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研究発表を行った学会；
・2015 International Workshop on Alport Syndrome
　2015年9月25日〜27日（Gettingen, Germany）

タイトル；Cellular-based assay clarifies the regulation of Alport syndrome-related protein COL4A5.
発表者；大町　紘平　氏
　　　（熊本大学　大学院生命科学研究部　遺伝子機能応用学分野）
要旨；
INTRODUCTION AND AIMS: Alport syndrome (AS) is caused by mutation in COL4A3, COL4A4 or COL4A5. Mutant COL4A3/4/5 cannot form heterotrimer. This loss of function causes abnormal GBM structure and renal dysfunction. Restoring the normal COL4A3/4/5 network in the GBM is important for AS therapy; hence the need to investigate the intracellular behavior of mutant COL4A5 such as degradation, heterotrimer formation and secretion. However, understanding COL4A3/4/5 regulation is poor due to a lack of cellular model for AS. Here, we established AS cellular model and investigated the regulation of wild type and mutant COL4A5 protein.
METHODS: We cloned and constructed human COL4A3/4/5 expression plasmids. Mutant COL4A5 plasmids were also generated by site-directed mutagenesis. These plasmids were transfected in 293T cells, and whole cell lysates were analyzed for protein expression by Western blotting. We investigated whether wild type and mutant COL4A5 are degraded by proteasome or lysosome by treating transfected cells with inhibitors. COL4A5 protein stability was also checked by chase experiments. Moreover, we examined the involvement of ER chaperones HSP47, BiP, GRP94 and PDI on the regulation of COL4A5 protein by overexpression or knockdown experiments.
RESULTS: COL4A3, COL4A4, COL4A5 and five mutant COL4A5 proteins were sufficiently expressed in 293T cells. Chase experiments revealed that the intracellular protein expression of both wild type and mutant COL4A5 were decreased at a relatively similar rate. Addition of proteasome inhibitor MG132 or lysosome inhibitor Bafilomycin A1 did not inhibit the decrease in COL4A5 protein during chase. Interestingly, treatment with Brefeldin A, an inhibitor of ER-Golgi transport, during chase experiment inhibited the decrease of intracellular protein expression of wild type and non-secreted mutant (C1567R) COL4A5. This result indicated that COL4A5 monomer was degraded at post-ER. Moreover, we found that HSP47 promoted the post-ER degradation of COL4A5. On further analysis, we showed that BiP and PDI inhibited while GRP94 promoted the secretion of COL4A5 monomer.
CONCLUSIONS: Here, we established an overexpression system of human COL4A3/4/5 proteins. This model is an important tool to study AS at the molecular level such as protein stability, degradation mechanism and heterotrimer formation of wild type and mutant COL4A5. Using this cellular expression system, we showed that while some ER chaperones are involved in the fine-tuning of the intracellular behavior of COL4A5, the COL4A5 monomer is degraded at post-ER. This study provides a novel insight into the molecular regulation of type IV collagen especially COL4A5, and clarifies the intracellular behavior of some COL4A5 mutant proteins, which could help to establish new therapeutic focus for Alport syndrome.