Submitted on January 5, 2008
Accepted on May 13, 2008
A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney
Yu-Jung Lee1, Hyo-Jung Choi1, Jung-Suk Lim1, Ji-Hyun Earm1, Byung-Heon Lee1, In-San Kim1, Jorgen Frokiaer2, Soren Nielsen2, and Tae-Hwan Kwon3*
1 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea, Republic of
2 The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark
3 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea, Republic of; The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark
* To whom correspondence should be addressed. E-mail: thkwon{at}knu.ac.kr.
Aquaporin-2 (AQP2), the vasopressin-regulated water channel in the collecting duct principal cells, plays a key role in the regulation of body water balance. We aimed to isolate high-affinity peptide ligands that bind to immunoisolated AQP2-expressing plasma membrane (PM) or intracellular vesicle (ICV) preparations from rat kidney by in vitro phage display technique. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV), compared with the non-immunoisolated or the preimmune IgG pulldown rat kidney samples. Moreover, AQP1 or H+-ATPase (B1-subunit) expression was minimal in the immunoisolated AQP2 membrane fractions, indicating the specificity of AQP2 membrane isolation. A phage peptide library based on T7 415-1b phage vector displaying CX7C was constructed. After three rounds of biopanning, seven phage clones of high frequency were selected, which showed high-affinity to the AQP2-containing PM or ICV fractions compared with non-recombinant T7 insertless phage clone. In contrast, these phage clones showed lower affinity to H+-ATPase-containing fractions. Fluorescein-conjugated peptide labeling was associated with intracellular compartment and PM of primary cultured IMCD cells, relative to absent or very weak labeling using fluorescein-conjugated control peptide. Library analyses demonstrated proteins which had homologous motifs to the peptide ligands, albeit it with a high probability of a random match due to short peptide sequences. In summary, we applied in vitro phage display technique to identify high-affinity peptide ligands to the AQP2-expressing membranes. Library analyses identified proteins having homologous motifs, which need to be examined for the involvement in the AQP2 trafficking and regulation.
