Monoclonal Antibodies to Human Epidermal Filaggrin, Some Not Recognizing Profilaggrin

Abstract

To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.

The Major Synovial Targets of the Rheumatoid Arthritis-Specific Antifilaggrin Autoantibodies Are Deiminated Forms of the α- and β-Chains of Fibrin

“…Our immunohistological and immunochemical analyses of synovial membranes from 9 RA patients with 29 anti-(pro)filaggrin mouse monoclonal IgG Abs that recognize at least 20 different epitopes borne by the various forms of (pro)filaggrin (Ref. 25 and our unpublished observations) definitively showed that (pro)filaggrins are not expressed in the tissue (data not shown). Therefore, since the epitopes targeted by AFA are generated by deimination of (pro)filaggrins (12, 13), we postulated that AFA were directed to one or several cross-reactive deiminated antigenic protein(s) present in the rheumatoid synovial membranes.…”

High Levels of Cytokeratin 19 Fragments But No Evidence of Cytokeratins 1, 2, 10/11, 14 or Filaggrin in the Serum of Squamous Cell Lung Carcinoma Patients

“…cytokeratin 1, 2, 10/11, 14 and filaggrin. Using antibodies previously produced in our laboratories [30][31][32] we developed immunoassays to identify these proteins in the serum of patients with SQCLC. However, less than 14% of sera from patients with SQCLC exhibited detectable signals.…”

“…AHF2 and AHF3 (IgG1Î) were obtained using basic filaggrin purified from human epidermis as the immunogen. They recognize two different epitopes borne by profilaggrin and the neutral-acidic and basic isoforms of filaggrin [32]. AHF16, AHF24 (IgG1Î) and AHF25 (IgG2bÎ) were produced after immunization with the neutral-acidic form of filaggrin purified from human epidermis.…”

The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations

“…The monoclonal antibody (mAb) AHF3 is specific to (pro)filaggrin as previously reported [27]. The mAb AHF11 was produced as previously described [27] except that mice were immunized with deiminated filaggrin purified from human epidermis.…”

“…The mAb AHF11 was produced as previously described [27] except that mice were immunized with deiminated filaggrin purified from human epidermis. These mAbs were used at 0.4 µg/ml for western blotting.…”