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      The folded and disordered domains of human ribosomal protein SA have both idiosyncratic and shared functions as membrane receptors

      research-article
      * , , * , , , * , , * , , 2
      Bioscience Reports
      Portland Press Ltd.
      cancer, epigallocatechin-gallate (EGCG), flavivirus, heparin, intrinsically disordered protein, laminin receptor 1 (LamR1), 37LRP, 37 kDa laminin receptor precursor, 67LR, 67 kDa laminin receptor, an alternative name for LamR1, DENV, dengue virus, ED1, ED2 and ED3, domains 1, 2 and 3 of the flaviviral envelope protein E, EGCG, epigallocatechin gallate, LamR1, laminin receptor 1 (an alternative name for RPSA), mAb, monoclonal antibody, pNP, p-nitrophenol, pNPP, pNP phosphate, RPS2, ribosomal protein S2, RPSA, ribosomal protein SA, SINV, Sindbis virus, VEEV, Venezualian equine encephalitis virus, WNV, West-Nile virus, YFV, yellow fever virus

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          Abstract

          The human RPSA [ribosomal protein SA; also known as LamR1(laminin receptor 1)] belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogens and the anticarcinogen EGCG (epigallocatechin-gallate). It contributes to the crossing of the blood–brain barrier by neurotropic viruses and bacteria, and is a biomarker of metastasis. RPSA includes an N-terminal domain, which is folded and homologous to the prokaryotic RPS2, and a C-terminal extension, which is intrinsically disordered and conserved in vertebrates. We used recombinant derivatives of RPSA and its N- and C-domains to quantify its interactions with ligands by in-vitro immunochemical and spectrofluorimetric methods. Both N- and C-domains bound laminin with K D (dissociation constants) of 300 nM. Heparin bound only to the N-domain and competed for binding to laminin with the negatively charged C-domain, which therefore mimicked heparin. EGCG bound only to the N-domain with a K D of 100 nM. Domain 3 of the envelope protein from yellow fever virus and serotypes-1 and -2 of dengue virus bound preferentially to the C-domain whereas that from West Nile virus bound only to the N-domain. Our quantitative in-vitro approach should help clarify the mechanisms of action of RPSA, and ultimately fight against cancer and infectious agents.

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          Most cited references44

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          A receptor for green tea polyphenol EGCG.

          The major polyphenol in green tea, (-)-epigallocatechin-3-gallate (EGCG), has been shown to prevent carcinogenesis. We have identified a receptor that mediates the anticancer activity of EGCG. Expression of the metastasis-associated 67-kDa laminin receptor confers EGCG responsiveness to cancer cells at physiologically relevant concentrations. Experiments using surface plasmon resonance demonstrate binding of EGCG to the 67-kDa laminin receptor with a nanomolar K (d) value.
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            Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

            A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.
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              NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells.

              Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve NK cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein; it also binds to WNV virus-like particles. These WNV-virus-like particles and WNV-domain III of WNV envelope protein directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK degranulation. Finally, WNV infection of cells results in increased binding of rNKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFN-gamma secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 Abs. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein.
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                Author and article information

                Journal
                Biosci Rep
                Biosci. Rep
                bsr
                BSR
                Bioscience Reports
                Portland Press Ltd.
                0144-8463
                1573-4935
                9 November 2012
                20 December 2012
                2013
                : 33
                : 1
                : e00011
                Affiliations
                *Institut Pasteur, Unit of Molecular Prevention and Therapy of Human Diseases, Department of Infection and Epidemiology, rue du Dr. Roux, F-75015 Paris, France
                †CNRS, URA3012, rue du Dr. Roux, F-75015 Paris, France
                ‡Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, rue du Dr. Roux, F-75015 Paris, France
                Author notes

                1These authors contributed equally to this work.

                2To whom correspondence should be addressed (email hugues.bedouelle@ 123456pasteur.fr ).
                Article
                BSR20120103
                10.1042/BSR20120103
                4098866
                23137297
                26841d43-24d5-465c-9829-71f25dbe6b74
                © 2013 The Author(s).

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence ( http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 12 October 2012
                : 16 October 2012
                Page count
                Figures: 7, Tables: 4, Equations: 13, References: 54, Pages: 12
                Categories
                Original Paper
                S2

                Life sciences
                cancer,epigallocatechin-gallate (egcg),flavivirus,heparin,intrinsically disordered protein,laminin receptor 1 (lamr1),37lrp, 37 kda laminin receptor precursor,67lr, 67 kda laminin receptor, an alternative name for lamr1,denv, dengue virus,ed1, ed2 and ed3, domains 1, 2 and 3 of the flaviviral envelope protein e,egcg, epigallocatechin gallate,lamr1, laminin receptor 1 (an alternative name for rpsa),mab, monoclonal antibody,pnp, p-nitrophenol,pnpp, pnp phosphate,rps2, ribosomal protein s2,rpsa, ribosomal protein sa,sinv, sindbis virus,veev, venezualian equine encephalitis virus,wnv, west-nile virus,yfv, yellow fever virus

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