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Am J Physiol Renal Physiol 295: F300-F309, 2008. First published May 14, 2008; doi:10.1152/ajprenal.00006.2008
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INNOVATIVE METHODOLOGY

A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney

Yu-Jung Lee,1 Hyo-Jung Choi,1 Jung-Suk Lim,1 Ji-Hyun Earm,1 Byung-Heon Lee,1 In-San Kim,1 Jørgen Frøkiær,2 Søren Nielsen,2 and Tae-Hwan Kwon1,2

1Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; and 2Water and Salt Research Center, University of Aarhus, Aarhus C, Denmark

Submitted 5 January 2008 ; accepted in final form 13 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

aquaporin; phage display; urine concentration

IN VITRO PHAGE DISPLAY represents an emerging and innovative technology for the rapid isolation of high-affinity peptide ligands (15, 39). Phage display technologies using phages comprising a vast library of peptides have become fundamental to the isolation of high-affinity binding ligands for diagnostic and therapeutic applications, e.g., ligand proteomics, discovery of novel protein-protein interactions, antibody engineering, targeted delivery of therapeutic agents, and development of imaging probes (23, 35, 36, 38). Phage display makes use of bacteriophages that propagate in Escherichia coli to express a wide variety of peptide ligands with high specificity and affinity in vitro. These ligands, displayed on the surface of the phage particle, can be selected against any given target (36, 38).

Regulation of the water permeability of the apical plasma membrane of kidney collecting duct principal cells is critical to the regulation of renal water excretion and body water balance (19). Aquaporin-2 (AQP2) is the predominant vasopressin-regulated water channel protein of the renal collecting duct principal cells (8), where it constitutes the major route of water movement across the apical plasma membrane (19, 31). Vasopressin rapidly increases the osmotic water permeability of the collecting duct epithelium by binding to V2 receptors in the basolateral plasma membrane and inducing the cAMP-dependent translocation of AQP2 from intracellular vesicles in the cytoplasm to the apical plasma membrane of collecting duct principal cells (19, 30, 31). Although this fundamental mechanism is established, the specific intracellular protein targeting pathways involved and protein-protein interactions of proteins in AQP2-expressing vesicles or plasma membrane are not clearly understood.

In the present study, we aimed to isolate high-affinity peptide ligands binding to AQP2-expressing plasma membrane (PM) fractions and AQP2-expressing intracellular vesicle (ICV) fractions from rat kidney by exploiting the in vitro phage display technique. Potential protein candidates were searched, based on motifs homologous to the sequence of isolated high-affinity binding peptide ligands, albeit with a high probability of a random match due to short peptide sequences. These proteins are hypothesized to play a role in vasopressin-induced intracellular trafficking of AQP2 and regulation of AQP2 expression, presumably through functional protein-protein interactions or other signaling mechanisms. For this purpose, 1) we utilized phage display random libraries to identify phage-displayed peptide ligands demonstrating high affinity to the AQP2-expressing PM and/or ICV from rat kidney; 2) phage-displayed peptide ligands of high frequency were selected and examined as to whether they have high affinity to the AQP2-expressing PM and/or ICV fractions compared with a nonrecombinant T7 insertless phage clone; 3) we further validated the high affinity of these selected phage-displayed peptide ligands to the AQP2-expressing PM and/or ICV fractions by control experiments with a binding assay of these selected phage clones to intercalated cell H+-ATPase (B1 subunit)-expressing PM and ICV fractions from rat kidney; 4) to study the subcellular localization of these peptide ligands, synthetic fluorescein-conjugated peptides were localized in primary cultured rat kidney inner medullary collecting duct (IMCD) cells and the response to DDAVP treatment (10–8 M for 20 min) was examined; and finally 5) potential protein candidates that contain motifs homologous to the identified high-affinity peptide ligands were selected by library analyses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation for plasma membrane fractions and intracellular vesicle fractions from rat kidney. Pathogen-free male Sprague-Dawley rats (200–250 g) were obtained from Charles River (Orient Bio, Seongnam, Korea). The animal protocols were approved by the Animal Care and Use Committee of Kyungpook National University, and all animal experiments were conducted according to the guidelines of Kyungpook National University. The rats were anesthetized under light enflurane inhalation, and both kidneys were rapidly removed. Whole kidneys were placed on an ice-cold petri dish, dissected, and homogenized in 10 ml of dissecting buffer (0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, containing protease inhibitors: 8.5 µM leupeptin and 1 mM phenylmethylsulfonyl fluoride). This homogenate was centrifuged at 4,000 g for 15 min at 4°C (Eppendorf 5810R, Hamburg, Germany) to remove nuclei, mitochondria, and any remaining large cellular fragments (20, 21, 25). The supernatants were collected, and low-speed (LS) PM fractions and high-speed (HS) ICV fractions were prepared consecutively by centrifugation (Beckman Optima L-100XP with Ti-90 rotor) of the supernatant at 17,000 g for 30 min (LS) and 200,000 g for 1 h (HS), respectively (25). The LS pellet represented fractions enriched for PM, and the HS pellet represented fractions enriched for ICV, as previously demonstrated (25, 41). The pellets were resuspended in dissecting buffer.

Immunoisolation of AQP2-expressing plasma membrane fractions and AQP2-containing intracellular vesicle fractions from whole kidney in rat. Membrane fractions enriched either for PM (LS) or ICV (HS) from rat whole kidney were prepared as described above. Both magnetic beads (Dynal M-280, Dynal Biotech ASA, Oslo, Norway; precoated with anti-rabbit IgG) and an affinity-purified anti-AQP2 antibody (H7661AP, ~2 µg/107 beads) (26, 29) were incubated with either the PM fractions or the ICV fractions overnight at 4°C with continuous agitation.

For immunoblotting to detect AQP2, samples were carefully washed in 0.1% BSA in PBS three times for 10 min each, and the complex of magnetic beads + AQP2 antibody + PM or ICV fractions was separated magnetically. Pellets (magnetically isolated) were mixed with Laemmli sample buffer (10 mM Tris, pH 6.8, 1.5% SDS, 6% glycerol), followed by heating to 60°C for 15 min to solubilize proteins. The beads were then removed magnetically, and immunoisolated samples were used for immunoblotting (Fig. 1). For control experiments to examine the specificity of AQP2 membrane isolation, preimmune rabbit IgG pulldown samples (PM and ICV fractions) were subjected to AQP2 immunoblotting (Fig. 1A). Moreover, immunoisolated AQP2-expressing membrane fractions (PM and ICV) and immunoisolated H+-ATPase (B1 subunit)-expressing membrane fractions (PM and ICV) were subjected to AQP2, AQP1, and H+-ATPase immunoblotting (Fig. 1B).


Figure 1
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  		Fig. 1. Immunoblotting of aquaporin-2 (AQP2) (A) or AQP2, AQP1, and H+-ATPase (B). A: membrane fractions enriched for either plasma membrane (PM) or intracellular vesicles (ICV) from rat whole kidney (WK; nonimmunoisolated) were prepared and immunoisolated with anti-AQP2 antibody or preimmune rabbit IgG. Gels were stained with Coomassie blue dye to ensure that loading in the lanes was consistent. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV) compared with the nonimmunoisolated (WK) and preimmune IgG pulldown rat kidney samples. B: membrane fractions enriched for either PM or ICV from rat whole kidney (nonimmunoisolated) were prepared and immunoisolated with anti-AQP2 antibody or anti-H+-ATPase (B1 subunit) antibody. Gels were stained with Coomassie blue dye to ensure that loading in the lanes was consistent. AQP1 or H+-ATPase (B1 subunit) expression was minimal in the immunoisolated AQP2 membrane fractions, indicating the specificity of AQP2 membrane isolation.

 
For in vitro phage display, samples were carefully washed in 0.1% BSA in PBS three times for 10 min each for removal of unbound protein, and the complex (i.e., magnetic beads + AQP2 antibody + PM or ICV fractions) was obtained magnetically (see Biopanning of a T7 phage library).

Electrophoresis and immunoblotting of AQP2, H+-ATPase, and AQP1. To load an equal amount of protein in each lane, gels were stained with Coomassie blue dye (GelCode Blue Stain Reagent, Pierce) and loading volume was adjusted according to the band density of each lane in Coomassie blue-stained gels (20, 21, 29). SDS-PAGE was performed on 12% polyacrylamide gels (20, 21, 29). One gel was stained with Coomassie blue dye to ensure that loading in the lanes was consistent, while the other was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h, and incubated with anti-AQP2 antibody (H7661AP, 1:500; Ref. 29), anti-H+-ATPase (LL615AP, 1:1,000; Ref. 18), or anti-AQP1 (2353AP, 1:1,000; Ref. 32). The AQP2 antibody was raised against a synthetic peptide corresponding to the COOH terminus of rat AQP2 (8) with an added NH2-terminal cysteine for conjugation (NH2-CEVRRRQSVELHSPQSLPRGSKA-COOH, amino acids 250–271). The labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P448, diluted 1:3,000; DAKO, Glostrup, Denmark) with an enhanced chemiluminescence (ECL) system and exposure to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech, Little Chalfont, UK).

Biopanning of a T7 phage library. A phage peptide library based on T7 415–1b phage vector displaying CX7C (randomly sequenced 7-amino acid residue inserted between 2 cysteines) was constructed according to the manufacturer's instructions (Novagen, Madison, WI) (23). The library had a diversity of ~5 x 108 plaque-forming units (PFU). Biopanning and validation of the phage library were performed (Figs. 2 and 3) as previously described (12, 23). Briefly, an aliquot (1 x 1011 PFU) of the T7 phage library (Fig. 2A) was allowed to bind to the complex (i.e., magnetic beads + AQP2 antibody + PM or ICV fractions) by rotating gently at 4°C overnight (Fig. 2B). For this, a T7 phage library was incubated with magnetic beads (Dynal M-280; Dynal Biotech ASA, Oslo, Norway) that were precoated with anti-rabbit IgG antibodies. The phages that nonspecifically bound to magnetic beads were removed magnetically, and then the remaining unbound phage library was used for incubation with the complex of magnetic beads + AQP2 antibody + PM or ICV fractions at 4°C overnight. The next day, after washing 10 times with 1 ml of M9/LB medium (10 g bactotryptone, 5 g yeast extract, 10 g NaCl, 1 g NH4Cl, 3 g KH2PO4, 3 g NaHPO4·7H2O per liter containing 0.4% glucose and 1 mM MgSO4) to remove nonspecifically bound phages (Fig. 2C), the phages bound to the complex were eluted (Fig. 2D) and were subjected to a plaque assay for counting the number of phage clones (Fig. 2E). Moreover, these phages were used to infect a log-phase culture of E. coli for amplification (Fig. 2F). The amplified phages were again subjected to binding to a newly prepared complex of magnetic beads + AQP2 antibody + PM or ICV fractions by an identical procedure (Fig. 2). After three rounds of panning in the same manner, 80 plaques from the PM fractions and 80 plaques from the ICV fractions were randomly picked up from LB plates (Fig. 3A). Each phage clone was dissolved in 10 µl of TBS (3.0285 g Tris, 4.3838 g NaCl per 500 ml, pH 7.5) and sequenced (Fig. 3B) as described below. Seven phage clones were chosen, which revealed amino acid sequences of high frequency and included similar ones listed alongside (Table 1). We examined the high-affinity binding of these seven phage clones onto the AQP2-immunoisolated fractions (both PM and ICV fractions), relative to that by the T7 insertless phage clone, which did not display peptide ligands on the surface of the phage particle (Fig. 3, C–E). Moreover, to validate the high affinity of these phage clones to the AQP2-expressing PM and/or ICV fractions, a binding assay of selected seven phage clones was performed to intercalated cell H+-ATPase (B1 subunit)-expressing PM and/or ICV of the whole kidney.


Figure 2
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  		Fig. 2. Flow diagram for biopanning of T7 phage and plaque assay. A and B: an aliquot (1 x 1011 plaque-forming units) of the T7 phage library was allowed to bind to the complex (i.e., magnetic beads + AQP2 antibody + PM or ICV fractions). C–E: after washing to remove nonspecifically bound phage (C), the phages bound to the complex were eluted (D) and subjected to a plaque assay for counting the number of phage clones (E). F: these phages were used to infect a log-phase culture of Escherichia coli for amplification. The amplified phages were again subjected to binding to a newly prepared complex of magnetic beads + AQP2 antibody + PM or ICV fractions by an identical procedure. Three rounds of panning were performed in the same manner.

 

Figure 3
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  		Fig. 3. Validation of the selected phage clones to the AQP2-expressing PM and/or ICV fractions. A: after 3 rounds of panning, 80 plaques from the PM fractions and 80 plaques from the ICV fractions were randomly picked up from LB plates. B: each phage clone was sequenced, and 7 phage clones were chosen, which revealed amino acid sequences of high frequency. C: high-affinity binding of these 7 phage clones was examined by incubating each phage clone to the AQP2-immunoisolated fractions (both PM and ICV fractions), relative to that by T7 insertless phage clone, which did not display peptide ligands on the surface of the phage particle. D and E: phages bound to the complex were eluted (D) and subjected to a plaque assay for counting the number of phage clones (E). Thus the high number of each eluted phage clone indicates the high affinity of each phage clone to the AQP2-immunoisolated fractions.

 

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  		Table 1. Incidence of frequently occurring phage clones and similar sequences of phage clones

 
PCR amplification of plaques and library analyses. The phage clones dissolved in TBS served as a template in a PCR reaction with primers 5'-AGCGGACCAGATTATCGCTA-3' and 5'-AACCCCTCAAGACCCGTTTA-3' (Genotech). PCR was performed with Top-Taq PreMix (2x) version 3.0 (Corebio) under the following conditions: 95°C for 3 min, followed by 35 cycles (94°C for 50 s, 50°C for 1 min, 72°C for 1 min) and final extension at 72°C for 6 min. Four microliters of the reaction product was loaded on a 2% agarose gel to check the proper PCR amplification. After the phage DNA was amplified by PCR, the fragments were purified and sequenced to determine the corresponding amino acid sequence displayed on the T7 phage capsid. The DNA insert of the selected phage clones were sequenced by Koma Biotech (Daejeon, Korea).

Library analyses identified the rat proteins (see Tables 4–Go6) containing the sequence homologous to a peptide ligand with the NCBI BLAST program against the SWISSPROT database accessible at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The Expect (E) value, 1 which describes the random background noise when searching a database of each peptide sequence, is demonstrated in Tables 4–6.


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  		Table 4. Selected phage clones enriched from ICV fractions and rat proteins containing motifs homologous to each peptide sequence

 

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  		Table 5. Selected phage clones enriched from PM fraction and rat proteins containing motifs homologous to each peptide sequence

 

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  		Table 6. Selected phage clones enriched from both PM and ICV fractions and rat proteins containing motifs homologous to each peptide sequence

 
Primary culture of rat kidney IMCD cells and immunolabeling of fluorescein-conjugated peptides. Primary cultures enriched in IMCD cells were prepared from pathogen-free male Sprague-Dawley rats (200–250 g, Charles River) as previously described (4, 24). The IMCD cell suspension was seeded in human fibronectin-coated chamber slides (Lab-Tek Chamber Slides System, catalog no. 177402, NUNC, Roskilde, Denmark). IMCD cells were fed every 24 h and were grown in hypertonic culture medium (640 mosmol/kg H2O) supplemented with 10% fetal bovine serum at 37°C in 5% CO2-95% atmospheric air for 3 days and then in fetal bovine serum-free culture medium for an additional day. At day 5, IMCD cells were subjected to treatment with vehicle or DDAVP (10–8 M) for 20 min and then fixed with 2.5% paraformaldehyde in PBS, pH 7.4 for 20 min at room temperature. After fixation, cells were washed twice in PBS and permeabilized with 0.3% Triton X-100 in PBS at room temperature for 15 min. Cells were washed, blocked with 1% BSA in 0.01 M PBS for 30 min, and labeled with a fluorescein-labeled peptide (10 µM concentration each) at 4°C overnight. Synthetic peptides (PKQRFWP, KNMRSSA, and SRSRNKT) were conjugated to FITC (Peptron, Daejeon, Korea). Control peptide (NSSVDK) conjugated to FITC was used for the control experiment. Cells were washed and mounted with a hydrophilic mounting medium containing antifading reagent (catalog no. P36930 [GenBank] , Molecular Probes, Eugene, OR). Fluorescent microscopy was carried out with a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Immunoisolation of AQP2-expressing PM and ICV membrane fractions. Membrane fractions enriched for either PM or ICV from rat whole kidney were prepared, and these membrane fractions were immunoisolated with anti-AQP2 antibody, anti-H+-ATPase (B1 subunit) antibody, or preimmune rabbit IgG. Immunoblotting revealed that AQP2 was predominantly expressed in the immunoisolated AQP2 membrane fractions (PM and ICV fractions), compared with its expression in the nonimmunoisolated membrane fractions from rat whole kidney and the preimmune IgG pulldown samples (Fig. 1A). Moreover, AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV fractions) compared with its expression in the nonimmunoisolated membrane fractions from rat whole kidney and the H+-ATPase membrane isolation (Fig. 1B). AQP1 was abundantly expressed in the nonimmunoisolated membrane fractions compared with the immunoisolated AQP2 and H+-ATPase membrane fractions (Fig. 1B). Similarly, H+-ATPase was strongly expressed in the H+-ATPase membrane fractions compared with the nonimmunoisolated membrane fractions and the immunoisolated AQP2 membrane fractions (Fig. 1B). These data therefore indicate the specificity of AQP2- and H+-ATPase-expressing membrane isolation.

Selection of phage clones showing high affinity to AQP2-containing PM fractions and/or AQP2-containing ICV fractions from rat kidney. A T7 phage library expressing CX7C random peptides was screened to enrich phage clones that selectively bind to AQP2-expressing PM and/or AQP2-containing ICV. During three rounds of screening, phage clones were significantly enriched in each step (for both PM and ICV fractions; data not shown). Eighty phage clones from an enriched phage library for AQP2-expressing PM fractions and another 80 phage clones from an enriched phage library for AQP2-expressing ICV fractions were picked up randomly. These were all sequenced after amplification of the phage DNA by PCR to determine the corresponding amino acid sequence displayed on the T7 phage capsid. Seven phage clones were chosen from these randomly selected 160 phage clones out of the enriched phage libraries for AQP2-expressing PM fractions and ICV fractions, since they demonstrated a high number of identical or similar amino acid sequences (Table 1). Among these, phage clones displaying the sequences of CPKQRFWPC (7 identical sequences/160 phage clones: 4.4%), CKRVTGRPC (5/160: 3.1%), and CKNMRSSAC (5/160: 3.1%) constituted ~11% of all the randomly selected phage clones (total 160). They were examined by a plaque assay for counting selective binding to the AQP2-immunoisolated fractions (either PM or ICV fractions) of rat whole kidney. Each binding was then compared with that exerted by the nonrecombinant T7 insertless phage clone, which displayed no peptide ligand on the surface of the phage particle (Fig. 3, Table 2). Seven phage clones showed high-affinity binding to the AQP2-expressing PM and/or ICV fractions (~50- to 5,600-fold) compared with the binding of the T7 insertless phage clone (Table 2). Moreover, to further examine the high affinity of these phage clones to AQP2-expressing PM and/or ICV fractions, another binding assay of seven phage clones was performed with intercalated cell H+-ATPase (B1 subunit)-containing PM and/or ICV fractions from rat whole kidney (Table 3). All of these showed lower affinity (0.4- to 3.5-fold compared with binding of nonrecombinant T7 insertless phage clone; Table 3) to H+-ATPase-expressing membrane fractions, relative to their high affinity to AQP2-expressing membrane fractions. This finding could indicate the high selectivity of these seven phage clones for AQP2-expressing membrane fractions.


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  		Table 2. Binding of seven phage clones to magnetic beads, AQP2-expressing PM fractions, and AQP2-expressing ICV fractions (in comparison to binding of nonrecombinant T7 insertless phage)

 

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  		Table 3. Binding of seven phage clones to H+-ATPase-expressing PM fractions and H+-ATPase-expressing ICV fractions (in comparison to binding of nonrecombinant T7 insertless phage)

 
Screening of a phage-displaying peptide library. As seen in Table 1, alignment analysis of the peptide sequences revealed 7 frequently occurring sequences out of the 80 phage clones from PM fractions and another 80 phage clones from ICV fractions. Three peptide sequences were obtained from the ICV fractions, one from the PM fractions, and three from both PM and ICV fractions. Rat protein candidates that have motifs homologous to each peptide sequence were searched by the BLAST program accessible at the National Center for Biotechnology Information (Tables 4 –6). It should be noted that the sequences identified did not match the proposed proteins exactly and a high probability of random match due to short peptide sequences was seen, as demonstrated by high E values (Tables 4–6).

Labeling of peptides conjugated with FITC in primary cultured IMCD cells. We previously demonstrated (24) significantly increased AQP2 targeting to the plasma membrane in response to short-term V2 receptor agonist DDAVP treatment (10–11 M, 15 min) in primary cultured IMCD cells. To examine the subcellular localization of the peptide ligands and whether short-term DDAVP treatment altered the subcellular localization of the peptide ligands, particularly for the phage clones that were from the enriched phage library for AQP2-expressing ICV (i.e., SRSRNKT and KNMRSSA), these peptides were conjugated with FITC and cytochemistry was done in primary cultured IMCD cells in the absence or the presence of DDAVP (10–8 M) treatment for 20 min (Fig. 4). In the absence of DDAVP treatment, FITC-SRSRNKT staining was exclusively seen intracellularly (Fig. 4A), and FITC-KNMRSSA staining was associated with both intracellular compartment (Fig. 4C) and plasma membrane (arrows in Fig. 4C). In response to short-term DDAVP treatment (10–8 M, 20 min), no changes of subcellular labeling patterns were seen in both FITC-SRSRNKT and FITC-KNMRSSA-labeled cells (Fig. 4, B and D). In contrast, control peptide conjugated with FITC (FITC-NSSVDK) was unlabeled or very weakly labeled in the primary cultured IMCD cells (Fig. 4, G and H). In addition, we also observed the labeling pattern of FITC-PKQRFWP, the phage clone that was from the enriched phage library of both AQP2-expressing PM and ICV, in both intracellular compartment and plasma membrane (Fig. 4E). This was consistent with the finding that this phage clone showed high affinity to both PM and ICV fractions (Table 2). However, the labeling pattern was unchanged in response to short-term DDAVP treatment (Fig. 4F).


Figure 4
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  		Fig. 4. Fluorescence microscopy of FITC-conjugated peptides in primary cultured inner medullary collecting duct (IMCD) cells. A and C: FITC-SRSRNKT staining was exclusively seen intracellularly, and FITC- KNMRSSA staining was associated with both intracellular compartment and PM in nonstimulated IMCD cells. B and D: in response to short-term DDAVP treatment (10–8 M, 20 min), no changes of subcellular labeling pattern were seen in both FITC-SRSRNKT- and FITC-KNMRSSA-labeled cells. E and F: FITC-PKQRFWP staining was associated with both intracellular compartment and PM, and this was not changed by short-term DDAVP treatment. G and H: control peptide conjugated to FITC (FITC-NSSVDK) was unlabeled or very weakly labeled in primary cultured IMCD cells in the absence (G) or presence (H) of DDAVP treatment (10–8 M, 20 min).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we aimed to perform ligand peptidomics by applying an in vitro phage display technique for isolating high-affinity peptide ligands that bind to immunoisolated AQP2- expressing PM and/or ICV fractions of rat kidney. 1) Seven phage clones of high frequency were selected, which showed high affinity to the AQP2-containing PM and/or ICV fractions compared with the binding of a nonrecombinant T7 insertless phage clone. Moreover, these phage clones showed lower affinity to H+-ATPase (B1 subunit)-containing membrane fractions, indicating the selectivity of these phage clones. 2) Fluorescein-conjugated peptide labeling also suggested high affinity to collecting duct cells with subcellular localization of the intracellular compartment and plasma membrane of primary cultured IMCD cells. 3) Library analysis identified proteins having motifs homologous to each high-affinity peptide ligand. This study, for the first time to our knowledge, demonstrated that in vitro phage display technology could be exploited as an experimental tool for the selective isolation of high-affinity peptide ligands to the AQP2-expressing membrane of the kidney tubule epithelia.

Phage libraries displaying a large number of random peptide sequences on the surface of bacteriophages have been used to define peptides that target specific binding sites (i.e., monoclonal antibodies, carbohydrates, receptors, specific proteins, cells, tissues, organs, or tumors; Refs. 5, 6, 10, 38, 39). Phage libraries can have as many as 1010 different peptides, and peptides have several advantages over antibodies as a targeting moiety, e.g., better tissue penetration and less chance of unintended immune reactions (22). A prominent example of a targeting peptide is the three-amino acid-sequence RGD motif, which homes to tumor vascular endothelial cells through high-affinity binding to the {alpha}vβ3-integrin exclusively expressed in the angiogenic endothelial cells (1, 34). Importantly, previous studies identified several peptide ligands selectively binding to kidney tubular segments (2, 33). Odermatt et al. (33) performed ex vivo screening of phage display peptide libraries on microdissected intact rat proximal convoluted tubules (PCT) and cortical collecting ducts (CCD). They found two distinct peptide motifs for CCD and PCT. In particular, a linear nonapeptide [ELRGD(R/M)AX(W/L)] containing an RGD sequence was highly selective for rat CCD, which was preferentially associated with basolateral cell surface as seen by fluorescence microscopy. Since an RGD sequence is known to mediate binding to the integrin family of receptors, integrin receptors expressed in the basolateral cell surface of the CCD could play a role in the interaction with extracellular matrix proteins and adhesion molecules. Moreover, we recently identified that CSNRDARRC peptide is the selective targeting peptide for bladder cancer cells by phage display technology (23). The specificity of the CSNRDARRC peptide was demonstrated by its selective binding to cultured HT-1376 human bladder cancer cells and to cell suspensions prepared from primary human tumors. When the FITC-conjugated peptide is instilled into the bladder lumen of rats bearing carcinogen-induced bladder cancers in vivo, the peptide selectively binds to bladder tumor epithelium. These results therefore suggest that identification of peptide ligands selectively binding to their corresponding receptors by phage display technology would be exploitable for the development of new diagnostic and therapeutic tools (e.g., development of vectors for cell- or tissue-specific drug delivery, gene transfer, or development of image probes).

In the present study, we take one step forward to identify high-affinity peptide ligands binding to the membrane expressing a specific protein of interest, i.e., AQP2, the vasopressin-regulated water channel protein in the kidney collecting duct principal cell. During three rounds of screening, phage clones were significantly enriched in each step. Finally, 7 phage clones, which demonstrated identical amino acid sequences of high frequency, were chosen from 160 randomly selected phage clones out of the enriched phage libraries for AQP2-expressing PM fractions and ICV fractions. Among these, phage clones displaying the sequences of CPKQRFWPC, CKRVTGRPC, and CKNMRSSAC constituted ~11% of all the randomly selected phage clones (total 160). In contrast, none of these seven phage clones had a sequence of RGD. This finding was in contrast to a previous observation that RGD sequence-containing phage clones were predominantly recovered from the microdissected rat CCD (33). The difference may be explained by the observation that the clones having an RGD sequence were selective for the basolateral cell surface of the CCD, where they have high affinity to integrin that plays a role in the interaction with extracellular matrix proteins and adhesion molecules. In contrast, peptide sequences of the identified phage clones in the present study were selective for the AQP2-expressing membrane fractions (i.e., apical plasma membrane and intracellular subapical vesicles).

In addition, we were tempted to select protein candidates that have motifs homologous to the identified high-affinity peptide ligands by library analyses, on the assumption that these proteins may be involved in the specific intracellular targeting pathways and protein-protein interactions of the AQP2-expressing vesicles. Overall, our results demonstrated a number of proteins known to populate the collecting duct proteome as shown in previous studies using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) of IMCD proteins (3, 37), e.g., serine/threonine protein kinase, phosphodiesterase, protein phosphatase, cAMP-dependent protein kinase, annexin, synaptotagmin-like protein, myosin, synaptic vesicle-related protein, ubiquitin-protein ligase, endothelin receptor, angiotensin AT1 receptor, bassoon protein, and Wiskott-Aldrich syndrome protein. Importantly, Yu et al. (44) recently identified proteins associated with the apical plasma membrane of the rat IMCD. They used surface biotinylation to label proteins with extracellular lysines, followed by LC-MS/MS to identify the labeled proteins and peripheral membrane proteins bound to labeled proteins. Consistent with our presumptive list of proteins shown in Tables 4–6, the examples of bound proteins were PKA β-catalytic unit, myosin 9, bassoon protein, and Wiskott-Aldrich syndrome protein, which have potential roles in AQP2 regulation and in mediation of cellular responses to vasopressin. The presumptive proteins listed in Tables 4–6 can be research subjects for further hypothesis-driven studies for the regulation of AQP2 trafficking and AQP2 protein expression. Some of these were previously studied for involvement in DDAVP-induced AQP2 trafficking and AQP2 regulation (4, 7, 9, 11, 13, 16, 17, 19, 21, 26, 28, 40). However, further studies are warranted to examine whether these proteins are involved in AQP2 targeting/regulation pathways and protein-protein interactions of AQP2-expressing vesicles and what the underlying mechanisms are for better understanding of urinary concentration at the molecular level.

On the other hand, it should be emphasized that there are limitations to identification of specific protein candidates by library analysis using short peptide sequence. Because the peptide sequences we identified were short (9 amino acids consisting of a randomly sequenced 7-amino acid residue between 2 cysteines) and did not match exactly the sequence of listed natural proteins, the probability of random match was high, which was revealed by high E values in the BLAST. Per NCBI BLAST definition the P value of random matches is calculated as P = 1 – e–E. Accordingly, E values higher than 15 yield a P value of 1, i.e., 100% of random match. As an example, the annexin A4 has an E value of 26. Thus the novelty of the method and its application for ligand proteomics are undermined by the specificity of identification issue. Because of the low specificity of identification, the results can only give us a presumptive list of proteins that might be involved in vasopressin-regulated AQP2 targeting and/or AQP2 expression. To confirm their expression in the kidney, further studies are needed. Another limitation in this study was that interacting partners or receptors in the membrane for each peptide ligand are currently unknown. Identification of these receptors should be pursued in future studies, where the identification of peptide ligand-receptor pairs could serve as an important target for the treatment of AQP2 dysregulation and abnormal cellular responses to vasopressin. Moreover, the phage-displayed peptide libraries were incubated with AQP2-expressing membranes that were already bound to AQP2 antibody-conjugated Dynabeads. Therefore, more specifically, it is not possible to pick up the specific phage clones that will target selectively to the site of AQP2 protein itself when the binding site is already blocked by the antibody. In addition, since the selected phage clones were selective for the AQP2-expressing membrane fractions of the collecting duct principal cells (not the AQP2 protein itself), potentially these clones may also have high affinity to the other principal cell protein-enriched membrane fractions, e.g., urea transporters. This point needs to be examined in further studies.

In vivo homing of phage clones to the kidney tubular epithelial cells would be of an interest. In general, viral vector delivery to kidney tubular epithelial cells, which are nonproliferating and terminally differentiated cells, is known to be inefficient, since high-affinity viral receptors are lacking on the cell surface (42, 43). Consistent with this, McDonald et al. (27) previously demonstrated that perfusion of kidney with modified adenovirus led to the transduction of kidney cortical vasculature but not parenchymal cells. However, recent findings of phage homing to tumor cells after systemic circulation (1) and direct exposure (i.e., bladder instillation to bladder carcinoma; Ref. 23) or to atherosclerotic plaques after systemic circulation (14) may enable us to develop an efficient targeting system for in vivo homing of phage clones displaying selective peptide ligands to the surface or inside kidney tubular epithelial cells. If this is attainable, particularly for kidney tubular epithelial cells, identification of specific phage clones could contribute to the development of improved vectors for kidney-specific drug delivery or gene transfer, e.g., for the treatment of AQP2 dysregulation and abnormal cellular responses to vasopressin.


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This study was supported by the Korea Research Foundation Grant funded by the Korean government (KRF-2006-521-E00007), a Korea Science and Engineering Foundation grant funded by the MOST (R01-2007-000-20441-0), the Brain Korea 21 Project in 2008, the Danish National Research Foundation (Danmarks Grundforskningsfond), the Danish Medical Research Council, the WIRED program (Nordic Centre of Excellence in Molecular Medicine), and European Commission FP6 via the RTN "AQUAGLYCEROPORINS" (project 035995-2).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T.-H. Kwon, Dept. of Biochemistry and Cell Biology, School of Medicine, Kyungpook National Univ., Dongin-dong 101, Taegu 700-422, Korea (e-mail: thkwon{at}knu.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The Expect (E) value is a parameter that describes the number of hits one can "expect" to see by chance when searching a database of a particular size. Essentially, the E value describes the random background noise. For example, an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. However, virtually identical short alignments have relatively high E values. This is because the calculation of the E value takes into account the length of the query sequence (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Back


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