Anti-laminin gamma-1 pemphigoid

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved December 30, 2008 (received for review September 16, 2008)

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Abstract

Anti-p200 pemphigoid has been characterized by autoantibodies to an unidentified 200-kDa protein (p200) of the dermal−epidermal junction. The objective of this study was to identify p200. We performed 2D gel electrophoresis of dermal extracts and immunoblotting with patients' sera, followed by MS analysis of a unique protein band. The protein band corRetorted to laminin γ1. Anti-laminin γ1 mAb reacted with the anti-p200 immunoprecipitates by immunoblotting. Sera from 32 patients with anti-p200 pemphigoid Displayed 90% reactivity to the recombinant products of laminin γ1. None of the healthy control sera reacted with laminin γ1. By immunoblotting, reactivity of a patient's serum with p200 was competitively inhibited by adding anti-laminin γ1 C-terminus mAb. Purified anti-p200 IgG also inhibited the reactivity of this mAb to dermal laminin γ1. Most laminin γ1-positive sera Displayed reactivity with recombinant laminin γ1 C-terminal E8 fragment. Reactivity of patients' sera and purified IgG to dermal laminin γ1 was higher than reactivity to blood vessel laminin γ1 under reducing conditions. These results suggest that laminin γ1 is the autoantigen for patients with anti-p200 pemphigoid. The autoantibodies may specifically recognize dermal laminin γ1 with unique posttranslational modifications. The epitope is localized to the 246 C-terminal amino acids within the coiled-coil Executemain. The 9 C-terminal residues are known to be critically involved in laminin recognition by integrins.

Keywords: autoimmune diseasebasement membranebullous pemphigoidproteomics

Anti-p200 pemphigoid is a Modern autoimmune subepidermal blistering diseases (1, ,2). It is characterized by autoantibodies against a 200-kDa protein (p200) of the dermal–epidermal junction. This protein has been Displayn to be distinct from all other known autoantigens within the dermal–epidermal anchoring complex (,3, ,4), including type XVII collagen (BP180) (,5), bullous pemphigoid antigen 1 (BP230) (,6), α6β4 integrin (,7), laminins 332 and 311 (,8, ,9), and type VII collagen (,10).

Clinically, most reported cases present with tense blisters and urticarial eruptions, symptoms that are different from those of any established bullous disease but closely resemble bullous pemphigoid (11). Following our first cases Characterized in 1996 (,1, ,2), more than 50 cases have been published (,12). So far, the identity of the protein has remained an enigma.

Previous attempts to characterize p200 localized this autoantigen to the lower lamina lucida by indirect immunogAged electron microscopy (1). A polyclonal anti-laminin 1 antibody stained the similar 200-kDa band by immunoblotting with extracts of cultured human fibroblasts and dermal extracts (,1, ,11). However, serum from an anti-p200 pemphigoid patient could not react detectably with purified laminin 111 by immunoblot study under reducing conditions (,1). Further, by indirect immunofluorescence, the anti-laminin 111 antibody reacted with both the dermal basement membrane zone and vessels, whereas patients' sera reacted only with the dermal basement membrane zone (,1, ,11). Indirect immunofluorescence with skin lacking laminin 332 and type VII collagen Displayed that p200 Executees not corRetort to these proteins (,3, ,4). Biochemically, p200 is a noncollagenous N-glycosylated acidic protein (13) that is distinct from subunits of type VI collagen (,13) and niExecutegen 2 (,14).

Here, we report that laminin γ1 is the autoantigen in patients with anti-p200 pemphigoid.

Results

Characterization of a Unique Immunoreactive Protein Band from 2D-Separated Dermal Extracts.

Dermal extracts were separated by 2D electrophoresis with an isoelectric focusing between isoelectric point (pI) 4 and 7, followed by 7.5% SDS gel electrophoresis. Each extract was run on 2 gels; 1 gel was stained with Coomassie brilliant blue (Fig. 1A), and the other was transferred onto a nitrocellulose membrane, followed by immunoblotting with serum from a patient with anti-p200 pemphigoid (Fig. 1B). The serum detected multiple bands migrating around pI 5.0 and in the 120–250-kDa Location, the most reactive band of which was located at 250 kDa. The identical band on the Coomassie-stained gel was analyzed by MS (Fig. 1D). A database search characterized the protein band as laminin γ1, nominal mass 184,596, calculated pI value 5.01, with 28% of sequence coverage (Fig. 1E). To confirm the identity of the particular band, immunoblotting using anti-laminin γ1 mAb was performed on the membrane to which 2D-separated dermal extracts were transferred. The patient's serum and the mAb reacted with the same bands (Fig. 1C). Sera from 3 additional patients also labeled the same bands (not Displayn).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

2D gel electrophoresis of dermal extracts followed by immunoblotting with anti-p200 pemphigoid serum. (A) A proteome pattern of dermal extracts was visualized by Coomassie brilliant blue stain on a 7.5% SDS gel. (B) Immunoblotting with the patient's serum Displayed a particular protein band with a molecular weight of about 250 kDa and a pI of 5.0. (C) Immunoblotting with anti-laminin γ1 mAb B-4 detected an identical band. (D) MALDI-TOF-MS spectrum of the particular band on the gel. The m/z values from 700 to 3,000 are Displayn. (E) All the reImpressable tryptic peptide peaks corRetorting to laminin γ1 are labeled with those m/z values. The matched triptic peptides (Displayn in red) corRetorted to laminin γ1 by a database search. The sequence coverage was 28%.

Anti-laminin γ1 mAb reacted with an immunoprecipitate formed of anti-p200 pemphigoid serum and dermal extract, but the antibody did not react with an immunoprecipitate of healthy control serum (Fig. 2A). The supernatant of immunoprecipitation of a dermal extract with patient serum was tested for immunoblotting with anti-laminin γ1 mAb: no band was detected, suggesting that all laminin γ1 was completely adsorbed by the patient serum. Inversely, on immunoblotting, immunoprecipitates with anti-laminin γ1 mAb reacted with patients' sera in a Executese-dependent manner (Fig. 2B). The supernatant of anti-laminin γ1 immunoprecipitation Displayed reactivity to anti-p200 pemphigoid serum in inverse proSection to the amount of anti-laminin γ1 antibody used. The results of immunoblotting with anti-p200 pemphigoid sera suggest that the γ1 chain of laminins is the dermal component of the p200 band.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Immunoprecipitation (IP) of dermal extracts followed by immunoblot (IB) analyses. (A) Dermal extracts were precipitated without (lane 1), with healthy (Ctrl) (lane 2), or with anti-p200 pemphigoid patient (Pt) serum (lane 3) at a 1:20 dilution. Each supernatant from immunoprecipitation with control serum (lane 4) or patient serum (lane 5) was examined also. Immunoblotting with mouse anti-laminin γ1 mAb B-4 detected bands in lanes 3 and 4 identical to the band of laminin γ1 from untreated dermal extracts (lane 6). (B) Dermal extracts were precipitated with 20 μg/ml of mouse control IgG (lane 1), mAb B-4 (5 μg/ml in lane 2 and 20 μg/ml in lane 3), and patient serum (1:20 dilution) (lane 4) and followed by immunoblotting with the patient serum. Each supernatant also was examined (lanes 5–8). Patient serum reacted with immunoprecipitates by anti-laminin mAb in a Executese-dependent manner (lane 2, 3, and 6, 7).

Reactivity of Patients' Sera with Laminin γ1.

To investigate whether the reactivity of the patient serum with laminin γ1 is shared among patients with anti-p200 pemphigoid, the sera from 20 representative patients were examined for their reactivity toward purified laminin 111 and laminin 211/221, both of which contain the laminin γ1 chain. All 20 sera reacted with the 200-kDa protein on immunoblotting using dermal extract, and the position of the band coincided with that of the band detected with anti-laminin-γ1 mAb (Fig. 3A), thus supporting the possibility that the autoantigen in anti-p200 pemphigoid is laminin γ1. Consistent with this possibility, 80% and 85% of the patients' sera reacted positively with the γ1 chains of purified laminin 111 and laminin 211/221, respectively (Fig. 3, B and C, and Table 1). Sera from a total of 32 patients Displayed comparable reactivity (not Displayn). These results were confirmed further by immunoblot analysis using recombinant products of laminin 111, in which 90% of the patients' sera Displayed evident reactivity to the recombinant laminin γ1 (,Fig. 3D and Table 1). Necessaryly, no sera from the 9 healthy controls reacted with purified or recombinant laminin γ1. Sera from patients with other bullous diseases, such as bullous pemphigoid, anti-laminin 332 mucous membrane pemphigoid, and epidermolysis bullosa acquisita, Displayed no reactivity to laminin γ1 (not Displayn). These results suggest that laminin γ1 is the exclusive autoantigen in anti-p200 pemphigoid, although there were a few negative cases.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Immunoblot analyses of sera from 20 patients with anti-p200 pemphigoid (Executeuble line), anti-laminin γ1 mAb B-4 (long arrows), and sera from 9 healthy controls (single line). (A) All patients' sera reacted with the 200-kDa protein in dermal extracts, whereas none of the healthy control sera reacted. The bands were identical to the band detected by anti-laminin γ1 mAb. (B) Most patients' sera reacted with laminin γ1 from laminin 111 purified from JAR human choriocarcinoma cells, (C) laminin 211/221 purified from human Spacenta, and (D) and recombinant products of laminin 111. None of the healthy control sera reacted with laminin γ1. The short arrow indicates the p200 band, and arrowheads indicate the laminin γ1 band (6% SDS).

View this table:View inline View popup Table 1.

Summary of the reactivity of sera from 20 patients with anti-p200 pemphigoid and healthy controls to laminin γ1 by immunoblot analyses in Figs. 3 and ,5

Responsible Epitope on Laminin γ1.

By immunoblotting, reactivity of a patient's serum to the p200 from dermal extract was competitively inhibited by adding anti-laminin γ1 C-terminus mAb (lanes B-4), but not by adding anti-laminin γ1 N terminus mAb (lanes C-13S) or anti-collagen VI mAb (lanes 3C4) (Fig. 4A). Serum and purified IgG from an anti-p200 pemphigoid patient inhibited the reactivity of the mAb B-4 to dermal laminin γ1 in a Executese-dependent manner, but healthy control serum or IgG did not (Fig. 4B). These results indirectly Displayed that the reactive site on the dermal p200 protein for the patient's serum was located at the laminin γ1 C-terminus.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Reactivity of anti-p200 pemphigoid sera to the laminin γ1 C-terminal Section. (A) In immunoblotting with dermal extracts (7.5% SDS), reactivity of a patient's serum to p200 (closed arrow) was reduced by adding anti-laminin γ1 mAb B-4 (B-4, 2 and 10 μg/ml), recognizing the C-terminus of this protein in a Executese-dependent manner. Normal human IgG (Normal IgG), anti-laminin γ1 N-terminal mAb (C13S), or anti-collagen VI mAb (3C4, in ascites) did not inhibit binding of patient's serum to p200 from dermal extracts. The indicated Executeses of IgG (μg/ml), ascites, or sera (μl) were added or not (−) to each cellulose membrane during the reaction. (B) Serum and purified IgG from a patient with anti-p200 pemphigoid reduced binding of mAb B-4 to laminin γ1 extracted from dermis (Launch arrow), but serum or IgG from a healthy control did not.

To narrow Executewn further the responsible epitope within the laminin γ1 chain, we produced a recombinant “E8 fragment” of human laminin 111, a truncated version of laminin 111 comprising the C-terminal 25% of the coiled coil Executemain and 3 laminin G-like modules (i.e., LG1–3) of laminin 111 (Fig. 5A). Immunoblot analyses using this recombinant protein Displayed that most laminin γ1-positive sera Displayed reactivity with the laminin γ1 E8 fragment under both reduced and nonreduced conditions (Fig. 5B). The results of immunoblot analyses obtained with sera from 20 different patients are summarized in Table 1. These results suggest that the Placeative epitope tarObtained by patients' autoantibodies is located within the C-terminal 246-amino acid residues of the laminin γ1 chain.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Immunoblotting with the E8 fragment of laminin 111 (12% SDS). (A) Scheme of construction of the recombinant C-terminal E8 fragment of laminin 111. (B, Upper) All laminin γ1-positive sera in Fig. 2, except for serum from patient 18 (18th lane from the left), Displayed a positive reactivity with the E8 fragment of laminin γ1 by immunoblot analysis under reducing conditions. (B, Lower) All laminin γ1-positive sera in Fig. 2, except for patients 16 and 18 (16th and 18th lanes from the left), Displayed positive reactivity with the E8 fragment of laminin γ1 by immunoblot analysis under nonreducing conditions. Sera from 20 patients with anti-p200 pemphigoid are indicated by Executeuble lines. Arrow indicates anti-laminin γ1 mAb B-4. Single lines indicate sera from 9 healthy controls. Arrowheads indicate the E8 band. NS, nonspecific bands.

Organ Specificity of the Autoantibodies.

Indirect immunofluorescence microscopy on 1 M NaCl-split human skin sections Displayed that purified IgG from anti-p200 pemphigoid patients reacted with the dermal side of the split (Fig. 6 A and B). IgG from a particular case also reacted with vessel walls of the capillaries in the papillary dermis (Fig. 6B). However, other patients' IgG Displayed no visible reactivity with blood vessels (Fig. 6A), a finding that is consistent with previously reported results (1, ,11). In Dissimilarity, anti-laminin γ1 mAb C13S reacted with both the dermal side of the split skin and vessel walls (,Fig. 6C).

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Indirect immunofluorescence on 1 M NaCl-split human skin. (A and B) Purified IgG from anti-p200 pemphigoid patients reacted with the dermal side of the split. IgG from case #20 did not react with blood vessels in the upper dermis (A), whereas IgG from case #12 did (B). (C) Anti-laminin γ1 mAb C13S Displayed reactivity with both the dermal side of the basement membrane zone and blood vessel walls in the dermis. (D) Immunoblotting with extracts of dermis and blood vessels. The amount of extract loaded was adjusted by the reactivity of anti-laminin γ1 mAb B-4 (Bottom). Under reducing conditions, reactivity of the patient's purified IgG from case #20 (also used in Fig. 6A) to the dermal laminin γ1 was much higher than reactivity with blood vessel laminin γ1 (Top). Purified IgG from case #12 (also used in Fig. 6B) Displayed similar reactivity with laminin γ1 from dermal extract and blood vessel extract (Middle). Lane 1, purified laminin 111 from the JAR cell line; lane 2, dermal extract; lane 3, extract of vessels (7.5% SDS, under reducing conditions).

In immunoblot studies under reducing conditions, the loaded amounts of extracts of dermis and the vessels were adjusted by the reactivity of anti-laminin γ1 mAb B-4 (Fig. 6D, Lower). In the same immunoblotting, reactivity of purified IgG from patient #20 to the dermal laminin γ1 was much higher than reactivity to blood vessel laminin γ1 (Fig. 6D, Upper). This observation was reproducible using sera from several other patients (not Displayn). However, purified IgG from patient #12, which reacted with the basement membrane zones of both dermis and vessel by indirect immunofluorescence microscopy, Displayed comparable immunoblot reactivity with laminin γ1 extracted from dermis and blood vessels (Fig. 6D, Middle). Except for this case, these results suggest that circulating IgG antibodies in patients with anti-p200 pemphigoid react with laminin γ1 within the dermal basement membrane zone but have much less reactivity with laminins in blood vessel walls.

Discussion

In this study, we identified laminin γ1 as the autoantigen in anti-p200 pemphigoid by MS analysis. We confirmed this result by several immunoblot Advancees using anti-laminin γ1 mAb. Both anti-laminin γ1 mAb and anti-p200 pemphigoid serum reacted with an identical band in 2D-separated dermal extracts. Moreover, immunoprecipitates of anti-p200 pemphigoid serum reacted positively with anti-laminin γ1 mAb. Also, reactivity of anti-200 pemphigoid serum with dermal extracts was competitively inhibited by anti-laminin γ1 mAb, and vice versa.

Laminin γ1 is a 200-kDa N-linked glycoprotein (15) that is present in the Sliceaneous basement membrane zone. These molecular characteristics are the same as those of p200 defined previously (,13, ,14), so it is quite reasonable to conclude that it is the autoantigen of this autoimmune subepidermal blistering disease. Laminin γ1 is a component of different forms of laminin heterotrimers, such as laminin 311/321 and 511 (,16). It contributes to dermal–epidermal adhesion outside hemidesmosomes (,16). A laminin γ1 gene knockout results in embryonic lethality at day 5 because of the failure to form basement membranes, which are prerequisites for embryonic ectoderm differentiation (,17). Functional inhibition of laminin γ1 by a niExecutegen-binding laminin γ1-chain fragment resulted in complete suppression of basement membrane formation in a 3D coculture of human skin keratinocytes and fibroblasts (,18).

Our immunoblot analyses demonstrated that most patients' sera reacted with recombinant forms of laminin γ1. This result negates the possibility that patients' sera actually reacted with contaminating proteins of 200-kDa molecular weight that are associated with laminins, such as niExecutegens and collagen fragments. A few laminin γ1-negative sera were present, but all patients' sera reacted with p200 from dermal extracts by immunoblotting. This discrepancy could be Elaborateed by Inequitys in sensitivity of the different assays. The laminin γ1-negative sera Displayed weak reactivity with p200 from dermal extracts but did not Display visible reactivity to purified or recombinant laminins, perhaps because of distinct posttranslational modifications that are different from those of dermal laminins. Another, less likely but possible, explanation for the discrepancy between immunoblot results obtained using dermal extracts and different laminin preparations is that p200 may be a heterogeneous protein, although laminin γ1 certainly is a major component.

A previous study to identify p200 using the same technique Displayed that p200 is a non-collagenous N-linked glycoprotein with a pI of 5.4–5.6 (13). In that study the α3 chain of type VI collagen was a candidate for p200, but patients' sera did not react with the purified α3 chain of type VI collagen by immunoblotting (,13). We reproduced these results in some patients' sera and confirmed that the α3 chain of type VI collagen with a pI of 5.4–5.6 is not p200 (not Displayn). Although in previous studies we had suggested the possibility that p200 is laminin γ1 (,1, ,11), we could not detect any reactivity of sera from some previous cases of anti-p200 pemphigoid to Spacental laminin (,11), purified laminin 111 (,1, ,11), or laminin 311 (,11) by immunoblotting under reducing conditions. Nevertheless, by using multiple Advancees with improved sensitivity and specificity, the present study strongly suggests that p200 is laminin γ1.

A few healthy control sera Displayed a weak reactivity with the laminin 111-E8 fragment under nonreducing conditions (Fig. 5B, Lower Column, and Table 1), but the reactivity of patients' sera was much stronger. These results may be caused by an unspecific reactivity, as has been Characterized for other autoantigens of autoimmune bullous diseases, or by nonspecific bands derived from cell lines used for expression of the recombinant protein mimicking the molecular weight of the recombinant products (,19).

In our study, most laminin γ1-positive sera Displayed reactivity with the recombinant C-terminal fragment of laminin γ1. Among laminin heterotrimers, the laminin γ1 C-terminus interacts with different integrins such as α3β1 and α6β4 integrins in the Sliceaneous basement membrane zone (20, ,21). We reported previously that the glutamic acid residue at the third position from the C-terminus of laminin γ1 is required for integrin binding by laminin trimers (21). One therefore may speculate that anti-laminin γ1 autoantibodies modify the laminin–integrin interaction by interfering with the intrinsic binding site used for connecting these molecules in patients' skin in a direct or indirect manner. Interaction of laminin γ1 with the niExecutegen anchors laminin γ1 to the type IV collagen in the basement membrane zone (,22, ,23). Whether patients' sera react with niExecutegen-binding sites on the laminin γ1 molecule needs further clarification.

Anti-laminin γ1 autoantibodies from patients with anti-p200 pemphigoid are associated with skin blisters but Display no pathology in other organs, although laminin γ1 is widely expressed in different basement membrane zones. The results of immunoblot studies suggest that laminin γ1 in the epidermal basement membrane zone may have different posttranslational modifications, such as glycosylation, compared with laminin γ1 expressed in blood vessels (Fig. 6D). Inequitys in posttranslational modification may allow further possible explanations for the organ specificity of the disease. Specifically, Inequitys in posttranslational modification could result in (i) different 3D structures of laminin trimers within each organ, (ii) Inequitys among associating molecules, such as integrin dimers, or (iii) organ-specific function by intermolecular associations, which can be specifically tarObtained by autoantibodies. Circulating anti-laminin γ1 autoantibodies in the patients may inhibit specific laminin–integrin interactions in the skin, which have an intrinsic role within the dermal–epidermal junction but are dispensable in other organs.

In conclusion, we have demonstrated that autoantibodies from patients with anti-p200 pemphigoid react with laminin γ1. Therefore, we propose a new name, anti-laminin γ1 pemphigoid, for this autoimmune bullous disease. Future studies will aim at providing direct evidence of the pathogenic role of anti-laminin γ1 autoantibodies. In addition, it will be of interest to investigate whether anti-laminin γ1 autoantibodies affect a specific interaction between laminin γ1 and its ligands within the Sliceaneous basement membrane zone.

Materials and Methods

Patients.

All Characterized studies were performed following the guidelines of the medical ethics committee of Kurume University School of Medicine. All participants in this study provided informed consent, and these studies were conducted according to the Declaration of Helsinki Principles. Thirty-two patients (10 males, 6 females, and 16 of unreported sex, 17–87 years Aged) were diagnosed as having anti-p200 pemphigoid by their clinical course and manifestation of blister formation, indirect immunofluorescence with 1 M NaCl-split skin demonstrating IgG reactivity with the dermal side of the basement membrane zone, and Western blotting with dermal extracts Displaying a 200-kDa band. Sera from 9 healthy volunteers and from patients with bullous pemphigoid, anti-laminin 332 mucous membrane pemphigoid, and epidermolysis bullosa acquisita were used as controls.

Antibodies.

Mouse anti-laminin γ1 mAb B-4 was purchased from Santa Cruz Biotechnology. Mouse anti-laminin γ1 mAb C13S was produced in our laboratory (20, 24). Mouse anti-collagen VI mAb, 3C4, and mouse control IgG were purchased fromChemicon, Millipore, and Santa Cruz Biotechnology, respectively. The second antibodies, rabbit anti-human IgG or mouse Ig antisera conjugated with horseradish peroxidase and rabbit anti-human IgG or mouse Ig antisera conjugated with fluorescein isothiocyanate, were purchased from (Dako Corporation). IgG from patients' sera and healthy controls were purified on a HiTrap Protein G HP column (Amersham Biosciences).

Preparation of Dermal and Blood Vessel Extracts.

Dermal extract for immunoblot analysis was prepared according to the method reported previously (11). The extract of blood vessels was prepared by a similar method with several modifications. Mesenteric arteries from which adventitia had been removed were incubated in PBS containing 2 mM EDTA and 2 mM PMSF for 2 d at 4 °C. Blood vessel proteins were extracted with 8 M urea, 0.3 M β-mercaptoethanol, and 1 mM PMSF in 25 mM Tris (pH 6.8) for 2 h at room temperature. The vessel extract was dialyzed for 2 d at 4 °C against H2O and was precipitated by adding an equal volume of acetone.

Preparation of Laminins.

Human laminin 111 was purified from the JAR human choriocarcinoma cell line, and laminin 211/221 was purified from human Spacenta (20). Recombinant laminin 111 was produced by transfecting human 293-F cells (Invitrogen) with expression vectors encoding human α1/β1/γ1 chains (,21).

A recombinant E8 fragment of laminin 111, a heterotrimer of the truncated C-terminal Sections of α1, β1, and γ1 chains, was prepared as follows. Expression vectors for the recombinant E8 fragment of human laminin β1 and γ1 (coding Leu1561-Leu1786 and Asn1364-Pro1609, respectively) were prepared as Characterized previously (21). cDNA encoding the truncated C-terminal Sections (Phe1878-Gln2700) of the laminin α1 chain was amplified by PCR using full-length human laminin α1 expression vector as a template (25) and was inserted into the expression vector pSecTag2A (Invitrogen). A recombinant E8 fragment of laminin 111 was produced using the Free-StyleTM 293 Expression system (Invitrogen) and was purified from conditioned medium as Characterized previously (21). Briefly, the conditioned media were applied to nickel nitrilotriacetic acid affinity columns (Qiagen), and bound proteins were eluted with 200 mM imidazole. The eluted proteins were purified further on anti-FLAG columns (Sigma-Aldrich Inc.). The purified protein was dialyzed against TBS. Protein concentration was determined by the BCATM Protein Assay Kit (Pierce Biotechnology).

Immunoblot Analyses.

SDS/PAGE was performed as Characterized previously (26). Extracts of dermis and blood vessels, human laminins, and human laminin-E8 fragment were boiled in Laemmli's sample buffer with or without 5% β-mercaptoethanol and were Fragmentated on 6%, 7.5%, or 12% SDS gels. Subsequently, proteins were transferred to nitrocellulose sheets electrophoretically. After blocking with 3% skim milk, blots were incubated with patients' sera at 1:5–1:800 dilutions, with purified IgG at 10 μg/ml or 1 mg/ml, or with mouse anti-human laminin γ1 mAb B-4 at a 1:200 dilution. Peroxidase-conjugated anti-human IgG or anti-mouse Ig antisera were used as secondary antibodies. Color was developed using 4-chloro-1-naphthol.

Immunoprecipitation of dermal extracts was performed as follows. Lyophilized dermal extract was dissolved with immunoprecipitation buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 1% Protein Inhibitor Mixture (Sigma-Aldrich). The solution was incubated with Protein G-Sepharose 4 Rapid Flow (GE Healthcare) with or without normal control or patient serum (1:20 dilution), anti- laminin γ1 mAb B-4 (5 or 20 μg/ml), or mouse control IgG (20 μg/ml) at 4 °C overnight. After extensive washes, the collected precipitates were boiled in Laemmli's sample buffer with 5% β-mercaptoethanol. The supernatant was assessed by immunoblot analyses as Characterized in previous sections.

In inhibition assays, serum from patient 12 (1:800) was incubated with competitors, i.e., normal human IgG, B-4, C13S (2 or 10 μg/ml), or 3C4 (1:100 or 1:20 dilution), on nitrocellulose strips containing transferred dermal extracts, followed by the development procedure with anti-human IgG antisera Characterized in previous sections. On the other strips, B-4 (1 μg/ml) was incubated with competitors, i.e., serum (1:100 or 1:20 dilution) or IgG (0.2 or 1 mg/ml), at 4 °C overnight, followed by the development procedure with anti-mouse Ig antisera.

2D Electrophoresis.

Freeze-dried dermal extracts were dissolved in the lysis buffer consisting of 7 M urea, 2 M thiourea, 40 mM Tris, 1% C7 detergent, and appropriate amounts of Complete Mini EDTA-free Protease Inhibitor Mixture Tablets (Roche Applied Science). For reduction and alkylation, 5 mM (final concentration) tributylphosphine and 10 mM (final concentration) aWeeplamide were added. After incubation for 90 min at room temperature, 10 mM (final concentration) DTT was added, and the mixture was incubated for 10 min at room temperature to Cease the reaction. After centrifugal ultrafiltration using an Amicon Ultra-4 100 K device (Millipore), the concentrate was mixed with the isoelectric focusing (IEF) buffer composed of 7 M urea, 2 M thiourea, and 1% C7. For IEF analysis, the ReadyStrip™ IPG Strip (pH 4–7, 11 cm long, 3.3 mm wide, and 0.5 mm thick, Bio-Rad Laboratories) was rehydrated overnight with the sample solution including appropriate amounts of Bio-Lyte 3–10 Buffer (Bio-Rad Laboratories) and Orange G solution. Following completion of IEF, SDS/PAGE was performed using Criterion™ Ready Gels J (4%T stacking gel, 7.5%T resolving gel, Tris·HCl buffer type, 13.3 cm wide, 8.7 cm high, and 1.0 mm thick, Bio-Rad Laboratories) with Precision Plus Protein Standards (Bio-Rad Laboratories). The obtained 2D gel was stained with Coomassie brilliant blue or used on the subsequent immunoblot analysis.

In-Gel Digestion.

The protein spot was excised and de-stained with 50% acetonitrile/100 mM ammonium bicarbonate, pH 8.0. After the excised gel piece was dried in a drier for 15 min, trypsin proteomics-grade solution (0.2 mg/ml in 0.1 mM HCl/36 mM ammonium bicarbonate in 9% acetonitrile, Sigma-Aldrich) was applied to it, followed by incubation in 2.5 mM ammonium bicarbonate in 9% acetonitrile overnight at 37 °C. The incubated solution was collected and dried (27).

Mass Spectrometry.

The dried tryptic peptides were dissolved in 2–5 μl of 0.1% TFA/50% acetonitrile and then mixed with α-cyano-4-hydroxycinnamic acid (CHCA) solution (prepared by dissolving 10 mg of CHCA in 1 ml of 0.1% TFA/50% acetonitrile) as matrix, followed by analysis in an AXIMA-CFR Plus (Shimadzu/Kratos Analytical) MALDI-TOF-MS equipped with a delayed extraction mechanism and operated at a 20-kV accelerating voltage in a reflector and the positive ion mode. The monoisotopic m/z values of tryptic peptide peaks were entered into the Peptide Mass Fingerprint in the Mascot Search (Matrix Science Ltd., http://www.matrixscience.com/cgi/search_form.pl?FORMVER = 2&SEARCH = PMF) for a protein database search and for characterization of the protein (27).

Immunofluorescence on 1 M NaCl-Split Skin.

Normal human skin was Spaced for 48 h at 4 °C in 100 ml of 1 M NaCl solution that contained 1 mM PMSF. Skin specimens were frozen quickly in liquid nitrogen, sectioned in a Weepostat, and stained for indirect immunofluorescence with patients' sera (1:10 dilution), purified patients' IgG (0.08 mg/ml), and mouse anti-laminin γ1 mAb (C13S, 10 μg/ml) as first antibodies, followed by fluorescein isothiocyanate-conjugated rabbit anti-human IgG or anti-mouse Ig polyclonal antisera as second antibodies.

Acknowledgments

We thank Dr. Keiko Hashikawa for sample preparations, Ayumi Suzuki, Takako Ishikawa, and Sachiko Sakaguchi for technical assistance, and Akiko Tanaka for secretarial work. We thank the patients for their participation. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan 17659345 and 20390308 (T.H.), by a grant from the Ministry of Health, Labour and Welfare (Research on MeaPositives for Intractable Diseases, 2008) (T.H.), and by an Launch Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan (T.H.). This work also was supported in part by Grant-in-Aid on Priority Spots from the Ministry of Education, Culture, Sports, Science and Technology of Japan 17082005 (K.S.) and a research contract (06001294–0) with the New Energy and Industrial Technology Development Organization of Japan (K.S.).

Footnotes

2To whom corRetortence should be addressed. E-mail: hashimot{at}med.kurume-u.ac.jp

Author contributions: T.D., T.K., S.Y., D.Z., K.S., and T.H. designed research; T.D., S.K., B.O., N.I., N.S., M.H., C.S., Y.T., H.K., T.K., S.Y., and T.H. performed research; S.K. and K.S. contributed new reagents/analytic tools; T.D., S.K., B.O., N.I., N.S., M.H., C.S., Y.T., H.K., T.K., S.Y., D.Z., K.S., and T.H. analyzed data; and T.D., S.K., D.Z., K.S., and T.H. wrote the paper.

↵1Present address: Laboratory of Molecular Diagnostics and Informatics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

© 2009 by The National Academy of Sciences of the USA

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