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The Thomsen-Friedenreich Antigen-Binding Lectin Jacalin Interacts with Desmoglein-1 and Abrogates the Pathogenicity of Pemphigus Foliaceus Autoantibodies In Vivo

      Pemphigus foliaceus (PF) is an autoimmune skin blistering disease mediated by pathogenic autoantibodies against the desmosomal core glycoprotein desmoglein-1 (Dsg1). This study demonstrated that the O-glycan-specific plant lectin jacalin binds Dsg1 and inhibits the interaction of Dsg1/PF IgG. N-glycosylation is not involved in the interaction of Dsg1/jacalin or Dsg1/PF IgG. Subcutaneous injection of jacalin into neonatal mice drastically reduced PF IgG deposition at the epidermal cell surface and blocked PF IgG-induced skin blisters, both clinically and histologically. Interestingly, another plant lectin, peanut agglutinin, which shares the same carbohydrate specificity toward the O-linked carbohydrate structure known as Thomsen-Friedenreich antigen (TF antigen, Galβ1-3GalNAcα-O-Ser/Thr), also bound Dsg1 and blocked the skin blistering. In contrast, the plant lectin vicia villosa-B4 (VVL-B4), which shares the carbohydrate specificity toward the O-linked monosaccharide known as Thomsen-nouveau antigen (GalNAc-α1-O-Ser/Thr), did not bind Dsg1 and did not show a protective effect against the disease induced by the autoantibodies. Collectively, these results suggest that the binding of jacalin to O-linked TF carbohydrate motifs on Dsg1 impairs the Dsg1/PF autoantibody interactions and abrogates its pathogenicity in vivo. TF-specific binding ligands may have a potential therapeutic value for PF.

      Abbreviations

      Dsg
      desmoglein
      PF
      pemphigus foliaceus
      PNA
      peanut agglutinin
      s.c.
      subcutaneous
      TF antigen
      Thomsen-Friedenreich antigen (Galβ1-3GalNAcα-O-Ser/Thr)
      Tn antigen
      Thomsen-nouveau antigen (GalNAc-α1-O-Ser/Thr)
      VVL-B4
      vicia villosa-B4 lectin

      Introduction

      The endemic and nonendemic forms of pemphigus foliaceus (PF) are autoimmune blistering skin diseases characterized by superficial blisters, epidermal cell detachment (known as acantholysis) at the level of the granular layer of the epidermis, and IgG autoantibodies bound to the surface of detached keratinocytes (
      • Lever W.F.
      • Smith P.A.
      • Hurley N.A.
      Effects of intravenous heparin on the plasma lipoproteins in primary hypercholesteremic xanthomatosis and idiopathic hyperlipemia.
      ;
      • Beutner E.H.
      • Prigenzi L.S.
      • Hale W.
      • et al.
      Immunofluorescent studies of autoantibodies to intercellular areas of epithelia in Brazilian pemphigus foliaceus.
      ). The nonendemic form of PF occurs sporadically in many regions of the world and the endemic form, also known as Fogo Selvagem (FS), is common in certain rural areas of Brazil (
      • Aoki V.
      • Millikan R.C.
      • Rivitti E.A.
      • et al.
      Environmental risk factors in endemic pemphigus foliaceus (fogo selvagem).
      ).
      The antiepidermal autoantibodies in both clinical forms of PF are predominantly and in a pathogenic manner IgG4 subclass, as demonstrated by passive transfer of the IgG fraction of these sera into mice (
      • Rock B.
      • Martins C.R.
      • Theofilopoulos A.N.
      • et al.
      The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem).
      ;
      • Futei Y.
      • Amagai M.
      • Ishii K.
      • et al.
      Predominant IgG4 subclass in autoantibodies of pemphigus vulgaris and foliaceus.
      ). Neonatal mice that are injected with PF IgG, IgG4, or Fab' monovalent fragment develop skin blisters and subcorneal vesicles, which recapitulate the clinical and histological features of the human disease in these animals (
      • Roscoe J.T.
      • Diaz L.
      • Sampaio S.A.
      • et al.
      Brazilian pemphigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive transfer.
      ;
      • Rock B.
      • Martins C.R.
      • Theofilopoulos A.N.
      • et al.
      The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem).
      ,
      • Rock B.
      • Labib R.S.
      • Diaz L.A.
      Monovalent Fab' immunoglobulin fragments from endemic pemphigus foliaceus autoantibodies reproduce the human disease in neonatal Balb/c mice.
      ;
      • Espana A.
      • Diaz L.A.
      • Mascaro Jr, J.M.
      • et al.
      Mechanisms of acantholysis in pemphigus foliaceus.
      ). The antigen targeted by pathogenic PF autoantibodies is desmoglein-1 (Dsg1), a desmosomal core glycoprotein expressed predominantly in the upper layers of the epidermis (
      • Koulu L.
      • Kusumi A.
      • Steinberg M.S.
      • et al.
      Human autoantibodies against a desmosomal core protein in pemphigus foliaceus.
      ;
      • Rappersberger K.
      • Roos N.
      • Stanley J.R.
      Immunomorphologic and biochemical identification of the pemphigus foliaceous autoantigen within desmosomes.
      ). Affinity-purified anti-Dsg1 autoantibodies from PF sera are able to induce epidermal blisters and subcorneal acantholysis in mice passively transferred with these fractions (
      • Amagai M.
      • Hashimoto T.
      • Green K.J.
      • et al.
      Antigen-specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus.
      ;
      • Arteaga L.A.
      • Prisayanh P.S.
      • Warren S.J.
      • et al.
      A subset of pemphigus foliaceus patients exhibits pathogenic autoantibodies against both desmoglein-1 and desmoglein-3.
      ).
      In spite of the remarkable advances in understanding the molecular mechanisms of autoantibody-induced acantholysis in pemphigus (
      • Rubenstein D.S.
      • Diaz L.A.
      Pemphigus antibody induced phosphorylation of keratinocyte proteins.
      ;
      • Kitajima Y.
      • Aoyama Y.
      A perspective of pemphigus from bedside and laboratory-bench.
      ;
      • Sharma P.
      • Mao X.
      • Payne A.S.
      Beyond steric hindrance: the role of adhesion signaling pathways in the pathogenesis of pemphigus.
      ;
      • Culton D.A.
      • Qian Y.
      • Li N.
      • et al.
      Advances in pemphigus and its endemic pemphigus foliaceus (Fogo Selvagem) phenotype: a paradigm of human autoimmunity.
      ;
      • Waschke J.
      The desmosome and pemphigus.
      ), these knowledge has not been translated into newer therapies of patients. Current therapy of PF patients mainly relies on the use of corticosteroids and immunosuppressive agents. Although these therapies significantly reduce the mortality of the disease, they result in significant morbidity because of side effects.
      Identifying means of interfering with the autoantibody–antigen interaction may represent an alternative approach to treat autoantibody-mediated autoimmune diseases. While investigating a possible case of IgA pemphigus, we noticed that jacalin-agarose, which was used to precipitate IgA, also precipitated the baculovirus-expressed recombinant Dsg1 ectodomain (rDsg1). Further studies demonstrate that binding of jacalin interferes with PF autoantibody binding and protects mice against PF IgG-induced skin blisters.

      Results

      Jacalin binds Dsg1

      Aliquots of conditioned culture medium containing baculovirus-expressed human rDsg1 were incubated with agarose beads that were conjugated with jacalin or protein G/A (negative control). Following extensive wash (see Materials and Methods), bound proteins were released by boiling in SDS sample buffer and subjected to immunoblotting (IB). As shown in Figure 1a, jacalin-agarose (lane 1), but not protein G/A-agarose (lane 2), was able to pull-down Dsg1. To demonstrate the carbohydrate-dependency of the binding, we tested the ability of jacalin-specific sugars to elute the bound Dsg1. We found that both melibiose (0.1M) and D-galactose (0.8M) were able to elute the bound Dsg1 (lanes 3 and 4). Moreover, preincubation of jacalin-agarose with D-galactose diminished its binding to Dsg1 (lane 5) as compared with those preincubated with tris-buffered saline (TBS)-Ca2+ buffer alone (lane 6). These results demonstrate that jacalin is able to bind rDsg1 and that the binding is galactose-dependent.
      Figure thumbnail gr1
      Figure 1Jacalin (Jac) binds desmoglein-1 (Dsg1). (a) Binding to recombinant Dsg1 ectodomain (rDsg1). rDsg1 was incubated with Jac-agarose (lanes 1, 3–6) or protein G/A-agarose beads (lane 2). Bound-Dsg1 was released by boiling in SDS sample buffer (lanes 1–2 and 5–6) or eluted with Jac-inhibiting sugars (lane 3: 0.1M melibiose; lane 4: 0.8M D-galactose). In one set of experiments, Jac beads were preincubated with 0.8M D-galactose (lane 5) or buffer (lane 6) before adding the pemphigus foliaceus serum to further show the sugar-dependency binding. Released Dsg1 was analyzed by immunoblotting (IB) using anti-histidine antibodies. (b) Binding to epidermal Dsg1. Tissue lysates from human (H) or mouse (M) epidermis were incubated with Jac-agarose (lanes 1 and 2) or protein G/A-agarose beads (lanes 3 and 4) followed by IB using anti-Dsg1 antibodies.
      Considering possible differences in glycosylation of proteins expressed by insect cells and mammalian epidermal keratinocytes, we examined the interaction of jacalin with Dsg1 extracted from the skin. Tissue lysates from human epidermis and mouse skin were incubated with jacalin-agarose or protein G/A-agarose beads. Proteins bound by the beads were released and analyzed by IB using antibodies to Dsg1. As shown in Figure 1b, the 160-kDa Dsg1 from mouse or human epidermal extracts was precipitated by jacalin-beads (lanes 1 and 2), but not by protein G/A-beads (lanes 3 and 4).

      Jacalin blocks the binding of PF autoantibodies to Dsg1

      Finding that jacalin interacts with the ectodomain of Dsg1 prompted us to investigate whether such interaction could interfere with the binding of PF autoantibodies to Dsg1. We tested this possibility by ELISA and immunoprecipitation (IP) assays using a test FS serum (PF-1). ELISA results showed a concentration-dependent inhibition of jacalin on the binding of FS autoantibodies (Figure 2a), and that the inhibition was reversible by D-galactose (Figure 2b). A similar dose-dependent blocking effect on the binding of Dsg1 to FS autoantibodies was also observed by IP analysis (Figure 2c). To assess whether the jacalin inhibition on FS autoantibody binding is applicable to other FS/PF sera, we tested additional samples. Representative IP data are shown in Figure 2d. Among 20 PF/FS sera tested, jacalin significantly reduced Dsg1 binding in 18 samples (90%), and in 2 sera the reduction was modest (one of such sera, PF-2, is shown in lanes 15 and 16).
      Figure thumbnail gr2
      Figure 2Jacalin (Jac) inhibits pemphigus foliaceus (PF) autoantibody binding to desmoglein-1 (Dsg1). (a) ELISA assay showing dose-dependent inhibitory effect of Jac. Dsg1-coated microplates were preincubated with Jac (100, 250, and 500μgml−1) followed by incubating with PF-1 serum. Cutoff value: 0.421. (b) Reverse effects of D-galactose on Jac inhibition. Recombinant Dsg1 ectodomain (Dsg1)-microplates were preincubated with tris-buffered saline-Ca2+ buffer (left column), Jac (100μgml−1) (center), or Jac (100μgml−1) plus D-galactose (0.8M) (right) before adding PF-1 serum. (n=3, *P<0.05 (Student's t-test)). (c) Immunoprecipitation (IP) results showing dose-dependent inhibitory effect of Jac. rDsg1 was preincubated with Jac (0, 10, and 100μgml−1) before IP. (d) Representative IP showing that 100μgml−1 of Jac (even-numbered lanes) inhibits the interaction of rDsg1 with eight PF/Fogo Selvagem sera compared with the controls that were preincubated with buffer (odd-numbered lanes).

      Interaction of Dsg1/jacalin and Dsg1/PF autoantibodies is N-glycosylation-independent

      Although jacalin is a well-known O-glycan-specific plant lectin (
      • Hortin G.L.
      Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose.
      ;
      • Tachibana K.
      • Nakamura S.
      • Wang H.
      • et al.
      Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography.
      ), a few studies have suggested that jacalin may also have affinity for N-glycan (
      • Do S.I.
      • Lee K.Y.
      Jacalin interacts with Asn-linked glycopeptides containing multi-antennary oligosaccharide structure with terminal alpha-linked galactose.
      ;
      • Bourne Y.
      • Astoul C.H.
      • Zamboni V.
      • et al.
      Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose.
      ). To evaluate whether jacalin binds to N-glycans on Dsg1, we produced the Dsg1 in High-Five cells in the presence of tunicamycin to inhibit N-glycosylations. As expected, Dsg1 expressed in the presence of tunicamycin showed a reduced molecular weight on SDS–PAGE (Figure 3a, lane 1 vs. lane 2), an indication of N-glycosylation inhibition. We then used the same amount of tunicamycin-treated and nontreated Dsg1 for lectin pull-down assay. As expected, immobilized concanavalin A reacted poorly with tunicamycin-treated Dsg1 (Figure 3a, lanes 3 vs. 4), a further indication of the N-glycosylation inhibition. In contrast, immobilized jacalin was able to precipitate both the tunicamycin-treated and nontreated Dsg1 (Figure 3a, lanes 5 vs. 6). This result demonstrates that N-glycans on Dsg1 are not involved in jacalin binding.
      Figure thumbnail gr3
      Figure 3N-glycosylation is not involved in desmoglein-1 (Dsg1)/jacalin (Jac) and Dsg1/pemphigus foliaceus (PF) IgG binding. Baculovirus expressed Dsg1 ectodomain produced in the presence or absence of tunicamycin (0.5mgml−1) was used for the experiments. (a) Immunoblotting shows that tunicamycin treatment reduced the molecular weight of Dsg1 (untreated in lane 1 vs. treated in lane 2). Concanavalin A (ConA)-beads precipitated poorly with Dsg1 produced in the presence of tunicamycin (lanes 3) compared with nontreated Dsg1 (lane 4). In contrast, Jac-agarose precipitated both the tunicamycin-treated (lane 6) and nontreated Dsg1 (lane 5). (b) Representative immunoprecipitation shows that five PF/Fogo Selvagem sera reacted with tunicamycin-treated Dsg1 (even-numbered lanes) as well as nontreated Dsg1 (odd-numbered lanes).
      We also examined the binding ability of tunicamycin-treated Dsg1 with PF autoantibodies, as the role of N-glycosylation of Dsg1 in PF autoantibody binding has been controversial (
      • Olague-Alcala M.
      • Diaz L.A.
      The epitopes on bovine pemphigus foliaceus antigen are calcium-dependent and located on the peptide backbone of this glycoprotein.
      ;
      • Amagai M.
      • Ishii K.
      • Hashimoto T.
      • et al.
      Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.
      ;
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      ). We tested 12 PF/FS sera (6 PF and 6 FS) by IP, and all reacted well with both tunicamycin-treated and nontreated Dsg1. Representative IP data are shown in Figure 3b.

      Jacalin binds the epidermis and inhibits PF autoantibodies binding

      We next examined the binding of jacalin to cryostat sections of human and murine skin by immunofluorescence techniques. Sections of human or mouse skin incubated with FITC-conjugated jacalin produced a “pemphigus-like” staining pattern (Supplementary Figure S1-A online). Preincubation of the human or mouse skin sections with jacalin significantly inhibited PF IgG binding to the epidermis (Supplementary Figure S1-B, panels b and d online) as compared with those preincubated with TBS-Ca2+ buffer alone (Supplementary Figure S1-B, panels a and c online).

      Jacalin abrogates the pathogenicity of PF autoantibodies in mice

      We further investigated the in vivo effect of jacalin in the PF mouse model using pathogenic IgG isolated from PF-1, a serum that was significantly inhibited by jacalin (see Figure 2a–c). Control mice (n=4) pretreated with vehicle alone developed blisters, clinically and histologically (Figure 4aA and B) 20hours after PF IgG injections. In contrast, mice pretreated with jacalin (80μgg−1 body weight, n=4; 160μgg−1 body weight, n=4) did not develop skin lesions (Figure 4aD and E). Direct immunofluorescence staining revealed a significant reduction of IgG binding to the epiderms in jacalin-treated mice (Figure 4aF) compared with those of control mice (Figure 4aC). The inhibition of jacalin on the induction of skin lesions in mice was dose dependent (Figure 4b). These results suggest that jacalin affects the in vivo binding of PF autoantibodies to the epidermis and thus inhibits disease.
      Figure thumbnail gr4
      Figure 4Jacalin (Jac) protects mice from developing pemphigus foliaceus (PF). (a) Representative results showing that mice preinjected with control buffer (n=4) developed clinical and histological blisters upon injection with IgG from PF-1 (A, B). In contrast, mice pretreated with Jac (80μgg−1 body weight, n=4; 160μgg−1 body weight, n=4) did not develop skin blisters clinically and histologically (D, E). Direct immunofluorescence staining showed a significant reduction of IgG binding to the epidermis in mice treated with Jac (F) compared with buffer-treated controls (C). Bars=100μm (B, E) and 10μm (C, F), respectively. (b) Jac inhibits PF blistering in a dose-dependent manner. Animals were pretreated (s.c.) with different doses of Jac and then injected (s.c.) with the same dose of pathogenic PF IgG. Clinical disease was scored for each group of experimental mice. (n=4; *P<0.05, **P<0.01 (Student's t-test).)

      Study of other plant lectins

      Jacalin is known to have binding specificities for the O-linked disaccharide Thomsen-Friedenreich antigen (TF antigen, Galβ1-3GalNAc-α1-O-Ser/Thr) and the monosaccharide Thomsen-nouveau antigen (Tn antigen, GalNAc-α1-O-Ser/Thr) in either the asialylated or sialylated form. It is known that other lectins have overlapping and distinct binding specificities as jacalin. For example, the peanut agglutinin (PNA) has binding affinity for TF antigen in its asialylated form only, whereas vicia villosa-B4 (VVL-B4) binds specifically to Tn antigen. Therefore, we tested the reactivity of these two lectins with Dsg1 by the lectin pull-down assay. The results showed that PNA, but not VVL-B4, interacted with the epidermal Dsg1 extracted from human or mouse skin (Figure 5a), suggesting that Dsg1 carries the asialylated form of TF antigen but not the Tn antigen. Moreover, preinjection of PNA (80μgg−1 body weight, n=9) abrogated the pathogenicity of PF IgG, whereas preinjection of the same dose of VVL-B4 (n=3) did not show any blocking effect (Figure 5b). Similar to jacalin, subcutaneous (s.c.) injection of PNA or VVL-B4 alone did not cause any abnormal appearance of the mouse skin. Table 1 summarizes the binding specificity of the lectins tested and their effect on PF IgG-induced skin blisters in mice.
      Figure thumbnail gr5
      Figure 5Peanut agglutinin (PNA), but not vicia villosa-B4 (VVL-B4), binds to Dsg1 and abolishes the pathogenicity of PF autoantibodies. (a) Lectin pull-down assay. Tissue lysates from human epidermis (lanes 1 and 2) or mouse skin (lanes 3 and 4) were incubated with PNA-agarose (lanes 1 and 3) or VVL-B4-agarose beads (lanes 2 and 4). Dsg1 was detected by immunoblotting using anti-Dsg1 antibodies. PNA but not VVL-B4 was able to pull-down the epidermal Dsg1. (b) IgG passive transfer. Neonatal mice pretreated with PNA (160μgg−1 body weight, n=9) or VVL-B4 (160μgg−1 body weight, n=3), followed by subcutaneous injection of pathogenic IgG from PF-1. Injected mice were examined 20hours after IgG injection. PNA (A and B), but not VVL-B4 (C and D), inhibits PF IgG-induced skin blisters in mice. Bar=50μm.
      Table 1In vivo effect of plant lectins in IgG passive transfer mouse model of PF
      IgGTreatmentOligosaccharide specificityNo. of mice testedNo. of mice with skin blisterNo. of mice without skin blister
      PF-1Vehicle15150
      JacalinGalβ1-3GalNAc-α (asialo or sialo); GalNAc808
      PNAGalβ1-3GalNAc-α or –β (asialo)91
      Minimum disease score (0.5+).
      8
      VVL-B4GalNAc330
      PF-2Vehicle660
      Jacalin65
      Overall lower disease score (1.25+0.36) compared with those preinjected with vehicle (2.58+0.20) (P<0.01, Student's t-test).
      1
      Abbreviations: Gal, galactose; GalNAc, N-acetylglactosamine; Glc, glucose; PF, pemphigus foliaceus; PNA, peanut agglutinin; s.c., subcutaneous; TBS, tris-buffered saline; VVL-B4, vicia villosa-B4 lectin.
      Neonatal mice were preinjected (s.c.) with vehicle (TBS-Ca2+) or lectins for 2hours followed by pathogenic PF IgG injection (s.c.). Pathogenic IgG from two patients' sera was used. PF-1 serum showed a remarkable inhibition in Dsg1 binding by jacalin, whereas PF-2 showed a marginal reduction in Dsg1 binding by jacalin. Animals were evaluated 20hours after PF IgG injection. Jacalin: 80–160μgg−1 body weight; PNA and VVL-B4: 160μgg−1 body weight.
      1 Minimum disease score (0.5+).
      2 Overall lower disease score (1.25+0.36) compared with those preinjected with vehicle (2.58+0.20) (P<0.01, Student's t-test).

      Effect of jacalin on the pathogenicity of an additional PF serum that showed a modest reduction in Dsg1 binding by jacalin

      As described above, two patients' sera, including PF-2, showed a modest reduction on the binding of Dsg1 by IP when preincubated with jacalin. We expected that jacalin would have a lesser inhibitory effect on the pathogenicity of these PF autoantibodies. We tested this hypothesis in mice using pathogenic IgG prepared from PF-2 serum. Out of six animals preinjected with jacalin, five developed skin blisters, but the disease score was significantly reduced (P<0.01, Student's t-test) compared with those pretreated with vehicle (Table 1).

      Discussion

      Jacalin is a plant lectin that is highly specific for the α-O-glycoside of the terminal disaccharide TF carbohydrate motif (asialylated or sialylated) and its monosaccharide precursor Tn antigen (
      • Mahanta S.K.
      • Sastry M.V.
      • Surolia A.
      Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study.
      ;
      • Kabir S.
      Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research.
      ;
      • Tachibana K.
      • Nakamura S.
      • Wang H.
      • et al.
      Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography.
      ). Immobilized jacalin is often used for isolation of IgA1 from human serum and purifying other O-glycosylated proteins (
      • Roque-Barreira M.C.
      • Campos-Neto A.
      Jacalin: an IgA-binding lectin.
      ;
      • Hortin G.L.
      Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose.
      ). In this study, we found that jacalin-agarose beads precipitated the rDsg1 (Figure 1a) as well as the native Dsg1 from human and mouse epidermal lysates (Figure 1b). We demonstrated that the binding between jacalin and Dsg1 is galactose-dependent ((Figure 1a), but N-glycosylation-independent (Figure 3a), supporting the notion that jacalin has no or extremely low affinity for N-glycans (
      • Hortin G.L.
      • Trimpe B.L.
      Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.
      ;
      • Arockia Jeyaprakash A.
      • Jayashree G.
      • Mahanta S.K.
      • et al.
      Structural basis for the energetics of jacalin-sugar interactions: promiscuity versus specificity.
      ). Although Dsg1 is known to be N-glycosylated (
      • Koch P.J.
      • Goldschmidt M.D.
      • Walsh M.J.
      • et al.
      Complete amino acid sequence of the epidermal desmoglein precursor polypeptide and identification of a second type of desmoglein gene.
      ;
      • Olague-Alcala M.
      • Diaz L.A.
      The epitopes on bovine pemphigus foliaceus antigen are calcium-dependent and located on the peptide backbone of this glycoprotein.
      ;
      • Amagai M.
      • Ishii K.
      • Hashimoto T.
      • et al.
      Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.
      ;
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      ), its possible modification by O-linked oligosaccharides is less defined. The observation that Dsg1 interacts with jacalin and PNA but not VVL-B4 (Figure 5a) indicates that Dsg1 carries O-linked disaccharide TF antigen (asialylated) but not the monosaccharide Tn antigen.
      By competition ELISA and IP, we showed that jacalin was able to inhibit the binding of PF autoantibodies to Dsg1 in concentration- and galactose-dependent manners (Figure 2a–c). A significant inhibitory effect was observed in the majority of patients' sera tested (90%, n=20, Figure 2d) including PF-1, which was used throughout the study. Jacalin also markedly inhibited the binding of PF autoantibodies to human and mouse epidermis (Supplementary Figure S1 online). Furthermore, preinjection (s.c.) of jacalin into mice diminished the in vivo binding of PF-1 autoantibody to the epidermis and blocked skin disease (Figure 4). Interestingly, injection (s.c.) of jacalin alone did not cause any abnormal effect on mouse skin (data not shown), suggesting that binding of jacalin does not impair the adhesive function of Dsg1. Another lectin PNA, which shares the same carbohydrate specificity for the O-linked asialylated TF antigen but not the sialylated form, interacted with Dsg1 and blocked PF-1 autoantibody-induced skin blisters in mice (Figure 5). In contrast, lectin VVL-B4, which shares the carbohydrate specificity for the O-linked Tn antigen, did not interact with Dsg1 and did not show any beneficial effect on the disease induced by PF-1 IgG (Figure 5). These data suggest that binding of the O-linked TF carbohydrate motif of Dsg1 by jacalin and PNA can abrogate the pathogenicity of PF autoantibodies. However, there are a few PF sera (2 out of 20), in which the Dsg1–PF autoantibody interaction was modestly inhibited by jacalin, and the in vivo protective effect of jacalin on disease was less effective as demonstrated with PF-2 serum (Table 1).
      The molecular mechanism involved in the inhibitory effects of jacalin on PF autoantibody binding to Dsg1 is not clear. It is known that pathogenic PF autoantibodies bind calcium-dependent conformational epitopes located in the NH2-terminal region (EC1-2 or 1-161 residues) of Dsg1 (
      • Eyre R.W.
      • Stanley J.R.
      Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.
      ;
      • Kowalczyk A.P.
      • Anderson J.E.
      • Borgwardt J.E.
      • et al.
      Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1.
      ;
      • Sekiguchi M.
      • Futei Y.
      • Fujii Y.
      • et al.
      Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.
      ;
      • Li N.
      • Aoki V.
      • Hans-Filho G.
      • et al.
      The role of intramolecular epitope spreading in the pathogenesis of endemic pemphigus foliaceus (fogo selvagem).
      ). These epitopes are unlikely to be composed of the carbohydrate moieties as the bacterial-expressed Dsg1 ectodomain, which had no glycosylation modifications, was still recognized by PF sera (
      • Dmochowski M.
      • Hashimoto T.
      • Amagai M.
      • et al.
      The extracellular aminoterminal domain of bovine desmoglein 1 (Dsg1) is recognized only by certain pemphigus foliaceus sera, whereas its intracellular domain is recognized by both pemphigus vulgaris and pemphigus foliaceus sera.
      ). It is possible that jacalin binds the TF carbohydrate motifs of Dsg1 and spatially masks the major epitopes recognized by PF autoantibodies, thereby inhibiting the subsequent binding of PF autoantibodies. Alternatively, the binding of jacalin to Dsg1 may induce conformational changes of Dsg1, which impairs its interaction with PF autoantibodies. Identification of the position of the O-glycosylation sites on Dsg1 may help to understand the mechanism of action of jacalin. Potential O-glycosylation sites are predicted to be located in the membrane-proximal EC5 domain and possibly in other domains (EC1-4), depending on which predicting programs/methods are used (http://www.turing.cs.iastate.edu/EnsembleGly; http://www.cbs.dtu.dk/services/netoglyc; http://www.comp.chem.nottingham.ac.uk/glyco). Further experimental determination of the O-glycosylation sites on Dsg1 may shed light into the inhibitory mechanism.
      Two putative N-glycosylation sites (residues 61 and 131) are identified within the NH2-terminal region of Dsg1, where PF autoantibodies bind. The influence of the N-lined glycosylation on PF autoantibody binding has been examined with conflicting results reported. Although earlier studies showed that the binding of PF autoantibodies to Dsg1 is N-glycosylation-independent (
      • Olague-Alcala M.
      • Diaz L.A.
      The epitopes on bovine pemphigus foliaceus antigen are calcium-dependent and located on the peptide backbone of this glycoprotein.
      ;
      • Amagai M.
      • Ishii K.
      • Hashimoto T.
      • et al.
      Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.
      ), a recent study by
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      suggested that most PF epitopes are determined by N-linked carbohydrates.
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      further showed that topically applied lectin wheat germ agglutinin, which binds the N-linked carbohydrates, was able to block most PF autoantibody binding to the epidermis and abrogated their pathogenicity in mice (
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      ).
      In contrast to the results by
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      , our IP data show that inhibition of N-glycosylation of Dsg1 by tunicamycin does not attenuate their binding to PF/FS sera (n=12) (Figure 3b). Our results support the notion that binding of PF IgG to Dsg1 is N-glycosylation-independent (
      • Olague-Alcala M.
      • Diaz L.A.
      The epitopes on bovine pemphigus foliaceus antigen are calcium-dependent and located on the peptide backbone of this glycoprotein.
      ;
      • Amagai M.
      • Ishii K.
      • Hashimoto T.
      • et al.
      Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.
      ). It should be noted that the conclusion of N-glycosylation-dependent PF autoantibody binding was mainly based on the observation that, by IB, the majority of PF sera (7 out of 12 sera tested) failed to react with human epidermal Dsg1 that has been digested with PNGase F (
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      ). However, it is known that IB analysis is not an optimal technique to reveal the binding of PF autoantibodies to Dsg1 because more than half of PF sera failed to react with Dsg1 by this technique, presumably due to the loss of the conformational epitopes under the denatured conditions inherent to IB (
      • Eyre R.W.
      • Stanley J.R.
      Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.
      ;
      • Hashimoto T.
      • Ogawa M.M.
      • Konohana A.
      • et al.
      Detection of pemphigus vulgaris and pemphigus foliaceus antigens by immunoblot analysis using different antigen sources.
      ;
      • Kowalczyk A.P.
      • Anderson J.E.
      • Borgwardt J.E.
      • et al.
      Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1.
      ;
      • Amagai M.
      • Hashimoto T.
      • Green K.J.
      • et al.
      Antigen-specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus.
      ). It is probable that the failure of recognizing Dsg1 by most of the PF sera in the study by
      • Ortiz-Urda S.
      • Elbe-Burger A.
      • Smolle J.
      • et al.
      The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.
      was due to the limited sensitivity of the IB analysis rather than the removal of N-linked carbohydrates. Furthermore, we could not confirm that wheat germ agglutinin was capable of blocking PF IgG-induced skin blistering in mice. Instead of a beneficial effect, we found that wheat germ agglutinin itself induces intraepidermal cell detachment (acantholysis-like) and skin blisters upon injection into mice (unpublished observation). The reason for this conflicting observation is not clear and requires further investigation.
      The TF disaccharide (α-anomer, asialylated), recognized both by jacalin and PNA, is a tumor-associated carbohydrate structure. This glycotope occurs on the cell surface of a few normal epithelial cells, but is overexpressed on many types of carcinomas as a result of incomplete O-glycosylation of cell membrane proteins (
      • Springer G.F.
      T and Tn, general carcinoma autoantigens.
      ;
      • Cao Y.
      • Stosiek P.
      • Springer G.F.
      • et al.
      Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study.
      ). Its cryptic form (sialylated TF), which interacts with jacalin but not PNA, is expressed on many normal tissues/organs including blood cells (
      • Cao Y.
      • Stosiek P.
      • Springer G.F.
      • et al.
      Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study.
      ;
      • Kabir S.
      Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research.
      ). The β-anomer of TF (Galβ1-3GalNAc-β), found in the termini of certain glycolipids such as GM1 ganglioside and asialo-GM1 ganglioside (
      • Rittenhouse-Olson K.
      Jaa-f11: extending the life of mice with breast cancer.
      ), may interact with PNA but not jacalin. Although jacalin and PNA are dietary lectins that can be eaten raw, and local administration of the two lectins into neonatal mice did not induce any obvious toxicity within 24hours duration, it is possible that systemic administration of these lectins, especially jacalin, may be toxic/harmful because of its agglutinating properties of erythrocytes and its mitogenicity for human CD4+ T cells (
      • Cao Y.
      • Stosiek P.
      • Springer G.F.
      • et al.
      Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study.
      ;
      • Kabir S.
      Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research.
      ). Our findings, however, may lead to a new therapeutic strategy for PF, that is, targeting the TF glycotope with other more specific TF-binding ligands, such as anti-TF antibodies (
      • Heimburg J.
      • Yan J.
      • Morey S.
      • et al.
      Inhibition of spontaneous breast cancer metastasis by anti-Thomsen-Friedenreich antigen monoclonal antibody JAA-F11.
      ) or peptides (
      • Landon L.A.
      • Zou J.
      • Deutscher S.L.
      Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides.
      ). These agents react only with the asialylated α-anomer TF disaccharide, but not with its more common cryptic form (sialylated TF) that are expressed on cell surface of many normal cells including erythrocytes. Further studies are required to test the potential therapeutic value of the TF-specific agents for PF.

      Materials and Methods

      Patient sera

      Sera from 20 PF/FS patients (11 PF sera from the United States and 9 FS sera from Brazil) were included in this study. A well-characterized FS serum (PF-1), which was available in a relatively large quantity, was used throughout this study. A second PF serum (PF-2) that showed a modest reduction in Dsg1 binding by jacalin was also used for IgG passive transfer experiments (Table 1). The diagnosis of PF/FS had previously been confirmed using established clinical, histological, and serological criteria. This study was approved by the Institutional Review Board at UNC.

      Plant lectins

      Jacalin (Artocarpus integrifolia agglutinin), PNA, VVL-B4, concanavalin A, PNA-agarose, and VVL-B4-agarose were purchased from Sigma (St Louis, MO). Agarose-bound jacalin was from ICN Pharmaceuticals (Irvine, CA). Concanavalin A-sepharose was from Amersham Phamacia Biotech (Piscataway, NJ). FITC-conjugated jacalin was purchased from Vector Laboratories (Burlingame, CA).

      Production of rDsg1

      The ectodomain of human Dsg1 with a carboxyl-terminal histidine tag was produced in a baculovirus expression system as described previously (
      • Li N.
      • Aoki V.
      • Hans-Filho G.
      • et al.
      The role of intramolecular epitope spreading in the pathogenesis of endemic pemphigus foliaceus (fogo selvagem).
      ). To produce Dsg1 without N-glycosylation, we expressed Dsg1 in High-Five cells in the presence of tunicamycin (0.5μgml−1) as described by
      • Amagai M.
      • Ishii K.
      • Hashimoto T.
      • et al.
      Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.
      . For ELISA, Dsg1 was purified by nickel affinity chromatography as described previously (
      • Ding X.
      • Diaz L.A.
      • Fairley J.A.
      • et al.
      The anti-desmoglein 1 autoantibodies in pemphigus vulgaris sera are pathogenic.
      ).

      Lectin pull-down assay

      Conditioned culture medium containing rDsg1 or skin/epidermal lysates were incubated with immobilized lectins for 1.5hours at 4°C. Negative control experiments were carried out using agarose beads conjugated with protein G/A. After centrifugation, the beads were washed three times for 10minutes, each with either of the following buffers: (a) 0.1% Triton X-100 in TBS-Ca2+ (10mM Tris-HCl, pH 7.4, 140mM NaCl, and 5mM CaCl2), (b) RIPA buffer (1% NP40, 0.25% sodium deoxycholate, 0.1% SDS, 50mM Tris-HCl, pH 7.4, and 150mM NaCl), or (c) high ionic buffer (100mM Tris-HCl, pH 7.4, and 500mM NaCl). Proteins bound to the beads were released by boiling in 2 × SDS sample buffer or eluted with 0.8M D-galactose or 0.1M melibiose. The eluates were fractioned by SDS–PAGE and then subjected to IB using anti-histidine or anti-Dsg1 antibodies (
      • Emery D.J.
      • Diaz L.A.
      • Fairley J.A.
      • et al.
      Pemphigus foliaceus and pemphigus vulgaris autoantibodies react with the extracellular domain of desmoglein-1.
      ).

      ELISA

      Dsg1-ELISA was performed as previously reported (
      • Warren S.J.
      • Lin M.S.
      • Giudice G.J.
      • et al.
      The prevalence of antibodies against desmoglein 1 in endemic pemphigus foliaceus in Brazil. Cooperative Group on Fogo Selvagem Research.
      ) with the following modifications. Various amounts of jacalin in TBS-Ca2+ buffer containing 0.2% BSA and 0.05% Tween 20 were added in duplicate to the wells. In some experiments, jacalin was preincubated with 0.8M D-galactose before adding to the plates. Following a 1-hour incubation at room temperature, the plate was washed five times with TBS-Ca2+ buffer (pH 7.2) containing 0.05% Tween 20. The plate was then subjected to ELISA assay using PF serum samples (1:200).

      IP

      Cold IP was performed as previously described (
      • Li N.
      • Aoki V.
      • Hans-Filho G.
      • et al.
      The role of intramolecular epitope spreading in the pathogenesis of endemic pemphigus foliaceus (fogo selvagem).
      ). In brief, aliquots of conditioned culture supernatant containing the histidine-tagged rDsg1 were preincubated with buffer or jacalin and then incubated with PF serum (1:200). The mixture was then subjected to IP, followed by IB using horseradish peroxidase-conjugated anti-histidine antibodies.

      Induction of experimental PF and testing the effect of plant lectins

      Neonatal BALB/c mice (1–2 days old) were used for experimental PF induction (
      • Anhalt G.J.
      • Labib R.S.
      • Voorhees J.J.
      • et al.
      Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.
      ;
      • Rock B.
      • Martins C.R.
      • Theofilopoulos A.N.
      • et al.
      The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem).
      ). Animal care/experiments were approved by the Animal Care Committee at the institute and were in accordance with NIH guidelines. To test the effect of lectins, animals were s.c. injected with lectins or vehicle (TBS-Ca2+ buffer); twohours later, animals were injected (s.c.) with pathogenic PF IgG (0.4mgg−1 body weight). Twenty hours after IgG injection, the extent of skin disease was evaluated and scored on a scale of 0 to 3+ as described previously (
      • Li N.
      • Zhao M.
      • Hilario-Vargas J.
      • et al.
      Complete FcRn dependence for intravenous Ig therapy in autoimmune skin blistering diseases.
      ).

      Statistical analysis

      Data were presented as mean±SD. The Student's t-test (unpaired, two-tailed) was used to evaluate for statistical significance. P<0.05 was considered significant.

      ACKNOWLEDGMENTS

      This work was supported in part by the US Public Health Service National Institutes of Health grants AR053313, AR052109 awarded to N Li, AI40768, AI61430 to Z Liu, and AR30281, AR32599 awarded to LA Diaz.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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