Girard D, Senécal J L
Hôpital Notre-Dame, Department of Medicine, University of Montreal School of Medicine, QUE, Canada.
Autoimmunity. 1995;20(4):237-45. doi: 10.3109/08916939508995701.
We have characterized a human IgG antibody present in the serum of a patient with an autoimmune undifferentiated connective tissue disease and reactive with PtK2 epithelial cell-cell adhesions. The fluorescent staining pattern is observed only at cell-cell contacts whether cells are permeabilized or not. The serum reacts with polypeptides of 90, 48 and 45 kD by immunoblotting. IgG affinity-purified from these bands failed to reproduce the original immunofluorescence staining pattern. Treatment with cycloheximide did not abolish the staining pattern suggesting that the recognized antigen is not a newly expressed protein. However, when EGTA was used for chelating calcium ions in the culture medium the original staining pattern observed at cell-cell adhesions was affected although some fluorescence was still present at cell periphery. This was reversible when cells were reincubated with fresh medium containing Ca2+. The recognized antigen colocalizes at cell-cell adhesions with actin, the microfilament-associated proteins vinculin, alpha-actinin and myosin light chain, and with Triton-insoluble uvomorulin (E-cadherin) material. We conclude that the antibody reacts with, at least, an extracellular portion of a Ca(2+)-dependent PtK2 antigen. The characterization of this antibody based on (1) its localization at cell-cell adhesions, (2) its sensitivity to EGTA-treatment and (3) its colocalization with the epithelial cellular adhesion molecule (CAM) uvomorulin, strongly suggest that the recognized Ag is a CAM or a CAM-associated protein.
我们鉴定了一名自身免疫性未分化结缔组织病患者血清中存在的一种人IgG抗体，该抗体与PtK2上皮细胞间黏附反应。无论细胞是否通透，荧光染色模式仅在细胞间接触处观察到。通过免疫印迹法，该血清与90kD、48kD和45kD的多肽发生反应。从这些条带中亲和纯化的IgG未能重现原始的免疫荧光染色模式。用放线菌酮处理并没有消除染色模式，这表明识别的抗原不是新表达的蛋白质。然而，当使用EGTA螯合培养基中的钙离子时，尽管细胞周边仍有一些荧光，但在细胞间黏附处观察到的原始染色模式受到影响。当细胞与含有Ca2+的新鲜培养基重新孵育时，这种影响是可逆的。识别的抗原在细胞间黏附处与肌动蛋白、微丝相关蛋白纽蛋白、α-辅肌动蛋白和肌球蛋白轻链以及与Triton不溶性桥粒芯糖蛋白（E-钙黏蛋白）物质共定位。我们得出结论，该抗体至少与一种Ca(2+)依赖性PtK2抗原的细胞外部分发生反应。基于（1）其在细胞间黏附处的定位，（2）其对EGTA处理的敏感性，以及（3）其与上皮细胞黏附分子（CAM）桥粒芯糖蛋白的共定位，对该抗体的鉴定强烈表明识别的抗原是一种CAM或一种与CAM相关的蛋白质。
