Ken Ishii

Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display

Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.

Cadherin function: Breaking the barrier

Three desmoglein isoforms collaborate with desmocollins to build the adhesive core of desmosomes. A recent study has shown that altering the ratio of desmoglein isoforms influences epidermal barrier function, suggesting distinct roles for these cadherins that extend beyond adhesion.

Antibodies to the desmoglein 1 precursor proprotein but not to the mature cell surface protein cloned from individuals without pemphigus

In pemphigus foliaceus (PF), autoantibodies against desmoglein 1 (Dsg1) cause blisters. Using Ab phage display, we have cloned mAbs from a PF patient. These mAbs, like those from a previous patient, were directed against mature Dsg1 (matDsg1) on the cell surface of keratinocytes and precursor Dsg1 (preDsg1) in the cytoplasm. To determine whether individuals without pemphigus have B cell tolerance to Dsg1, we cloned mAbs from two patients with thrombotic thrombocytopenic purpura and a healthy person. We found mAbs against preDsg1, but not matDsg1. All but 1 of the 23 anti-preDsg1 mAbs from PF patients and those without PF used the VH3-09 (or closely related VH3-20) H chain gene, whereas no PF anti-matDsg1 used these genes. VH cDNA encoding anti-preDsg1 had significantly fewer somatic mutations than did anti-matDsg1 cDNA, consistent with chronic Ag-driven hypermutation of the latter compared with the former. These data indicate that individuals without PF do not have B cell tolerance to preDsg1 and that loss of tolerance to matDsg1 is not due to epitope shifting of anti-preDsg1 B cells (because of different VH gene usage). However, presentation of peptides from Dsg1 by preDsg1-specific B cells may be one step in developing autoimmunity in PF.

Update on the cloning of monoclonal anti‐desmoglein antibodies from human pemphigus patients: implications for targeted therapy

Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.

Identification of Desmoglein as a Cadherin and Analysis of Desmoglein Domain Structure.

In the 1980s and early 1990s, the ability to clone complementary DNA (cDNA) resulted in great advances in cell biology, including our understanding of cell adhesion. Cloning the cDNA encoding desmosomal molecules, and the determination of their deduced amino-acid sequences, provided novel insights into their structure and function. It had been shown by various biochemical and immunochemical techniques that desmosomal proteins consist of plaque proteins inside the cell and transmembrane glycoproteins. For desmosomal transmembrane glycoproteins, Koch et al. (1990) first isolated and characterized the cDNA encoding desmoglein in 1990. Using monoclonal antibodies against desmoglein, they screened cDNA expression libraries constructed from bovine muzzle epithelial mRNA. The major scientific advance from this work was that the deduced amino-acid sequences of their isolated clones showed high homology to cadherins, which had already been shown to be calciumdependent cell adhesion molecules. Thus, they identified desmoglein as a member of the cadherin supergene family of cell adhesion molecules. Similar to classic cadherins such as Eand N-cadherins, they, and subsequently others, showed that desmoglein is a type I transmembrane protein with an amino-terminal extracellular domain, a single transmembrane spanning region, and a carboxy-terminal cytoplasmic domain (Figure 1). The extracellular domain was shown to contain four subdomain repeating units that have homology to similar extracellular subdomains in cadherins and that, similar to those units, contained putative calcium-binding sites. The cytoplasmic domain of desmoglein, although it contained a subdomain (termed the intracellular cadherin segment or ICS) that was homologous to a cytoplasmic subdomain of cadherin, significantly differed in that it was longer than that of cadherins. In addition to the ICS subdomain, desmoglein contained a proline-rich linker (IPL) and a terminal repeating unit domain (RUD). Although the ICS domain has been shown to bind various intracellular molecules, such as plakoglobin, the function of the additional subdomains of desmoglein remains unclear. Progress, by cDNA cloning, was also made in characterizing the other major transmembrane molecule of the desmosome, desmocollin (Collins et al., 1991). cDNA cloning of desmocollin indicated that it also belonged to the cadherin family. The unique aspect of desmocollin was that the cytoplasmic domain contained a longer “a” form and a shorter “b” form, produced by alternative splicing of mRNA. To date, four isoforms of desmoglein (Dsg1–4) and three isoforms of desmocollin (Dsc1–3) have been identified, each arising from a different gene. The genes are clustered on the q arm of chromosome 18 for humans (Kljuic et al., 2003). Because of their homologies to classical cadherins, these glycoproteins are now termed “desmosomal cadherins”. The cDNA cloning of desmogleins and, subsequently, desmocollins,