Kidney Week

Abstract: FR-PO591

Structural Analysis of the Extracellular Portion of Polycystin-1

Session Information

• Cystic Kidney Diseases: Mechanisms and Models
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Baldauf, Sabrina, University of Regensburg, Regensburg, Germany

Sabrina Baldauf, MD

• Gaines, Matthew, University of Exeter, Exeter, United Kingdom

Matthew Gaines, PhD

• Pilsl, Michael, University of Regensburg, Regensburg, Germany

Michael Pilsl, PhD

• Madej, M. Gregor, University of Regensburg, Regensburg, Germany

M. Gregor Madej, PhD

• Maier, Olga, University of Regensburg, Regensburg, Germany

Olga Maier

• Rachel, Reinhard, University of Regensburg, Regensburg, Germany

Reinhard Rachel, PhD

• Ziegler, Christine Maria, University of Regensburg, Regensburg, Germany

Christine Maria Ziegler

• Daum, Bertram, University of Exeter, Exeter, United Kingdom

Bertram Daum, DrPH

• Witzgall, Ralph, University of Regensburg, Regensburg, Germany

Ralph Witzgall, MD

Background

PKD1 and PKD2 code for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Mutations in both of these genes lead to autosomal dominant polycystic kidney disease, yet the precise mechanism of cyst formation is still not completely understood. To learn more about its biological function, we have begun with the structural analysis of the extracellular NH₂-terminus of PC1 that is cleaved through cis-autoproteolysis.

Methods

HeLa cells were stably transfected with an expression plasmid encoding the StrepII-tagged extracellular NH₂-terminus of PC1 (amino acids 1-3074). For recombinant protein purification, the cell culture supernatant was loaded onto Streptactin affinity columns. Fractions of interest were concentrated for further purification by gel filtration, finally the recombinant protein was subjected to mass spectrometry, polyacrylamide gel electrophoresis combined with silver staining, and negative staining with subsequent electron microscopy. The fraction with the highest purity and concentration was plunge frozen for cryo-EM and the micrographs processed with cryoSPARC. Further characterization included deglycosylation, mass photometry and CD spectroscopy.

Natural PC1 was purified from urine by sucrose density gradient ultracentrifugation. Fractions containing PC1 were identified by Western blotting and again further purified by gel filtration. Finally, PC1 was frozen for cryo-EM, and single-particle analysis was performed with Relion and cryoSPARC.

Results

The recombinant extracellular NH₂-terminus of PC1 exists as highly glycosylated monomers. PC1 particles are filamentous and consist mostly of β-sheets as secondary structure elements. In contrast, natural PC1 purified from urine forms a very peculiar structure which can be found in different species. This peculiar structure is also present in recombinant PC1 samples although at a much lower frequency.

Conclusion

The structural characterization of the NH₂-terminus of PC1 opens new opportunities for a better understanding of its biological functions. The COOH-terminus of PC1 requires PC2 for transport, therefore the lower frequency of the peculiar PC1 structure observed in recombinant PC1 may be explained by the absence of PC2 in HeLa cells.

Funding

• Government Support – Non-U.S.