Basement membrane proteins kalinin and nicein are structurally and immunologically identical.
BACKGROUND
The purpose of this investigation was 2-fold: (a) to compare two recently described proteins, the anchoring filament protein, kalinin and the hemidesmosome-associated protein, nicein (formerly called BM-600) which are both absent in junctional epidermolysis bullosa (JEB) Herlitz's disease; (b) to further define the structural defect in JEB Herlitz's disease.


METHODS
Cultured keratinocytes were analyzed with monoclonal antibodies (mAbs) against kalinin and nicein by indirect immunofluorescence. These mAbs were also used to immunoprecipitate radiolabeled proteins from keratinocyte cultures and to immunoaffinity purify proteins from keratinocyte conditioned culture medium. The precipitated or purified products were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, partial V8 protease digestion, and rotary shadowing.


RESULTS
Kalinin and nicein mAbs show identical immunofluorescent staining patterns on cultured 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 nicein and kalinin mAbs are identical. Kalinin (like nicein) is absent from JEB Herlitz keratinocyte conditioned medium although K-laminin, another anchoring filament component, is present in these cultures. Kalinin, purified from conditioned keratinocyte medium by antibody affinity chromatography with K140-Sepharose (mAb against kalinin) and nicein purified from conditioned keratinocyte medium with GB3-Sepharose (mAb against nicein) are electrophoretically identical by sodium dodecyl sulfate-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 demonstrate that kalinin and nicein are identical by biochemical and immunologic analysis. We also verify that kalinin, like nicein, is absent in the conditioned medium of cultured JEB Herlitz keratinocytes, although another anchoring filament protein, K-laminin, is secreted by these cultures. These results correlate with previous immunofluorescent findings that show that while kalinin or nicein is absent in basement membranes of individuals with JEB Herlitz's disease, K-laminin appears to be present.
Marinkovich
M P
MP
Department of Dermatology, Oregon Health Sciences University, Shriner's Hospital for Crippled Children, Portland.
Verrando
P
P
Keene
D R
DR
Meneguzzi
G
G
Lunstrum
G P
GP
Ortonne
J P
JP
Burgeson
R E
RE
D000911:Antibodies, Monoclonal; D001485:Basement Membrane / Q000166:cytology; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology*; D008297:Male; D008970:Molecular Weight; D015531:Radioimmunoprecipitation Assay; D012867:Skin / Q000166:cytology; D000097794:Kalinin
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D000097794:Kalinin