The Thomsen-Friedenreich antigen-binding lectin jacalin interacts with desmoglein-1 and abrogates the pathogenicity of pemphigus foliaceus autoantibodies in vivo

Abstract

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.

Figure 1. Jacalin binds Dsg1

(A) Binding to rDsg1. rDsg1 was incubated with jacalin-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 jacalin-inhibiting sugars (lane 3: 0.1 M melibiose; lane 4: 0.8 M D-galactose). In one set of experiment, jacalin beads were preincubated with 0.8 M D-galactose (lane 5) or buffer (lane 6) before adding the PF serum to further show the sugar-dependency binding. Released Dsg1 was analyzed by IB using anti-His antibodies. (B) Binding to epidermal Dsg1. Tissue lysates from human (H) or mouse (M) epidermis were incubated with jacalin-agarose (lanes 1 and 2) or protein G/A-agarose beads (lanes 3 and 4) followed by IB using anti-Dsg1 antibodies.

Figure 2. Jacalin inhibits PF autoantibody binding to Dsg1

(A) ELISA assay showing dose-dependent inhibitory effect of jacalin. Dsg1-coated microplates were preincubated with jacalin (100, 250, and 500 μg/ml) followed by incubating with PF-1 serum. Cut-off value: 0.421. (B) Reverse effects of D-galactose on jacalin inhibition. Dsg1-microplates were preincubated with TBS-Ca²⁺ buffer (left column), jacalin (100 μg/ml) (center), or jacalin (100 μg/ml) plus D-galactose (0.8 M) (right) before adding PF-1 serum. [n=3, *p<0.05 (Student's t-test)]. (C) IP results showing dose-dependent inhibitory effect of jacalin. rDsg1 was preincubated with jacalin (0, 10 and 100 μg/ml) before IP. (D) Representative IP showing that 100 μg/ml of jacalin (even-numbered lanes) inhibits the interaction of rDsg1 with 8 PF/FS sera compared with the controls that were preincubated with buffer (odd-numbered lanes).

Figure 3. N-glycosylation is not involved in Dsg1/jacalin and Dsg1/PF IgG binding

Baculovirus expressed Dsg1 ectodomain produced in the presence or absence of tunicamycin (0.5 mg/ml) was used for the experiments. (A) IB shows that tunicamycin treatment reduced the molecular weight of Dsg1 (untreated in lane 1 vs treated in lane 2). ConA-beads precipitated poorly with Dsg1 produced in the presence of tunicamycin (lanes 3) compared with nontreated-Dsg1 (lane 4). In contrast, jacalin-agarose precipitated both the tunicamycin-treated (lane 6) and nontreated-Dsg1 (lane 5). (B) Representative IP shows that 5 PF/FS sera reacted with tunicamycin-treated Dsg1 (even-numbered lanes) as well as nontreated-Dsg1 (odd-numbered lanes).

Figure 4. Jacalin protects mice from developing 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 jacalin (80 ug/g b. w., n=4; 160 ug/g b. w., n=4) did not develop skin blisters clinically and histologically (d, e). Direct IF staining showed a significant reduction of IgG binding to the epidermis in mice treated with jacalin (f) compared to buffer-treated controls (c). Scales bars = 100 μm (b, e) and 10 μm (c, f), respectively. (B) Jacalin inhibits PF blistering in a dose-dependent fashion. Animals were pretreated (s.c) with different doses of jacalin 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)].

Figure 5. PNA, but not 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 IB using anti-Dsg1 antibodies. PNA but not VVL-B4 pull-down the epidermal Dsg1. (B) IgG passive transfer. Neonatal mice pretreated with PNA (160 μg/g b.w. n=9) or VVL-B4 (160 μg/g b.w., n=3), followed by s.c. injection of pathogenic IgG from PF-1. Injected mice were examined 20 h post IgG injection. PNA (panels a and b), but not VVL-B4 (panels c and d), inhibits PF IgG-induced skin blisters in mice. Scale bar = 50 μm