BASEMENT-MEMBRANE PROTEINS KALININ AND NICEIN ARE STRUCTURALLY AND IMMUNOLOGICALLY IDENTICAL

BACKGROUND: The purpose of this investigation was 2-fold: (a) to compa re two recently described proteins, the anchoring filament protein, ka linin and the hemidesmosome-associated protein, nicein (formerly calle d BM-600) which are both absent in junctional epidermolysis bullosa (J EB) Herlitz's disease; (b) to further define the structural defect in JEB Herlitz's disease. EXPERIMENTAL DESIGN: Cultured keratinocytes wer e analyzed with monoclonal antibodies (mAbs) against kalinin and nicei n by indirect immunofluorescence. These mAbs were also used to immunop recipitate radiolabeled proteins from keratinocyte cultures and to imm unoaffinity purify proteins from keratinocyte conditioned culture medi um. The precipitated or purified products were compared by sodium dode cyl sulfate-polyacrylamide gel electrophoresis, Western blotting, part ial V8 protease digestion, and rotary shadowing. RESULTS: Kalinin and nicein mAbs show identical immunofluorescent staining patterns on cult ured keratinocytes. Kalinin and nicein mAbs immunoprecipitate peptides from radiolabeled normal human keratinocyte cell and medium fractions that are electrophoretically identical. Partial V8 protease digestion patterns of the reduced 140 kilodalton peptides precipitated by nicei n and kalinin mAbs are identical. Kalinin (like nicein) is absent from JEB Herlitz keratinocyte conditioned medium although K-laminin, anoth er anchoring filament component, is present in these cultures. Kalinin , purified from conditioned keratinocyte medium by antibody affinity c hromatography with K140-Sepharose (mAb against kalinin) and nicein pur ified from conditioned keratinocyte medium with GB3-Sepharose (mAb aga inst nicein) are electrophoretically identical by sodium dodecyl sulfa te-polyacrylamide gel electrophoresis and immunologically identical by immunoblotting using kalinin and nicein mAbs. Rotary shadowed images of kalinin and nicein molecules are identical. CONCLUSIONS: We demonst rate that kalinin and nicein are identical by biochemical and immunolo gic analysis. We also verify that kalinin, like nicein, is absent in t he conditioned medium of cultured JEB Herlitz keratinocytes, although another anchoring filament protein, K-laminin, is secreted by these cu ltures. These results correlate with previous immunofluorescent findin gs that show that while kalinin or nicein is absent in basement membra nes of individuals with JEB Herlitz's disease, K-laminin appears to be present.