Potential cellular conformations of the CCN3(NOV) protein.
Potential cellular conformations of the CCN3(NOV) protein.
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DOI：
10.1186/1478-811x-2-9
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发表时间：
2004-09-10
期刊：
影响因子：
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通讯作者：
Perbal, Bernard
中科院分区：
文献类型：
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作者：
Kyurkchiev, Stanimir;Yeger, Herman;Perbal, Bernard
目的：研究CCN3（NOV）的细胞分布，确定CCN3的羧基端是否由于与其他伙伴的高亲和力相互作用而被隐藏或掩盖。采用亲和纯化抗体（anti-K19M- af）和Protein a纯化的抗K19M抗体（anti-K19M IgG）对人CCN3蛋白c端19氨基酸肽（K19M）进行检测。将抗体应用于神经胶质肿瘤细胞系G59及其转染ccn3的变体G59/540和肾上腺皮质细胞系NCI-H295R的间接免疫荧光试验和免疫酶测定。结果：ELISA检测抗K19M- af抗体与K19M肽反应，免疫印迹法识别H295R（肾上腺皮质癌）细胞培养上清中51 kDa和30 kDa两条条带。免疫印迹显示含有CCN3的H295R培养上清液在液相中与抗CCN3抗体不发生反应。抗ccn3抗体染色非通透性H295R细胞的表面膜和通透性H295R细胞的细胞质。同样，抗ccn3染色G59/540的表面膜，但不与G59细胞反应。G59/540细胞质染色明显，G59/540和H295R的细胞足迹被强烈标记。结论：针对CCN3 c端19氨基酸肽的K19M-AF抗体可识别变性条件下的分泌蛋白。然而，分泌的CCN3的c端基序在液相中不能被K19M-AF接近。这些抗CCN3抗体染色了定位于细胞质储存、细胞膜和细胞外基质的CCN3蛋白。这表明细胞质和细胞膜结合的CCN3有一个暴露的c端，而分泌的CCN3有一个隔离的c端，这可能是由于与其他蛋白质或自身的相互作用（二聚化）。因此，K19M-AF抗体揭示了天然CCN3蛋白的至少两种构象状态。
AIM: To study the cellular distribution of CCN3(NOV) and to determine if the carboxyterminus of CCN3 is hidden or masked due to high affinity interactions with other partners. CCN3 was detected using affinity purified antibodies (anti-K19M-AF) as well as a Protein A purified anti-K19M antibodies (anti-K19M IgG) against a C-terminal 19-aminoacid peptide (K19M) of human CCN3 protein. The antibodies were applied in indirect immunofluorescence tests and immunoenzyme assays on glial tumor cell line, G59, and its CCN3-transfected variant G59/540 and the adrenocortical cell line, NCI-H295R. RESULTS: Anti-K19M-AF antibodies reacted against K19M peptide in ELISA and recognized two bands of 51 kDa and 30 kDa in H295R (adrenocortical carcinoma) cell culture supernatants by immunoblotting. H295R culture supernatants which contained CCN3 as shown by immunoblotting did not react with anti-CCN3 antibodies in liquid phase. Anti-CCN3 antibodies stained the surface membranes of non-permeabilized H295R and cytoplasm in permeabilized H295R cells. Similarly, anti-CCN3 stained surface membranes of G59/540, but did not react with G59 cells. Prominent cytoplasmic staining was observed in G59/540, as well as the cell footprints of G59/540 and H295R were strongly labeled. CONCLUSIONS: The K19M-AF antibody directed against the C-terminal 19-aminoacid peptide of CCN3 recognized the secreted protein under denaturing conditions. However, the C-terminal motif of secreted CCN3 was not accessible to K19M-AF in liquid phase. These anti-CCN3 antibodies stained CCN3 protein which was localized to cytoplasmic stores, cell membranes and extracellular matrix. This would suggest that cytoplasmic and cell membrane bound CCN3 has an exposed C-terminus while secreted CCN3 has a sequestered C-terminus which could be due to interaction with other proteins or itself (dimerization). Thus the K19M-AF antibodies revealed at least two conformational states of the native CCN3 protein.