Induction of Pemphigus Phenotype by a Mouse Monoclonal Antibody Against the Amino-Terminal Adhesive Interface of Desmoglein 3 (1)
Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease that is caused by IgG autoantibodies against the cadherin-type adhesion molecule desmoglein (Dsg)3. The titers of serum anti-Dsg3 IgG autoantibodies are not absolute indicators for the severity of the disease among groups of patients, and it is sometimes the case that patients with low titers of anti-Dsg3 IgG autoantibodies show severe phenotypes, while patients with high titers show mild phenotypes.
The hybridomas were screened by ELISA using mDsg3; positive clones were further screened by live staining of mouse keratinocyte PAM212 cells. The second screening selected mAbs that could bind to the native Dsg3 on keratinocyte cell surfaces in vivo. Eight independent clones were isolated and designated as AK mAbs. Some mAbs (AK1, 15, 18, 19, 20, and 23) cross-reacted with human skin or mucosa. All of the mAbs reacted exclusively, as assessed by ELISA, with the recombinant extracellular domain of the mDsg3, except AK1, which cross-reacted with mDsg1. The mAbs that had detectable cross-reactivity by immunofluorescence with human tissues also reacted with hDsg3 in the ELISA.
Judging from the number of mice with gross blisters, AK23 appeared to be more potent than AK19. the AK19 and AK23 mAbs cause the loss of cell-cell adhesion of keratinocytes in neonatal mice, while the other AK mAbs do not have apparent blister-inducing activities. Hybridoma cells were inoculated into Rag2−/− immunodeficient mice, and evaluated the appearance of the PV phenotype, which was manifested by weight loss, patchy hair loss, and mucosal erosions.
Mice that received AK23 hybridoma cells showed patchy hair loss and sudden mortality between days 7 and 9, which preceded ascites formation. Skin biopsies around the area of patchy hair loss showed intense IgG depositions on the cell surfaces of keratinocytes that surrounded the telogen hair club. The development of oral erosions probably inhibited food and water intake, which led to dehydration and eventual death. AK1, 7, 9, 15, 18, 19, and 20 are not sufficiently potent to induce the loss of cell-cell adhesion of keratinocytes in adult mice that have ascites formation.
When a mDsg3-coated ELISA plate was treated with EDTA, the binding of AK19 and AK23 was also abolished, while the binding of the other AK mAbs was not affected significantly. Therefore, the AK19 and AK23 mAbs recognize calcium-dependent epitopes, while the other AK mAbs recognize calcium-independent epitopes. Recombinant Dsg3-His is produced by baculoviruses as a doublet protein, in which the lower and upper bands are the mature (cleaved prosequence) and immature (uncleaved prosequence) forms, respectively. Only the lower band was immunoprecipitated by AK23, suggesting that AK23 mAbs, but not the other AK mAbs, bind preferentially to an epitope that only becomes available after cleavage of the prosequence.
The AK7, AK9, and AK20 mAbs precipitated residues 195–565 and 403–565, but not residues 1–162 and 1–402. Therefore, the epitopes of AK7, AK9, and AK20 appear to reside in residues 403–565 of the mDsg3, which represents the C-terminal portion of the extracellular domain. The AK15 and AK18 mAbs reacted with residues 1–402 and 195–565, but not with residues 1–162 and 403–565. Therefore, the epitopes of AK15 and AK18 appear to be present in residues 195–402 of the mDsg3, which represents the middle portion of the extracellular domain. The AK19 and AK23 mAbs recognized residues 1–162 and 1–402, but not residues 195–565 and 403–565. Only 22 aa residues of amino-terminal residues 1–87 were not conserved between the hDsg3 and hDsg1 proteins. The introduction of V3, K7, and P8 from Dsg3 onto the Dsg1 backbone (hDsg1-M7) was not sufficient to restore AK23 binding, which suggests that additional Dsg3-specific residues are necessary to form the AK23 epitope. When D59 was introduced into hDsg1-M7 (hDsg1-M7–8), hDsg1-M7–8 showed strong reactivity with AK23. The substitution of D59 with E59 (hDsg3-M8) did not abolish reactivity with AK23. Thus, V3, K7, and P8 are essential residues for AK23 binding, and the combination of V3, K7, P8, and D59 is sufficient to generate the AK23 epitope on the Dsg1 backbone.
When the amino acid sequences of Dsg3 were superimposed on the predicted structure of C-cadherin, the predicted residues for the adhesive interface of the W2 donor side were E1 to P8, P20, K23, T25, S26, D27, and D59.