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,1
* Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, Tokyo, Japan;
PRESTO, Japan Science and Technology Agency, Saitama, Japan;
Department of Bacteriology, Nara Medical University, Kashihara, Japan;
Department of Medical Ecology and Informatics, Research Institute, International Medical Center of Japan, Tokyo, Japan;
|| Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
1Correspondence: Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, 1–21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: knishika{at}ri.imcj.go.jp
SPECIFIC AIMS
Shiga toxin 2 (Stx2), a major virulent factor of Stx-producing Escherichia coli (STEC) infections, binds to target cells via multivalent interactions between its B-subunit pentamer and globotriaosylceramide (Gb3). After binding, it is first retrogradely transported to the Golgi, then to the endoplasmic reticulum (ER). The aim of this study was to identify an inhibitor that directly blocks the multivalent interactions between Stx2 and Gb3 and provide biochemical and mechanistic insights into the intracellular transport of Stx2 using the identified Stx2 inhibitor.
PRINCIPAL FINDINGS
1. Identification of a novel Stx2 inhibitor using a multivalent peptide library approach
Each B-subunit monomer of Stx2 contains three distinct binding sites (i.e., sites 1, 2, and 3) for the trisaccharide moiety of Gb3. Multivalent interactions mediated through these sites enable Stx2 to bind to target cells with a high affinity, and this is sometimes referred to as the "clustering effect." In the present study, we have improved on a previously described affinity-based peptide library technique so as to make use of the "clustering effect" by synthesizing a tetravalent peptide library.
The library was comprised of compounds containing a polylysine core bifurcating at both ends with four randomized peptides (Fig. 1
A). The core structure of the compounds, including the length of the spacers, was optimized for its high-affinity binding to the Stx2 B-subunit pentamer. Design of the core structure was based on structural requirements that had already been established using a series of dendritic Stx inhibitors carrying the trisaccharides as an Stx binding unit (referred to as SUPER TWIGs). The tetravalent peptide library was screened for compounds capable of binding to recombinant histidine-tagged Stx2 B-subunit (2BH), but not to a mutant 2BH that had an Ala residue substituted for Trp33 (2BH-W33A) in trisaccharide binding site 3. The mutation of this site abolishes receptor binding activity. In addition, all of the SUPER TWIGs that bind to the B-subunit with high affinity bind exclusively to site 3, making this site an excellent target for inhibitor design. As shown in Fig. 1B
, Arg and Asn were strongly selected at positions 1 and 3, respectively, and basic amino acids were preferred at all positions. Based on this result, a second set of tetravalent peptide libraries containing Arg and/or Asn fixed in these positions were constructed and screened to further refine peptide selection (Fig. 1B
). In all three of these "secondary" libraries, Arg was strongly selected at positions 6 and 7, whereas a Pro cluster was selected at amino-terminal positions.
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Peptides with the amino acid sequences obtained from the second library screening, as well as a consensus peptide, MAFRRNRRNA (FRR-mono), were synthesized for kinetic analysis. All of these peptides, but not the free trisaccharide analog (n-hexenyl trisaccharide), effectively bound to 2BH, clearly indicating that these peptides are superior Stx binding units compared with the natural binding unit (trisaccharide) (Fig. 1C
). Tetravalent forms of these peptides with the same polylysine core, referred to as PPR-tet, PPP-tet, KRR-tet, and FRR-tet, bound to the B-subunit with high affinities (Fig. 1D
). In contrast, (MA-AU) 4-3Lys (MA-tet), which has the same core structure but lacks any Stx binding motifs, did not bind to the B-subunit. Furthermore, using structural and mutational analyses, we confirmed that the tetravalent peptides bound to the B-subunit in site 3-oriented configurations.
2. Tetravalent peptides inhibit Stx2 cytotoxicity by inducing aberrant cellular transport of Stx2
We found that all of the tetravalent peptides, but not MA-tet, protected Vero cells against the cytotoxic effects of Stx2. However, they did not inhibit the binding of Stx2 to the cells, indicating that their cytoprotective effects are not associated with the binding inhibition. To elucidate the mechanism by which the tetravalent peptides neutralize Stx2, we examined the effects of PPP-tet on the intracellular transport of Stx2. Time-dependent localization of Alexa-Stx2 to the Golgi, which was confirmed by colocalization with Golgi markers, merged well with that of Alexa-PPP-tet. In contrast, Alexa-PPP-tet was diffusely distributed in the absence of Alexa-Stx2. These results indicate that the PPP-tet forms a complex with Stx2, which is then incorporated into the cells and transferred to the Golgi. They also indicate that PPP-tet itself is cell permeable. Colocalization of Stx2 with Hsp47, an ER marker, was completely inhibited by the presence of PPP-tet, indicating that the transport of Stx2 from the Golgi to the ER was blocked by PPP-tet. Furthermore, degradation of 125I-Stx2, which was measured by the production of small degradation products released into the culture medium, was enhanced by 3-fold in the presence of PPP-tet. This degradation was blocked by the addition of chloroquine, a lysosome inhibitor. Taken together, these results indicate that PPP-tet inhibits the cytotoxic activity of Stx2 by inducing its aberrant cellular transport from the Golgi to an acidic compartment rather than the ER, resulting in its degradation.
3. Each Gb3 binding site on the B-subunit has specific role in the retrograde transport of Stx2
To further investigate the mechanism by which the tetravalent peptides neutralize Stx2, we examined the effect of PPP-tet on the intracellular transport of a double mutant of 2BH, 2BH-W29A/G61A. This mutant has amino acid substitutions in site 1 (Trp29) and site 2 (Gly61), and is unable to bind to target cells. PPP-tet formed a complex with 2BH-W29A/G61A via site 3, the only residual trisaccharide binding site, and this complex was substantially incorporated into Vero cells. However, this mutant did not bind to cells in the absence of PPP-tet. Of note, the PPP-tet/2BH-W29A/G61A complex that had been incorporated into cells was faintly detected and the complex was not transferred to the Golgi, whereas the PPP-tet/ 2BH complex was efficiently incorporated and transferred to the Golgi. These results demonstrate that each Gb3 binding site on the B-subunit has a specific role. That is, the binding of Gb3 through sites 1 and 2 is required for efficient uptake of Stx2 and its subsequent retrograde transport to the Golgi, whereas the binding of Gb3 through site 3, which is specifically blocked by PPP-tet, plays an essential role in its transfer from the Golgi to the ER.
CONCLUSIONS AND SIGNIFICANCE
In the present study, using a multivalent peptide library approach we have identified four tetravalent peptides that exhibit high affinities for the Stx2 B-subunit pentamer and markedly inhibit Stx2-cytotoxicity. Each tetravalent peptide has a novel Stx binding motif that is superior to the natural Stx binding unit, the trisaccharide. Since the B-subunit pentamer is known to bind to the trisaccharide assembled into multimers with a 106-fold greater efficacy than its free form, it is conceivable that the multivalent nature of the library enabled the efficient identification of such high-affinity motifs. Other widely used techniques, including screening of chemical compounds and phage display libraries, have failed to identify such inhibitors because they are not based on the "clustering effect." Moreover, the clinical application of SUPER TWIGs has been substantially hampered by the synthetic complexity of its trisaccharide moiety. The tetravalent peptides identified here overcome this problem. Thus, this technique is widely applicable to the development of selective inhibitors for a variety of disease-related molecules, such as influenza hemagglutinins and selectins, which recognize their intended carbohydrates based on the clustering effect.
We found that PPP-tet inhibits Stx2 by inducing its aberrant cellular transport from the Golgi to an acidic compartment rather than to the ER, resulting in the effective degradation of Stx2. So far, there has been no information regarding the specific role of the individual trisaccharide binding site in the intracellular transport of Stx2. This is due to the fact that introduction of a mutation in each site results in a marked reduction of Stx2 binding to target cells. In contrast, due to its cell-permeable nature, PPP-tet can attach itself, in complex with 2BH or even with its double mutant having mutations at sites 1 and 2 (2BH-W29A/G61A), to the plasma membrane, and thus can permit the complex to be incorporated into cells. By investigating the intracellular transport of these complexes, we found that the binding of Gb3 through sites 1 and 2 is required for the efficient uptake of Stx2 and its subsequent retrograde transport to the Golgi, whereas the binding of Gb3 through site 3, which is specifically blocked by PPP-tet, plays an essential role in its transfer from the Golgi to the ER.
Based on these observations, we propose the following model by which PPP-tet exerts inhibitory activity against Stx2 (Fig. 2
). PPP-tet forms a complex with Stx2 through site 3, and the complex associates with target cells due to the cell-permeable nature of PPP-tet. On the cell surface, Stx2 and Gb3 form a complex via interactions mainly at site 1, but also at site 2. The complex is then effectively incorporated and transported to the Golgi in a retrograde manner. However, because of the lack of a functional interaction between Stx2 and Gb3 through site 3, the complex is transported from the Golgi to an acidic compartment rather than to the ER, resulting in the degradation of Stx2. Although the precise molecular mechanism by which each trisaccharide binding site exerts its specific function remains to be elucidated, our present results indicate that the transport of Stx2 from the plasma membrane to the Golgi and from the Golgi to the ER are independently regulated.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6572fje
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