organ culture studies for autoimmune blistering diseases. The neonatal mouse, while offering an in vivo environment, does not mimic or represent the human immune system, because it cannot mount an innate immune response to the injected sera.

The validity of our subcloning experiments comes from the observations of our immunoperoxidase studies. Abs to cloned fragments of integrin {alpha}6 subunit bind to the BMZ in a pattern identical with that observed in OP sera and immunoaffinity-purified OP sera, but not in normal human sera.

The in vitro organ culture model has been effective in demonstrating the functional capacity of human Abs in patient sera, and those produced in rabbits to putative antigenic proteins, to produce BMZ separation (3, 40). In our earlier experiments, MMP sera produced BMZ separation of oral and conjunctival mucosa, and PV sera produced acantholysis, serving as a positive control. NHS did not produce any change, demonstrating the utility of the model to study the interaction of BMZ proteins and the Abs against them (3, 40).

Interestingly, the mAb GoH3 also bound to fragment A2, and caused BMZ separation of normal human oral mucosa in organ culture in our experiments. The mAb BQ16 bound to the cloned fragment B. Rabbit Abs to fragment B and mAb BQ16 did not produce BMZ separation of normal human oral mucosa. Earlier studies indicate that mAb GoH3 does inhibit binding of epithelial cells to laminin (41), whereas BQ16 does not (42). These observations may be explained in part by the differences in the Ab binding sites on the integrin {alpha}6 subunit. Because commercially available rat mAb GoH3, produced against the mouse integrin {alpha}6 subunit, reacts with cloned human integrin {alpha}6 subunit fragments A and A2, the epitope to which it binds is common to mouse and human.

OP autoantibodies present in the sera of OP patients and mAb GoH3 that target the integrin {alpha}6 subunit upon binding to the epitope may induce conformational changes, resulting either in its dissociation from the beta subunit or reducing its affinity for its ligand, laminin 5, or both. Change in the conformation of the {alpha}6 subunit, either because of the binding of the autoantibodies or the subsequent intracellular signals, can modify its functional properties. The integrin {alpha}6 subunit preferentially binds the beta4 subunit in cells that coexpress the integrin beta1 and beta4 subunits, such as was seen in the MCF-10A cell line (43). The mAb GoH3 impacts the function of hemidesmosome associated with the integrin {alpha}6beta4 heterodimer in MCF-10A cell lines (43). A biological consequence of disrupting the integrin {alpha}6beta4 heterodimer may be the loss of a dominant adhesive structure between the basal lamina and basal cells, resulting in detachment of the cells and the resultant BMZ separation.

The transition of the integrins from a low affinity to a high affinity state is accompanied by conformational change in the molecule (44). In its low affinity state, the extracellular domain of the integrin is in a bent conformation, with the N-terminal ligand-binding pocket close to the membrane (45, 46). On activation, a "switchblade knife" opening of the integrin is seen, with the extracellular domain straightening out, and the ligand-binding pocket moves away from the membrane (47, 48). This may be one of the mechanisms involved, but there is no evidence to prove it. Steric hindrance is another possible mechanism of ligand dissociation (49, 50).

In earlier murine studies, the addition of OP sera resulted in disruption of hemidesmosomal assembly, in cells in culture (7). The data in this study would suggest that disruption of hemidesmosomal assembly may in part be due to Abs to {alpha}6 subunit of the integrin, in the OP patient sera.

Our in vitro data identify a 14-aa peptide A2.1 (292–305 aa) as one of the epitopes for the binding site for autoantibodies in the sera of patients with OP. The mAb GoH3 binds to the same epitope. Organ culture studies demonstrate that Ab to this epitope can cause BMZ separation. Confocal microscopy studies reveal that in OP oral biopsies, the integrin {alpha}6beta4 is dissociated from its ligand, laminin 5. In a recent review (51), it has been reported that seven fibronectin repeats are present at the N-terminal extracellular domain of integrin {alpha}6 subunit from aa 1 to 470. This coincides with fragment A of our study. Domain between fibronectin repeats III and IV mediates the ligand binding. The OP autoantibody binding domain between aa 292 and 305 lies between fibronectin domains IV and V. This provides initial and preliminary evidence that binding of OP autoantibody to its target site, on integrin {alpha}6 subunit molecule, may influence ligand binding. Thus, it would appear that this region of the integrin {alpha}6 is important in adherence of the basal layer cells to the extracellular matrix proteins. Studies that focus on the molecular events after the binding of OP autoantibody to its epitope(s) will enhance our understanding of epithelial biology and the adhesion of cells to the BMZ of the oral and other mucosae.

Yancey and colleagues (52) have described a subset of MMP patients, who produce Abs to laminin 5 also known as epiligrin. This subset is referred to as AECP. Hence, it is apparent that Abs to the two components of the {alpha}6beta4 heterodimer produce two distinct disease processes, within two different clinical profiles, outcomes, and prognosis, creating subsets of MMP based on Ag specificity (Table I). Interestingly, the ligand of integrin {alpha}6beta4 is laminin, and patients with autoantibodies to laminin have a clinical profile very similar to that in patients with Abs to the beta4 subunit. However, they frequently have poor prognosis. A high incidence of malignancy has been reported in AECP patients with Abs to laminin 5 (53). In contrast, patients with MMP with Abs to beta4 integrin have lower than expected incidence of cancer (our unpublished results).


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  		Table I. Clinical spectrum in MMP patientsa

 
In two studies, investigators have identified novel Ags in the BMZ that play a role in MMP and OCP. Ghohestani and colleagues (54) identified a 168-kDa protein in buccal mucosa to which sera of MMP patients bound. However, their patients had MMP, and not OP only. Moreover, their sera did not bind to dermal or epidermal extracts (54). Smith et al. (55) described a 45-kDa protein in epidermal extracts that bound to an IgA Ab in sera of OCP patients. It did not bind to an IgG Ab, and all of the patients had disease limited to the eye. In both studies, proteins were identified as not being similar or degraded products of bullous pemphigoid Ag 1, bullous pemphigoid Ag 2, laminin, or collagen 7 (54, 55). Furthermore, neither protein has been characterized as a specific molecule or protein of a specific molecule with a defined function in the BMZ.

Thus, it is becoming apparent that autoantibodies to two components of a heterodimer ({alpha}6beta4) and their ligand (laminin) can become autoantigens. The autoimmune diseases they produce have a wide and different clinical spectrum that has significant differences in clinical outcome, prognosis, and association with malignancy. Thus, nature has produced a unique intersection and association of molecules that mediate cell and extracellular matrix adhesion, autoimmunity, and association with malignancy. The importance of these molecules in maintaining homeostasis of basement membranes and their role in autoimmunity and malignancy will help us better understand the balance between health and disease.


   Acknowledgments
 
We are grateful to Dr. Jeffery Casaglia for assistance with the confocal microscopy photographs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by National Institutes of Health Grant ROI EY014228 and in part by a grant from the Pemphigus Foundation (both to A.R.A.). Back

2 Address correspondence and reprint requests to Dr. A. Razzaque Ahmed, Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, 190 Longwood Avenue, Boston, MA 02115. E-mail address: razzaque_ahmed{at}hms.harvard.edu Back

3 Abbreviations used in this paper: MMP, mucous membrane pemphigoid; AECP, antiepiligrin cicatricial pemphigoid; BMZ, basement membrane zone; BP, bullous pemphigoid; NHS, normal human serum; OCP, ocular cicatricial pemphigoid; OP, oral pemphigoid; PF, pemphigus foliacious; PV, pemphigus vulgaris; RT, room temperature; SSS, salt split skin. Back

Received for publication September 21, 2005. Accepted for publication November 9, 2005.


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 Introduction
 Materials and Methods
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 Disclosures
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