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Genetics and Pathogenesis of Polycystic Kidney Disease

Genetics and Pathogenesis of Polycystic Kidney Disease. Maria Abreu. What is polycystic kidney disease?. Autosomal dominant disease One of the most common hereditary disorders.

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Genetics and Pathogenesis of Polycystic Kidney Disease

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  1. Genetics and Pathogenesis of Polycystic Kidney Disease Maria Abreu

  2. What is polycystic kidney disease? • Autosomal dominant disease • One of the most common hereditary disorders. • Characterized by cyst formation in ductal organs: kidneys and the liver, and by GI, cardiovascular, and musculoskeletal abnormalities.

  3. Normal Kidney vs. Polycystic kidney

  4. Identification and Characterization of Polycystin-2, the PKD2 Gene product Cai, Y., Y. Maeda, A. Cedzich, V.E. Torres, G. Wu, T. Hayashi, T. Mochizuki, J.H. Park, R. Witzgall, and S. Somlo. Identification and characterization of polycystin-2, the PKD2 gene product. 1999. Journal of Biological Chemistry 274 (40): 28557-28565.

  5. Autosomal Dominant Polycystic Kidney Disease (ADPKD) • ADPKD is caused by mutations in two genes, PKD1 and PKD2. • Mapped to chromosomes 16p13.3 and 4q13-p23 respectively

  6. PKD2 gene • Expressed in many tissues and organs • Mutated in 15% of affected families • Encodes for polycystin-2 • Highly conserved in multicellular organisms • Loss of functional polycystin-2 in renal tubule and biliary epithelial cells causes cells to proliferate, loss their normal structure within tissue architecture, and go on to form cysts.

  7. What is the focus of the paper? • Want to identify and characterize polycystin-2 as an integral membrane glycoprotein whose predominant subcellular location is in the ER.

  8. Glycoproteins are complexes in which carbohydrates are attached covalently to asparagine (N-glycans) or serine/threonine (O-glycans) residues of peptides. http://www.glycoforum.gr.jp/science/word/glycoprotein/GPA00E.html

  9. Methods

  10. Use of polyclonal antisera against intracellular domains of polycystin-2 • Protein C2 (a.a. 687-962) contains most of the cytoplasmic COOH-terminal portion of polycystin-2 • B9(a.a. 103-203) contains part of the NH2- terminal portion of polycystin-2

  11. http://www.biocenter.helsinki.fi/bi/peranen/Kotisivunlinkit/Vectors/pGEX-2TL.htmhttp://www.biocenter.helsinki.fi/bi/peranen/Kotisivunlinkit/Vectors/pGEX-2TL.htm

  12. Expression Plasmids • Full length PKD2 cDNA (TM4-FL) was constructed. • Introduced naturally occuring mutations (R742X and R872X) into TM4-FL by site-directed mutagenesis. • Truncation constructs: introducing stop codons. (E877X and G821X)

  13. Primers designed R742X5’-GTTAAACTTTGACGAACTTTGACAAGATCTCAAAGGGAAG-3’ E787X 5’-GACTTGGAGAAAGAGAGGTAAGACCTGGATTTGGATC-3’

  14. Cell culture and Transfections • HEK293 cell lines expressing TM4-FL were selected with Zeocin (antibiotic). • Individual colonies selected and tested by immunoblotting and immocytochemistry for expression of polycystin-2 using YCC2 and YCB9 antisera. • Resistant colonies were cultured individually and tested for the expressin of the full-length and truncated PKD2 proteins.

  15. Western Blots and Immunoprecipitation • Proteins subjected to electrophoresis. • Fractioned proteins were electrotransfered to membrane and detected with YCC2 and YCB9 polyclonal antisera.

  16. Results

  17. Identification of polycystin-2

  18. Polycystin-2 is an integralmembrane protein

  19. Analysis of N-linked glycosylation of polycystin-2

  20. Glycoproteins are complexes in which carbohydrates are attached covalently to asparagine (N-glycans) or serine/threonine (O-glycans) residues of peptides. http://www.glycoforum.gr.jp/science/word/glycoprotein/GPA00E.html

  21. Subcellular distribution of polycystin-2

  22. Conclusion • PKD2 is an integral membrane protein that is widely expressed and highly conserved in mammalian cells and tissues tested. • NH2 terminus appears to be intracellular • Polycystin-2 is an Endo-H sensitive glycoprotein • Polycystin-2 is predominantly present in the endoplasmic reticulum.

  23. Products of PKD1 & PKD2 are transmembrane proteins & a membrane-bound calcium channel

  24. Thank you! Questions?

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