Marinkovich M P, Verrando P, Keene D R, Meneguzzi G, Lunstrum G P, Ortonne J P, Burgeson R E
Department of Dermatology, Oregon Health Sciences University, Shriner's Hospital for Crippled Children, Portland.
Lab Invest. 1993 Sep;69(3):295-9.
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.
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.
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.
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.
本研究有两个目的：（a）比较两种最近描述的蛋白质，即锚定细丝蛋白卡利宁和半桥粒相关蛋白尼斯因（以前称为BM - 600），这两种蛋白在交界性大疱性表皮松解症（JEB）赫利茨病中均不存在；（b）进一步明确JEB赫利茨病的结构缺陷。
用抗卡利宁和尼斯因的单克隆抗体（mAb）通过间接免疫荧光分析培养的角质形成细胞。这些mAb还用于从角质形成细胞培养物中免疫沉淀放射性标记的蛋白质，并从角质形成细胞条件培养基中免疫亲和纯化蛋白质。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳、蛋白质印迹、部分V8蛋白酶消化和旋转阴影法比较沉淀或纯化的产物。
卡利宁和尼斯因mAb在培养的角质形成细胞上显示相同的免疫荧光染色模式。卡利宁和尼斯因mAb从放射性标记的正常人角质形成细胞细胞和培养基组分中免疫沉淀出电泳相同的肽。尼斯因和卡利宁mAb沉淀的还原140千道尔顿肽的部分V8蛋白酶消化模式相同。尽管另一种锚定细丝成分K - 层粘连蛋白存在于这些培养物中，但JEB赫利茨角质形成细胞条件培养基中不存在卡利宁（与尼斯因一样）。用K140 - 琼脂糖（抗卡利宁mAb）通过抗体亲和层析从条件角质形成细胞培养基中纯化的卡利宁和用GB3 - 琼脂糖（抗尼斯因mAb）从条件角质形成细胞培养基中纯化的尼斯因，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳电泳相同，使用卡利宁和尼斯因mAb进行免疫印迹时免疫相同。卡利宁和尼斯因分子的旋转阴影图像相同。
我们通过生化和免疫学分析证明卡利宁和尼斯因是相同的。我们还证实，尽管另一种锚定细丝蛋白K - 层粘连蛋白由这些培养物分泌，但在培养的JEB赫利茨角质形成细胞的条件培养基中不存在卡利宁，与尼斯因一样。这些结果与先前的免疫荧光结果相关，该结果表明，虽然在JEB赫利茨病患者的基底膜中不存在卡利宁或尼斯因，但K - 层粘连蛋白似乎存在。
