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Abstract: FR-PO1002

Identification of Novel Secretion-Defective Mutations of Type IV Collagen α5 Genes by Split NanoLuc-Based α345(IV) Trimer Formation Assay

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Kamura, Misato, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
  • Yamamura, Tomohiko, Kobe University Graduate School of Medicine, Kobe, Japan
  • Omachi, Kohei, Washington University, St. Louis, Missouri, United States
  • Shuto, Tsuyoshi, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
  • Nozu, Kandai, Kobe University Graduate School of Medicine, Kobe, Japan
  • Iijima, Kazumoto, Kobe University Graduate School of Medicine, Kobe, Japan
  • Kai, Hirofumi, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
Background

Alport syndrome (AS) is a hereditary glomerular disease caused by a mutation in type IV collagen α3, α4, or α5 (α3/α4/α5(IV)), components of the glomerular basement membrane (GBM). Several hundred different mutations have been found in AS patients and the phenotype differs depending on the mutation. Generally, immunostaining of renal biopsy specimens for type IV collagen is useful for predicting prognosis; however, its invasiveness and incompleteness on the prediction accuracy are clinically problematic. Here, by utilizing the cell-based system to assess α345(IV) trimer formation (Omachi K., et al., Cell Chemical Biology 2018), we evaluate the capability of α345(IV) trimer formation of several α5 missense mutants that are clinically relevant but poorly understood.

Methods

Split nanoLuc-fusion α3/ α5 and α4 were transfected into HEK293T cells, and luminescence was assessed in the cell lysate for intracellular trimer and in culture media for secreted trimer. Scatterplot analysis of the intracellular/secreted RLU ratio from cells expressing the WT or several α5 missense mutants classifies the character of mutants.

Results

Trimer formation assay revealed that G509R, G805R, G953V, G1000V, G1030S, G1140V, G1143S α5 mutants have normal properties of intracellular trimerization (>98%) and extracellular secretion (>65%). Scatterplot analysis showed that most of the mutants (G509R, G805R, G953V, G1143S) with mild phenotype (no obvious proteinuria) showed the similar pattern as the WT, while some mutants (G1000V, G1030S, G1140V) exhibited secretion-dependent defect. Particularly, G1140V with severe phenotype (obvious proteinuria) showed significantly increased intracellular accumulation of trimer (>130%).

Conclusion

Trimer formation assay reveals that three α5 mutants (G1000V, G1030S, G1140V) had defect in secretion. Because patient with G1140V mutation had relatively severe clinical phenotype, our system may, at least in part, reflect genotype-phenotype correlation. Further phenotypic characterization would also be needed for G1000V and G1030S mutations. Moreover, because adjacent mutations G1140V and G1143S had severe and mild phenotypes, respectively, structure and molecular bases on differential phenotypes is now under investigation.

Funding

  • Government Support - Non-U.S.