Isolation of plasma membranes from keratinocytes: a newly developed method allows the sensitive detection of the membrane antigen pattern in normal and psoriatic skin.
Distinct differences of proteins in the plasma membranes have been described in psoriatic keratinocytes as compared with normal epidermis. These changes are presumably involved in the pathogenesis of psoriasis. To identify and distinguish glycosylated proteins of the plasma membranes of keratinocytes, a method for mild preparation was developed that avoids the use of degrading or digestive enzymes. After cell lysis, three steps of centrifugation were performed, including the use of a sucrose step gradient. A fine-vesicular membrane fraction was obtained. Using marker enzymes for cell compartments, no contamination of cell nuclei or mitochondria and only 0.4% of endoplasmic reticulum was detectable in the final membrane fraction. Based on this preparation, disc-polyacrylamide-gel electrophoresis, followed by Western blotting, were performed. Staining with five different lectins to visualize glycosylated proteins allowed 75 different membrane glycoproteins to be distinguished. The patterns of normal, transformed (HaCaT), foreskin and psoriatic keratinocytes after cell culture with one passage were compared. Up to six proteins per lectin staining were expressed differently in psoriatic as compared to normal keratinocytes. Psoriatic cells shared similarities with highly proliferative foreskin cells, but not with transformed HaCaT cells. Main alterations of glycosylation were detected in the fucose content. In conclusion, the method developed for isolation of plasma membranes allows selective and sensitive examination of plasma membranes of normal and pathological keratinocytes. The glycosylation patterns observed suggest that distinct membrane proteins may be involved in the pathogenesis of psoriasis.
Department of Dermatology, Medical Center Minden, Germany.
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Licht
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Bauer
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Stadler
D000954:Antigens, Surface / Q000032:analysis*; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology*; D002478:Cells, Cultured; D003584:Cytological Techniques; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology / Q000276:immunology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D011565:Psoriasis / Q000276:immunology* / Q000473:pathology; D012016:Reference Values; D012680:Sensitivity and Specificity; D012867:Skin / Q000201:enzymology / Q000276:immunology* / Q000473:pathology
D000954:Antigens, Surface; D008565:Membrane Proteins