@article{f0dc3f2f980b49a0b48a0dabf6072556,
title = "Analysis of the polycystin complex (PCC) in human urinary exosome–like vesicles (ELVs)",
abstract = "The polycystin–1 (PC1), polycystin–2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome–like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C–terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C–terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C–terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is > 2 MDa in size and that N–terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.",
author = "Lea, {Wendy A.} and Kerri McGreal and Madhulika Sharma and Parnell, {Stephen C.} and Lesya Zelenchuk and Charlesworth, {M. Cristine} and Madden, {Benjamin J.} and Johnson, {Kenneth L.} and McCormick, {Daniel J.} and Hogan, {Marie C.} and Ward, {Christopher J.}",
note = "Funding Information: This study was supported by the PKD Biomarkers and Biomaterials Core in the Kansas PKD Research and Translational Core Center (National Institutes of Health (NIH): National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK] grant P30 DK106912). Additional support was from National Institutes of Health NIDDK grants R01DK080688 (“Functional analysis of PKD proteins in urinary exosomes”) and RC1DK086161 (“Analysis of the proteome of PKD–ELVs in polycystic kidney disease and controls”). E8 monoclonal antibody was provided by the NIDDK sponsored Baltimore Polycystic Kidney Disease Research and Clinical Core Center, P30DK090868”. The address of the Core is {\textquoteleft}University of Maryland School of Medicine Division of Nephrology, 655 W. Baltimore St. Baltimore, MD 21201{\textquoteright}. Funding Information: Antibodies.? Antibodies used were anti–N–terminal PC1 7e12 (IgG1κ anti–LRR)35, anti–C–terminal PC1 PKS–A (161F, IgG1κ)) epitope mapped between amino acids (4070..4199)36. PC2 antibodies (H-280): sc-25749 rabbit polyclonal to 689..968 aa (Santa Cruz), YCE2 Polycystin-2 Antibody (YCE2): sc-47734 (IgG2aκ)37. Fibrocystin antibody, Ab 18 (IgG2aκ) to the extracellular portion of the molecule and epitope mapped between residues 143..238 aa38. Anti–CEMIPS_TMEM2 polyclonal antibody (HPA044889 Sigma) produced in rabbit to the cytoplasmic region of CEMIPS_TMEM2 1..69 aa. The E8 antibody, rat monoclonal polycystin-1 (E8) Antibody (E8A-8C3C10 and E8B-8C3D11) was provided by the NIH NIDDK sponsored Baltimore Polycystic Kidney Disease Research and Clinical Core Center, P30DK090868 fqian@medicine.umaryland.edu http://www. baltimorepkdcenter.org/. Publisher Copyright: {\textcopyright} 2020, The Author(s).",
year = "2020",
month = dec,
day = "1",
doi = "10.1038/s41598-020-58087-3",
language = "English (US)",
volume = "10",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",
}