IDENTIFICATION OF A KERATIN-ASSOCIATED PROTEIN THAT LOCALIZES TO A MEMBRANE COMPARTMENT
IDENTIFICATION OF A KERATIN-ASSOCIATED PROTEIN THAT LOCALIZES TO A MEMBRANE COMPARTMENT
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DOI：
10.1042/bj2980457
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发表时间：
1994-03-01
影响因子：
4.1
通讯作者：
OMARY, MB
中科院分区：
文献类型：
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作者：
CHOU, CF;RIOPEL, CL;OMARY, MB
我们描述了一种酸性糖蛋白(分子质量类似于85 kDa)的特征，它与‘简单’型上皮的角蛋白中间丝有关。用多种抗角蛋白的单抗与人结肠上皮细胞株HT29和其他几种上皮细胞株的角蛋白多肽8和18(K8/18)进行免疫共沉淀，鉴定出85 kDa的糖蛋白。这种角蛋白相关蛋白(称为KAP85)在体外对来自有丝分裂抑制细胞的K8/18免疫沉淀物进行半乳糖化后很容易被检测到。它的增溶和检测依赖于所使用的洗涤剂，在异步化生长的G0/G1期细胞体外半乳糖化后几乎检测不到。它在G0/G1期细胞中较差的体外半乳糖基化可能反映了缺乏可用的末端N-乙酰氨基葡萄糖残基，因为它可以用NaIO4/(NaBH4)-H-3在G0/G1期和G2/M期细胞中进行类似程度的标记。糖苷酶分析表明，KAP85含有较高的甘露糖和复杂的低聚糖。将细胞总K8/18分成可溶池和细胞骨架不溶池，结果表明KAP85只与细胞骨架K8/18池结合。亚细胞分离表明，KAP85与包括转铁蛋白受体和KS-1抗原的质膜富集组分共定位。我们的结果在体外证明了一种膜相关糖蛋白(KAP85)，它可能是丝状K8/18的附着部位。
We describe the characterization of an acidic glycoprotein (molecular mass similar to 85 kDa) that associates with keratin intermediate filaments of 'simple'-type epithelia. Using a number of anti-keratin monoclonal antibodies, the 85 kDa glycoprotein was identified by co-immunoprecipitation with keratin poly-peptides 8 and 18 (K8/18) from the human colonic epithelial cell line HT29 and several other epithelial cell lines. This Keratin-Associated Protein (termed KAP85) was readily detected after in vitro galactosylation of K8/18 immunoprecipitates obtained from mitosis-arrested cells. Its solubilization and detection were dependent on the detergent used, and it was barely detected after in vitro galactosylation of asynchronously growing G0/G1-phase cells. Its poor in vitro galactosylation in G0/G1-phase cells is likely a reflection of the lack of available terminal N-acetyl-glucosamine residues, since it can be labelled to a similar extent in G0/G1- and G2/M-phase cells using NaIO4/(NaBH4)-H-3. Glycosidase digestion showed that KAP85 contains high mannose and complex oligosaccharides. Fractionation of total cellular K8/18 into soluble and cytoskeletal insoluble pools showed that KAP85 associates exclusively with the cytoskeletal K8/18 pool. Subcellular fractionation showed that KAP85 co-localizes with a plasma-membrane-enriched fraction that includes the transferrin receptor and KS-1 antigen. Our results demonstrate in vitro evidence of a membrane-associated glycoprotein (KAP85) which may serve as an attachment site for filamentous K8/18.