A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney.
Aquaporin-2 (AQP2), the vasopressin-regulated water channel in 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 the in vitro phage display technique. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV), compared with the nonimmunoisolated or 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 a nonrecombinant 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 inner medullary collecting duct cells, relative to absent or very weak labeling with fluorescein-conjugated control peptide. Library analyses demonstrated proteins that had motifs homologous to the peptide ligands, albeit with a high probability of a random match due to short peptide sequences. In summary, we applied the in vitro phage display technique to identify high-affinity peptide ligands to AQP2-expressing membranes. Library analyses identified proteins having homologous motifs, which need to be examined for involvement in AQP2 trafficking and regulation.
Lee
Yu-Jung
YJ
Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea.
Choi
Hyo-Jung
HJ
Lim
Jung-Suk
JS
Earm
Ji-Hyun
JH
Lee
Byung-Heon
BH
Kim
In-San
IS
Frøkiaer
Jørgen
J
Nielsen
Søren
S
Kwon
Tae-Hwan
TH
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008024:Ligands; D008297:Male; D008856:Microscopy, Fluorescence; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism
C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D008024:Ligands; D019151:Peptide Library; D010455:Peptides