Research Article: Green Tea Polyphenol EGCG Sensing Motif on the 67-kDa Laminin Receptor

Date Published: May 29, 2012

Publisher: Public Library of Science

Author(s): Yoshinori Fujimura, Mami Sumida, Kaori Sugihara, Shuntaro Tsukamoto, Koji Yamada, Hirofumi Tachibana, Maxim Antopolsky. http://doi.org/10.1371/journal.pone.0037942

Abstract

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.

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, K166 and H169, 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.

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.

Partial Text

Green tea has been shown to have cancer preventive activity in a variety of organ sites in animal models [1]–[3] and humans [4]. Among the green tea constituents, (–)-epigallocatechin-3-O-gallate (EGCG) is the most abundant and most active constituent in inhibiting experimental carcinogenesis and related reactions. Although many mechanisms for the anti-cancer activities of EGCG at concentrations of more than 10 µM, concentrations that are much higher than those observed in plasma or tissues, have been proposed based mainly on studies in cell lines, it is still not clear which EGCG-induced molecular events and potential molecular targets are responsible for its cancer-preventive activity in vivo[5]. Recently we have identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that mediates the anti-cancer action, cancer cell growth inhibition, of a physiologically relevant EGCG [6], and others showed that RNAi-mediated silencing of 67LR results in abrogation of EGCG-induced apoptosis in myeloma cells [7].

The evolutionary analysis of 67LR has suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, corresponding to residues 173–178 [11]. Intriguingly, this segment is proximate to the EGCG-functional domain, residues 161-170. We have previously reported that a laminin retreatment partially inhibited the binding of EGCG to the 67LR and treatment with the cell-surface LR-specific antibody MLuC5, capable of inhibiting laminin binding to 67LR, prevented EGCG’s abilities such as its cell-surface binding and inhibition of cancer cell growth [6]. Thus, the position proximity between the EGCG- and laminin-binding motifs may provide a possible explanation, steric hindrance caused by laminin or antibody, for the inhibition of the functions of EGCG. In spite of the proximity between both motifs, laminin did not show an inhibitory effect on cancer cell growth in a similar fashion as EGCG [16]. These facts imply that our proposed EGCG sensing motif may not be easily regulated by a proximal region-specific ligand. From the crystal structure of 67LR, residues 161-180 have been shown to comprise the linker between β-sheet strand 7 (β7) and α helix E (αE) [23]. The only portion of this sequence that was solvent-accessible was suggested to be residues 165-169 in the β7-αE linker. It was speculated that such residues might be exposed by a conformational change of the C-terminal tail of the receptor. Although these observations may provide a possible rationale for the accessibility of EGCG to the 161-170 region of 67LR, further structural studies are required to elucidate the details of the EGCG-67LR interaction.

Source:

http://doi.org/10.1371/journal.pone.0037942