Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus

Abstract

Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the selfdesigned cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment. Autoantibodies in pemphigus vulgaris (PV)4 are mainly directed against the adhesion molecules desmoglein (Dsg) 3 and Dsg1 (1, 2). Recently, we provided direct evidence that PV autoantibodies directly block Dsg3 transinteraction (3). In contrast, Dsg1 autoantibodies in pemphigus foliaceus (PF) were found to disrupt Dsg1 transinteraction most likely via cellular signaling events rather than by direct inhibition (3, 4). However, it still remained unanswered whether direct inhibition of Dsg3 transinteraction is the sole pathogenic mechanism in mucosaldominant PV where autoantibodies against Dsg3 but not against Dsg1 are present (1). Alternatively, direct inhibition could act in concert with autoantibody-induced cellular signaling or may just be an epiphenomenon secondary to skin blistering (1, 5-7).The idea of direct interference with Dsg transinteraction by pemphigus autoantibodies has been proposed when Dsg3 was discovered to be a cadherin-type cell adhesion molecule (8, 9). From structural and mutational analyses, it was concluded that classical cadherins are able to form adhesive dimers through their N-terminal cadherin extracellular domain (EC1) via different interaction schemes (10 -13). Investigations of desmosomes using electron tomography from tissue sections also indicated various interaction models for desmosomal cadherins (14). Some of these interaction models resembled the interaction schemes found in the high resolution crystal structure analyses of N-and E-cadherin.This study was designed to modulate Dsg3 and Dsg1 transinteraction by using peptides fitting the most likely Dsg adhesive interface revealed by three-dimensional modeling. In an earlier study, it was reported that peptides targeting the putativ...

Desmocollin 3-mediated Binding Is Crucial for Keratinocyte Cohesion and Is Impaired in Pemphigus

“…In AFM experiments we previously obtained evidence for homophilic trans-interaction of both Dsg1 and Dsg3 (3,15,17,24). The deflection of a flexible cantilever attached to the AFM head is monitored during its approach and retrace to and from a fixed mica plate and recorded as force distance curves (Fig.…”

“…Because laser trapping is based on binding of Dsc3-coated microbeads to desmosomal cadherins located on the keratinocyte cell surface, this approach does not only allow us to detect direct autoantibody-mediated interference but also to observe changes in cadherin binding mediated by cellular signaling pathways (3,15,17). Settling of cadherin-coated microbeads for 30 min on HaCaT cells induced formation of cell-to-bead contacts as described previously for Dsg1-coated beads (17).…”

“…Desmosomal cadherins share a common domain organization with five N-terminally located extracellular subdomains (EC1-5). The membrane-distal EC1 domain is thought to contain the adhesive interface necessary for trans-interaction as could be concluded from structural analysis and blocking studies using peptides and antibodies (3)(4)(5). By establishing trans-and cisinteracting adhesive complexes, desmosomal cadherins participate in providing mechanical strength to stratified epithelia (6).…”

Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

“…The fact that cell dissociation by Dsg3 depletion was prevented partially by p38 MAPK inhibition indicates a dual role for Dsg3. It serves as an adhesion molecule mediating trans-interaction to desmogleins on adjacent cells (30,31,38). In addition, Dsg3 controls p38 MAPK signaling, which is also relevant for cell adhesion.…”

Desmosomal Cadherins and Signaling: Lessons from Autoimmune Disease

“…Direct steric inhibition apparently mimics the so-called tryptophan-swap between interacting Dsg3 molecules which hampers cis-and trans-interaction (Di Zenzo et al, 2012;Spindler et al, 2013;Yamagami et al, 2010). As inhibition of Dsg3 binding contributes to loss of cell cohesion in vivo and in vitro (Heupel et al, 2009b(Heupel et al, , 2008Spindler et al, 2013), it is possible that this mechanism alone is suffi cient to cause blister formation in some cases, especially when blistering is mechanically induced (Mao et al, 2013;Saito et al, 2012). However, direct inhibition of Dsg1 binding in PF may not be required for loss of cell cohesion (Waschke et al, 2005) and binding of PV autoantibodies to the cell surface alone is not suffi cient to cause cell dissociation (Calkins et al, 2006).…”