ISOLATION AND CHARACTERIZATION OF HEMIDESMOSOMES FROM BOVINE CORNEAL EPITHELIAL-CELLS
ISOLATION AND CHARACTERIZATION OF HEMIDESMOSOMES FROM BOVINE CORNEAL EPITHELIAL-CELLS
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DOI：
10.1016/0014-4827(91)90084-8
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发表时间：
1991-02-01
影响因子：
3.7
通讯作者：
FRANKE, WW
中科院分区：
文献类型：
--
作者：
OWARIBE, K;NISHIZAWA, Y;FRANKE, WW
半桥粒（HD）是复层和复杂上皮细胞的特化细胞与基底层连接，其特征在于中间丝（IF）锚定的细胞质斑块。为了系统地鉴定和表征HD组分，我们开发了一种分离和压裂HD的方法。当牛角膜上皮从细胞外基质基质中剥离时，附着在基底层上的HD与附着在细胞质HD斑块上的细胞角蛋白IF簇一起留下。用EDTA冲洗这些残留的基底细胞成分后，可以将HD从基质机械分离并通过离心收集。生化和电子显微镜检查获得的馏分，显示丰富的HD结构，以及一个突出的230-kDa的多肽，“类天疱疮抗原”已知位于HD斑块。此外，HD馏分显示，除了残留量的角膜细胞角蛋白，主要多肽ofMr 120，180，200，230，和480 kDa，其中前三个似乎是糖蛋白。使用分离的HDs免疫，我们制备的230-和180-kDa的多肽，分别特异性的单克隆抗体，并表明，这两个都是专门位于HDs。这种分离HD的方法和HD蛋白抗体的可用性将有助于HD分子组织的研究，使HD研究独立于人类自身免疫抗体。
The hemidesmosome (HD) is a specialized cell-to-substratum junction of stratified and complex epithelia which is characterized by a cytoplasmic plaque to which intermediate filaments (IFs) are anchored. To identify and characterize HD constituents systematically, we have developed a procedure to isolate and fractionate HDs. When bovine corneal epithelium is peeled off from the extracellular matrix stroma, HDs attached to the basal lamina are left behind, together with tufts of cytokeratin IFs attached to the cytoplasmic HD plaques. After rinsing these residual basal cell elements with EDTA, the HDs could be mechanically detached from the stroma and collected by centrifugation. The fraction obtained was examined biochemically and electron microscopically, showing enrichment of HD structures as well as of a prominent 230-kDa polypeptide, the “pemphigoid antigen” known to be located in the HD plaque. In addition, the HD fraction revealed, besides residual amounts of corneal cytokeratins, major polypeptides ofMr120, 180, 200, 230, and 480 kDa, of which the first three appeared to be glycoproteins. Using the isolated HDs for immunization, we prepared monoclonal antibodies specific for the 230- and 180-kDa polypeptides, respectively, and showed that both were exclusively located in HDs. This method for isolating HDs and the availability of antibodies to HD proteins will be useful in studies of the molecular organization of HDs and make HD research independent from human autoimmune antibodies.