REVIEW Proteomic knowledge of human aquaporins Fulvio Magni 1 , Cecilia Sarto 2 , Davide Ticozzi 1 , Monica Soldi 2 , Niccolò Bosso 1 , Paolo Mocarelli 1, 2 and Marzia Galli Kienle 1 1 Department of Experimental, Environmental Medicine and Medical Biotechnologies, University of Milano-Bicocca, Monza, Italy 2 University Department of Laboratory Medicine, University Milano-Bicocca, Hospital of Desio, Desio (MI), Italy Aquaporins (AQPs) are an ubiquitous family of proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs. At present, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), or also glycerol and other small solutes (AQP3, AQP7, AQP9, AQP10, AQP12). The genomic, structural, and functional aspects of this family are briefly described. In particular, proteomic approaches to identify and characterize the most studied AQPs, mainly through SDS-PAGE followed by MS analysis, are discussed. Moreover, the clinical importance of the best studied aquaporin (AQP1) in human diseases is also provided. Received: March 29, 2006 Revised: June 20, 2006 Accepted: June 20, 2006 Keywords: Aquaporin / Post-translational modifications Proteomics 2006, 6, 5637–5649 5637 1 Introduction The discovery of water transporter aquaporin-1 (AQP1) in red cells and renal tubules dates back to the 1990s, and the struc- ture of several aquaporins (AQPs) has now been determined by studies of site-directed mutagenesis and by 2-D and 3-D electron crystallography [1–5]. Recently, the interest in the AQP family has increased, as emerges from the numerous reviews published over the last few years, with several studies suggesting a role for AQP in various diseases [6]. AQPs belong to the ubiquitous major intrinsic protein (MIP) family, which includes more than 200 membrane channel proteins in plants, microbes, and other members of the animal kingdom. These membrane proteins are characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs [7]. Currently, 13 human AQPs (Table 1, Fig. 1) are known and they are divided into two subgroups according to their ability to transport only water molecules (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), or also glycerol and other small solutes (AQP3, AQP7, AQP9, AQP10, AQP12). 2 Genomic aspects Phylogenetic analysis of aquaporins recognized two major clusters in relation to water channel function and amino acid sequence, including aquaporins and glycerol facilitator-like proteins, respectively [8]. A comprehensive evolutionary anal- ysis that classified the eukaryotic members of the MIP family and considered also proteins not related to the aquaporin functions, came to divergent conclusions and assigned four major clades and two minor clusters to all of these proteins [9]. The 13 known human AQPs can be clustered according to their chromosomal location, tissue distribution, and function: AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6 can be grouped into one gene cluster; AQP3, AQP7 located on 9p13, and AQP9 into another. The remaining AQP8 [10], AQP10 [11], AQP11 [12], and AQP12, encoded on diverse chromo- somes, belong to different clusters (Table 1). The first aquaporin to be discovered was AQP1, encoded on chromosome 7p14, the ancestor of a group of tissue-spe- cific AQPs. Its gene is expressed in a variety of tissues Correspondence: Dr. Cecilia Sarto, Department of Laboratory Medicine, Desio Hospital, Via Mazzini 1, Desio MI 20033, Italy E-mail: sarto@uds.unimib.it Fax: 139-0362-383464 Abbreviations: AQP , aquaporin; ICAT ; isotope-coded affinity tag; MIP , major intrinsic protein; TFE, trifluoroethanol DOI 10.1002/pmic.200600212  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com