TY - JOUR
T1 - Differential expression of AQP1 in microdomain-enriched membranes of renal cell carcinoma
AU - Ticozzi-Valerio, Davide
AU - Raimondo, Francesca
AU - Pitto, Marina
AU - Rocco, Francesco
AU - Bosari, Silvano
AU - Perego, Roberto
AU - Sarto, Cecilia
AU - Di Fonzo, Andrea
AU - Bosso, Niccolò
AU - Mocarelli, Paolo
AU - Galli-Kienle, Marzia
AU - Magni, Fulvio
PY - 2007/6
Y1 - 2007/6
N2 - Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex.
AB - Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex.
KW - Aquaporin-1
KW - Clear cell renal cell carcinoma
KW - Microdomain-enriched membranes
KW - MS
KW - Subcellular fractions
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U2 - 10.1002/prca.200601048
DO - 10.1002/prca.200601048
M3 - Article
C2 - 21136710
AN - SCOPUS:38149008944
VL - 1
SP - 588
EP - 597
JO - Proteomics - Clinical Applications
JF - Proteomics - Clinical Applications
SN - 1862-8346
IS - 6
ER -