Gu X, Ko M K, Kay E P
Doheny Eye Institute and the University of Southern California School of Medicine, Los Angeles 90033, USA.
Invest Ophthalmol Vis Sci. 1999 Feb;40(2):289-95.
Previous studies by the current investigators showed that type I collagen was posttranslationally regulated in corneal endothelial cells (CECs). These cells synthesize type I procollagen and degrade it intracellularly; however, when CECs are modulated with fibroblast growth factor-2 and/or corneal endothelium modulation factor, they synthesize and secrete type I collagen. Heat shock protein 47 (Hsp47), an endoplasmic reticulum resident protein, is known to function as a molecular chaperon in regulating procollagen folding and/or assembly. The interaction of Hsp47 with type I procollagen synthesis in CECs was also studied.
Expression of proteins was analyzed by radioactive labeling or immunoblot analysis. The steady state level of type I collagen and Hsp47 mRNAs was determined by northern blot analysis. Coprecipitation using immunoprecipitation followed by immunoblotting was performed to determine the association profile between Hsp47 and type I procollagen. Subcellular localization of Hsp47 and type I procollagen was determined by immunofluorescent staining.
Normal and modulated cells expressed Hsp47 and Hsp70. The relative amount of Hsp47 produced by modulated cells was much higher than that of control cells, but the expression level of Hsp70 was the same in control and modulated cells. The steady state levels of type I collagen transcripts were higher in normal cells than in modulated cells, whereas modulated cells contained a much higher steady state level of Hsp47 mRNA. Type I procollagen was found to be associated with Hsp47 when analyzed by coprecipitation or cross-linking. The cytoplasmic localization profile of Hsp47 and type I collagen was different in normal cells, although a colocalization profile was observed to some degree. These two proteins were predominantly colocalized in the Golgi area in modulated CECs.
Hsp47 may be involved in the synthesis and/or intracellular transport of type I collagen in CECs. Modulated cells that secrete type I collagen into the extracellular matrix express a higher level of Hsp47 than do control cells.
当前研究人员之前的研究表明，I型胶原蛋白在角膜内皮细胞（CECs）中受到翻译后调控。这些细胞合成I型前胶原蛋白并在细胞内将其降解；然而，当用成纤维细胞生长因子-2和/或角膜内皮调节因子对CECs进行调节时，它们会合成并分泌I型胶原蛋白。热休克蛋白47（Hsp47）是一种内质网驻留蛋白，已知其在调节前胶原蛋白折叠和/或组装过程中起分子伴侣的作用。本研究还探讨了Hsp47与CECs中I型前胶原蛋白合成的相互作用。
通过放射性标记或免疫印迹分析来分析蛋白质的表达。通过Northern印迹分析确定I型胶原蛋白和Hsp47 mRNA的稳态水平。采用免疫沉淀后免疫印迹的共沉淀法来确定Hsp47与I型前胶原蛋白之间的关联情况。通过免疫荧光染色确定Hsp47和I型前胶原蛋白的亚细胞定位。
正常细胞和经调节的细胞均表达Hsp47和Hsp70。经调节的细胞产生的Hsp47相对量远高于对照细胞，但对照细胞和经调节的细胞中Hsp70的表达水平相同。正常细胞中I型胶原蛋白转录本的稳态水平高于经调节的细胞，而经调节的细胞中Hsp47 mRNA的稳态水平则高得多。通过共沉淀或交联分析发现I型前胶原蛋白与Hsp47有关联。在正常细胞中，Hsp47和I型胶原蛋白的细胞质定位情况不同，尽管在一定程度上观察到了共定位情况。在经调节的CECs中，这两种蛋白主要共定位于高尔基体区域。
Hsp47可能参与CECs中I型胶原蛋白的合成和/或细胞内运输。向细胞外基质分泌I型胶原蛋白的经调节细胞比对照细胞表达更高水平的Hsp47。
