split NanoLuc評価系を用いた Alport 症候群治療薬探索と分子動力学シミュレーションによる病態解析 : 原因タンパク質 Type Ⅳ Collagen に着目して

熊本大学博士（薬科学）thesi

Trimerization and genotype-phenotype correlation of COL4A5 mutants in Alport syndrome

INTRODUCTION: Alport syndrome is a hereditary glomerulonephritis that results from the disruption of collagen α345(IV) heterotrimerization caused by mutation in METHODS: We selected 9 α5(IV) missense mutants with typical glycine substitutions that clinically differed in disease progression. To quantify the trimerization of each mutant, split nanoluciferase-fused α3/α5 mutants and α4 were transfected into the cells, and intracellular and secreted heterotrimer were detected by luminescence using an assay that we developed previously. RESULTS: Trimer formation and secretion patterns tended to be similar to the wild type in most of the mutations that did not show proteinuria at a young age. On the other hand, trimer secretion was significantly reduced in all the mutations that showed proteinuria and early onset of renal failure. One of these mutants has low ability of intracellular trimer formation, and the others had the defect of low-level secretion. In addition, the mutant that is assumed to be nonpathogenic has similar trimer formation and secretion pattern as wild-type α5(IV). CONCLUSION: The result of cell-based α345(IV) heterotrimer formation assay was largely correlated with clinical genotype-phenotype. These trimerization assessments provide additional phenotypic considerations and may help to distinguish between pathogenic and nonpathogenic mutations