Screening of a specific peptide binding to VPAC1 receptor from a phage display peptide library

Abstract

Background/purpose: The VPAC1 receptor, a member of the vasoactive intestinal peptide receptors (VIPRs), is overexpressed in the most frequently occurring malignant tumors and plays a major role in the progression and angiogenesis of a number of malignancies. Recently, phage display has become widely used for many applications, including ligand generation for targeted imaging, drug delivery and therapy. In this work, we developed a panning procedure using a phage display peptide library to select a peptide that specifically binds to the VPAC1 receptor to develop a novel targeted probe for molecular imaging and therapy.

Methods: CHO-K1 cells stably expressing VPAC1 receptors (CHO-K1/VPAC1 cells) were used to select a VPAC1-binding peptide from a 12-mer phage peptide library. DNA sequencing and homologous analysis of the randomly selected phage clones were performed. A cellular ELISA was used to determine the most selectively binding peptide for further investigation. Binding specificity to the VPAC1 receptor was analyzed by competitive inhibition ELISA and flow cytometry. The binding ability of the selected peptide to CHO-K1/VPAC1 cells and colorectal cancer (CRC) cell lines was confirmed using fluorescence microscopy and flow cytometry.

Results: A significant enrichment of phages that specifically bound to CHO-K1/VPAC1 cells was obtained after four rounds of panning. Of the selected phage clones, 16 out of 60 shared the same peptide sequence, GFRFGALHEYNS, which we termed the VP2 peptide. VP2 and vasoactive intestinal peptide (VIP) competitively bound to the VPAC1 receptor. More importantly, we confirmed that VP2 specifically bound to CHO-K1/VPAC1 cells and several CRC cell lines.

Conclusion: Our results demonstrate that the VP2 peptide could specifically bind to VPAC1 receptor and several CRC cell lines. And VP2 peptide may be a potential candidate to be developed as a useful diagnostic molecular imaging probe for early detection of CRC.

Figure 1. Stable expression of the recombinant human VPAC1 receptor in CHO-K1 cells.

(A) Reverse transcription PCR of the VPAC1 gene expression. M: DNA marker DL 5000 bp, lane 1 and lane 2: VPAC1 gene expression in CHO-K1 cells transfected with pcDNA3.1(+)/VPAC1 plasmid, lane 3: VPAC1 gene expression in CHO-K1 cells, lane 4 and lane 5: GAPDH in CHO-K1 cells transfected and non-transfected with pcDNA3.1(+)/VPAC1 plasmid. (B) Western blot analysis of VPAC1 expression. Migration of molecular weight marker is indicated on the left of the blot. CHO-K1 cells transfected with pcDNA3.1(+)/VPAC1 plasmid yielded a single prominent band at approximately 58 kDa. CHO-K1 cells as a negative control. (C) Immumofluorescence analysis of VPAC1 expression. VPAC1 receptor was expressed on the cell membrane and accumulated in the cytoplasm of positive CHO-K1/VPAC1 cells (a), (b). CHO-K1 cells as the negative control (c), (d). (b), (d) represents the merged image.

Figure 2. Specific enrichment of recovered phages.

A specific enrichment of phages binding to CHO-K1/VPAC1 cells was seen after four rounds of panning. The titers of the recovered phages from each round were evaluated by the blue plaque-forming assay on LB/IPTG/X-gal plates. Here, Mp represents phages recovered from an acid elution fraction, INp represents phages recovered from a lysate fraction and CHO-K1 denotes phages recovered from CHO-K1 cells.

Figure 3. Identification of the binding selectivity of the 18 clones by cellular ELISA.

Phage clones binding to CHO-K1/VPAC1 cells (blue bars) and wild-type CHO-K1 cells (red bars) were detected by the HRP-conjugated anti-M13 phage antibody. PBS and URps (unrelated phage, an amplified phage randomly selected from the original phage peptide library) were used as negative controls. Triplicate determinations were done at each data point, and average OD_{450 nm} of two types of cells are shown. Single asterisk denotes p<0.01(OD_{450 nm} of each clone binding to CHO-K1/VPAC1 cells versus CHO-K1 cells).

Figure 4. Competitive inhibition of binding of the positive phage clone VP2 to CHO-K1/VPAC1 cells by the synthetic peptide VP2.

The average inhibition rates at different concentrations of the VP2 peptide were shown. When the concentration of VP2 peptide was increased above 0.001 µg/ml, a significant inhibition occurred. An unrelated peptide displayed by the unrelated phage was used as a negative control.

Figure 5. Binding specificity of the VP2 peptide to the VPAC1 receptor.

(A) Competitive inhibition ELISA by VIP. The average inhibition rates at different concentrations of VIP were shown. When the concentration of VIP was increased above 0.001 µg/ml, a significant inhibition occurred. An unrelated peptide displayed by the unrelated phage was used as a negative control. (B) Flow cytometry analysis of the inhibition effect of VIP on binding of VP2 peptide to CHO-K1/VPAC1 cells. Here, a,d: blank control, b: VIP+FITC-VP2, e: Unrelated peptide+FITC-VP2, c,f: FITC-VP2.

Figure 6. Binding of VP2 peptide to CHO-K1/VPAC1 cells and colorectal cancer cell lines (×200).

The FITC-conjugated VP2 (FITC-VP2) was incubated with CHO-K1/VPAC1 (A), HT29 (B), SW480(C), SW620 (D) and control CHO-K1 cells (E). At the same time, the control FITC-conjugated unrelated peptide (FITC-URp) was incubated with CHO-K1/VPAC1 (a), HT29 (b), SW480 (c), SW620 (d) and CHO-K1 cells(e). The cells were observed under a fluorescence microscope.

Figure 7. FACS analysis of VP2 peptide binding to CHO-K1/VPAC1 cells and colorectal cancer cell lines.

The FITC-VP2 and the control FITC-URp were incubated with CHO-K1/VPAC1(A), HT29(B), SW480(C), SW620 (D) and control CHO-K1 cells(E), respectively. PBS was used as a blank control. Triplicate determinations were done at each data point.