Green tea polyphenol EGCG sensing motif on the 67-kDa laminin receptor

Abstract

Background: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities.

Methodology/principal findings: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K(166) and H(169), have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG.

Conclusions/significance: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.

Figure 1. The relationship between the responsiveness of EGCG to the HepG2 cells and 67LR expression.

A) Chemical structure of green tea polyphenol EGCG. B) Western blot analysis of whole cell lysate from HepG2 cells using anti-LR antiserum (I) and anti-LR antibody F-18 (II). C) To examine the expression of 67LR on cell membrane in HepG2 cells, both cytosolic and membrane fractions were prepared, and the 67LR were detected by western blot analysis using anti-LR antiserum. This test was performed under reducing (2-Me (+)) or non-reducing (2-Me (−)) conditions. 2-Me indicates 2-mercaptoethanol. The lower panel displays protein levels from the same filter blotted again with the anti-β-actin antibody used as a quantitative loading control. D) The cells transfected with either the empty vector (−) or the 67LR gene expression vector (+) were lysed and total cellular protein was subjected to western blot analysis using the cell-surface LR-specific antibody MLuC5. The lower panel displays protein levels from the same filter blotted again with the anti-β-actin antibody used as a quantitative loading control. E) Both transfected cells were fixed on the sensor chip. The cell-surface binding of EGCG to immobilized 67LR-overexpressed or control HepG2 cells were measured using a surface plasmon resonance (SPR) biosensor. EGCG was injected at a concentration of 10 µM for the time indicated interval in the figure. F) Both types of cells were treated with 1 µM EGCG for 5 days. The results are shown as the relative cell number to untreated control and the data presented are means ± S.D. (n = 3) (Student’s t-test, **, p<0.01).

Figure 2. The neutralization of the cell-surface binding of EGCG by peptides deduced from the extracellular domain of 67LR.

A) Synthetic peptide sequences deduced from the extracellular domain of 67LR. The neutralizing activity of these peptides for the cell-surface binding was assayed. After incubation of EGCG (1 µM) with each peptide (1 µM): (B) 20-amino acid residue peptides, (C) 10-amino acid residue peptides, or (D) 9-amino acid residue peptides (single amino acid deletion form of the N- or C-terminus of the peptide LR 161-170), interactions between these EGCG-peptide mixtures and the 67LR-overexpressed HepG2 cells were measured by a SPR assay. Sensorgrams of the net binding of EGCG, which is the value obtained from subtracting the peptide-binding signal from the total mixture-binding signal, are shown in panels B-D. The results are represented as EGCG alone (blue line) and EGCG plus peptide (red line).

Figure 3. The neutralization of the inhibitory effect of EGCG on cancer cell growth by peptides deduced from the extracellular domain of 67LR.

After pre-incubation of EGCG (1 µM) with each peptide (1 µM): (A) 20-amino acid segment peptides, (B) 10-amino acid segment peptides, or (C) 9-amino acid segment peptides (single amino acid deletion form of the N- or C-terminus of the peptide LR 161-170), the 67LR-overexpressed HepG2 cells were treated with these mixtures for 5 days and the cell number was assessed. The results, EGCG plus peptide (closed bar), are shown as the relative cell number to the EGCG-nontreated control (open bar), and the data presented are the means ± S.D. (n = 3) (Student’s t-test, *, p<0.05, **, p<0.01, ***, p<0.001). D) SDS-PAGE of recombinant LR (r-hLR_102-295 and the mutant r-hLR_102-295Δ161-171 expressed in E. coli). The lanes of Wild and Δ161-171 are the wild-type r-hLR_102-295 and the mutant r-hLR_102-295Δ161-171, respectively. Molecular mass markers (in kDa) are indicated at the left. E) Western blot analysis of the recombinant LR was performed by using the anti-LR antibody F18. F) The effect of r-hLR_102-295 and the mutant r-hLR_102-295Δ161-170 on the cancer cell growth inhibition by EGCG. After incubation of each r-hLR protein or LR161-170 peptide with or without EGCG, HepG2 cells were treated with these mixtures for 5 days and the cell number was assessed. The results are shown as the relative cell number of EGCG-, EGCG plus LR peptide-, or EGCG plus LR protein-treated cells (closed bar) to the EGCG-nontreated control cells (open bar) under each mixture condition (none, LR peptide, or LR protein), and the data presented are the means ± S.D. (n = 3) (Student’s t-test, ***, p<0.001).

Figure 4. Importance of basic amino acid resides in the EGCG sensing motif for EGCG’s activity.

A) Basic amino acid replacements of LR161-170. Replaced peptide sequences are shown in the list. B) The neutralizing activity of several basic amino acid-replaced LR161-170 segments for the cell-surface binding of EGCG. After incubation of EGCG with each peptide at a molar ratio of 1∶1 in PBS, interactions between these EGCG-peptide mixtures and the cells were measured by a SPR assay. Sensorgrams of net binding of EGCG, which is the value of the subtracted peptide-binding signal from the total mixture-binding signal, are shown. The results are represented as EGCG alone (blue line) and EGCG plus deletion mutant of LR161-170 (red line). C) The neutralizing activity of several basic amino acid-replaced LR161-170 segments on the EGCG-induced inhibition of cancer cell growth. After incubation of EGCG with each peptide, the 67LR-overexpressed HepG2 cells were treated with the mixtures for 5 days and the cell number was assessed. The results, EGCG plus peptide (closed bar), are shown as the relative cell number to the EGCG-nontreated control (open bar), and the data presented are the means ± S.D. (n = 3) (Student’s t-test, *, p<0.05, **, p<0.01).

Figure 5. A possible role of LR161-170 motif derived from other organisms for exerting of EGCG’s activities.

A) LR161-170 motif of other organisms. B) The neutralizing activity of LR161-170 derived from other organisms for the cell-surface binding of EGCG. After incubation of EGCG with each peptide at a molar ratio of 1∶1 in PBS, interactions between these EGCG-peptide mixtures and the 67LR-overexpressed HepG2 cells were measured by a SPR assay. Sensorgrams of the net binding of EGCG, which is the value of the subtracted peptide-binding signal from the total mixture-binding signal, are shown. The results are represented as EGCG alone (blue line) and EGCG plus deletion mutant of LR161-170 (red line). C) The neutralizing activity of LR161-170 derived from other organisms on the EGCG-induced inhibition of cancer cell growth. After incubation of EGCG with each peptide, HepG2 cells were treated with the mixtures for 5 days and the cell number was assessed. The results, EGCG plus peptide (closed bar), are shown as the relative cell number to the EGCG-nontreated control (open bar), and the data presented are the means ± S.D. (n = 3) (Student’s t-test, *, p<0.05).

Figure 6. The nature of the EGCG-LR peptide interactions.

Electrospray ionization mass spectrum of peptide (A) or peptide-EGCG mixture (B, C). The peptide solution was prepared by incubating each peptide (5 µM) with or without EGCG (5 µM) in water at room temperature for 15 min. Solutions of the mixture were analyzed in the positive ion mode. The arrows in the figure show the observed ion peaks. A) The Cys residue-containing peptide human LR161-170 and (B) its mixture with EGCG. B) An inset is the figure zooming in the relevant peak to see the difference between non-covalent (m/z 749.7 [LR161-170+EGCG+2H]²⁺) and covalent binding (m/z 748.7 [(LR161-170+EGCG-2H)+2H]²⁺). C) Mass spectrum of the mixture of the Cys residue-lacking peptide soy LR168-177 with EGCG. [LR161-170+2H]²⁺ or [Soy LR168-177+2H]²⁺ represents the doubly protonated form of each peptide. [LR161-170+EGCG+2H]²⁺ or [Soy LR168-177+EGCG+2H]²⁺ represents the peptide ion with added EGCG (EGCG-peptide complexes).