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V. B. Loktev - One of the best experts on this subject based on the ideXlab platform.
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c terminal fragment of human Laminin Binding Protein contains a receptor domain for venezuelan equine encephalitis and tick borne encephalitis viruses
Biochemistry, 2009Co-Authors: A A Malygin, E V Protopopova, E I Bondarenko, V A Ivanisenko, G G Karpova, V. B. LoktevAbstract:Polyclonal and monoclonal antibodies (MABs) to human Laminin-Binding Protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for Binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 Protein and tick-borne encephalitis virus (TBEV) E Protein. Mapping of Binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP Protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral Proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the Laminin-1 molecule has been proposed.
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Immunochemical and single molecule force spectroscopy studies of specific interaction between the Laminin Binding Protein and the West Nile virus surface glycoProtein E domain II.
Journal of molecular recognition : JMR, 2008Co-Authors: M. V. Bogachek, V. B. Loktev, B. N. Zaitsev, Mélanie Favre, Sergey K. Sekatskii, Giovanni DietlerAbstract:ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoProtein E domain II (DII) of the tick born encephalitis and Dengue viruses with the Laminin Binding Protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoProtein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific Binding between these species is confirmed by both methods. Copyright (c) 2007 John Wiley & Sons, Ltd.
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cellular Laminin Binding Protein and venezuelan equine encephalomyelitis virus replication in vero 293 and 9s2 cells
Tsitologiia, 2007Co-Authors: M M Gridina, A. V. Kachko, E V Protopopova, E I Bondarenko, A V Ivanova, V. B. LoktevAbstract:The level of Laminin-Binding Protein (LBP) expression on cellular membranes was studied in three cell lines including 293 cells transformed by plasmide with human LBP gene. Vero cells show a high level of LBP on the cell surfaces and demonstrate a high level of the Venezuelan equine encephalomyelitis (VEE) virus replication. The inhibition of VEE virus replication was more than 200 times as much after treatment of Vero cell surfaces with monoclonal antibodies to human LBP. 293 cells have more low level of LBP on their surfaces but being transformed by plasmide with LBP human gene these cells showed an increase in the level of cellular LBP. The VEE virus replication in transformed cells (9S2) was more than 2000 times higher compared to 293 cells. The results obtained demonstrate a principal role of cellular LBP in VEE virus entry into mammalian cells. It can be proposed that LBP is a key cellular Protein at the early stage of VEE virus replication in cells. So, LBP might be a target Protein for development of some new generation of antiviral drugs that would be able to inhibit (enhance) the alphavirus replication in human cells.
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inhibition of replication of venezuelan equine encephalomyelitis with polyclonal antibodies to Laminin Binding Protein
Voprosy virusologii, 2004Co-Authors: E I Bondarenko, E V Protopopova, Alexander N Shvalov, V. B. LoktevAbstract:A study of temporal and quantitative characteristics of inhibition of replication of Venezuelan equine encephalomyelitis (VEE) virus, strain TC-83, in Vero and CPE on PK cells showed purified polyclonal rabbit antibodies to human recombinant Laminin-Binding Protein (LBP) to be able to block completely the development of cytopathic effect (CPE) in such cells, when infected with 10(7) CPE60. The extent of VEE infection inhibition in Vero was in direct proportion to a concentration of specific antibodies within a range of 0.44-3 microg/100 microl. When antibodies were added to Vero cells after they were infected, there was a gradual attenuation of the inhibition effect, which stopped almost completely 9 hours after the antibodies were placed. Inhibition was effective at 4 degrees C and 37 degrees C. A lack of synthesis of viral glycoProtein E2 in Vero cells infected in the presence of antibodies to LBP is an extra argument proving that the VEE replication is inhibited at early infection stages. The data obtained demonstrated the general LBP significance for the penetration of VEE into mammalian cells and the related importance of designing new antiviral drugs against alpha-viral infection, which are based on blocking the mechanism of receptor penetration of the virus into the cell.
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monoclonal antibodies to recombinant human Laminin Binding Protein production and immunochemical description
Vestnik Rossiĭskoĭ akademii meditsinskikh nauk Rossiĭskaia akademiia meditsinskikh nauk, 2004Co-Authors: E I Bondarenko, E V Protopopova, Vyacheslav M Nekrasov, V. B. LoktevAbstract:Thirteen murine hybridoma lines producing monoclonal antibodies (Mabs) to recombinant human Laminin-Binding Protein (rLBP) were developed. All 13 Mabs reacted with affinity purified 43 kDA rLBP in ELISA and Western blotting. Mab class determination showed 9 Mabs as belonging to IgM class, 2 Mabs--to IgG2 subclass, 1 Mab--to IgG1 and 1 Mab--to IgG2b. Ten Mabs of different classes were capable to react with LBP on the surface of Vero cells. Mabs displayed a high and simultaneously varying affinity to rLBP (10(8) 10(9) M(-1)). The Mab affinity was found to be comparable with the mean affinity of mouse and rabbit antibodies isolated from hyperimmune sera. The possibility of using the produced Mabs in mapping the LBP domains involved in virus attachment, cell differentiation and cancer metastases progression as well as in the systemic response to bacterial protozoan and parasitic infection is under discussion.
E V Protopopova - One of the best experts on this subject based on the ideXlab platform.
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force induced globule coil transition in Laminin Binding Protein and its role for viral cell membrane fusion
Journal of Molecular Recognition, 2014Co-Authors: B. N. Zaitsev, Fabrizio Benedetti, Andrey Mikhaylov, Denis V Korneev, S K Sekatskii, Tanya Karakouz, P A Belavin, N A Netesova, E V ProtopopovaAbstract:The specific interactions of the pairs Laminin Binding Protein (LBP)-purified tick-borne encephalitis viral surface Protein E and certain recombinant fragments of this Protein, as well as West Nile viral surface Protein E and certain recombinant fragments of that Protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human V3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70pN) sharp globule-coil transition for LBP and LBP-fragments of Protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright (c) 2014 John Wiley & Sons, Ltd.
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c terminal fragment of human Laminin Binding Protein contains a receptor domain for venezuelan equine encephalitis and tick borne encephalitis viruses
Biochemistry, 2009Co-Authors: A A Malygin, E V Protopopova, E I Bondarenko, V A Ivanisenko, G G Karpova, V. B. LoktevAbstract:Polyclonal and monoclonal antibodies (MABs) to human Laminin-Binding Protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for Binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 Protein and tick-borne encephalitis virus (TBEV) E Protein. Mapping of Binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP Protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral Proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the Laminin-1 molecule has been proposed.
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complex studies of interactions between flaviviruses and Laminin Binding Protein
34th Congress of the Federation-of-European-Biochemical-Societies, 2009Co-Authors: M. V. Bogachek, B. N. Zaitsev, Mélanie Favre, Valery B. Loktev, E V Protopopova, S K Sekatskii, Giovanni DietlerAbstract:Reference LPMV-TALK-2009-001View record in Web of Science Record created on 2010-02-09, modified on 2017-12-03
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cellular Laminin Binding Protein and venezuelan equine encephalomyelitis virus replication in vero 293 and 9s2 cells
Tsitologiia, 2007Co-Authors: M M Gridina, A. V. Kachko, E V Protopopova, E I Bondarenko, A V Ivanova, V. B. LoktevAbstract:The level of Laminin-Binding Protein (LBP) expression on cellular membranes was studied in three cell lines including 293 cells transformed by plasmide with human LBP gene. Vero cells show a high level of LBP on the cell surfaces and demonstrate a high level of the Venezuelan equine encephalomyelitis (VEE) virus replication. The inhibition of VEE virus replication was more than 200 times as much after treatment of Vero cell surfaces with monoclonal antibodies to human LBP. 293 cells have more low level of LBP on their surfaces but being transformed by plasmide with LBP human gene these cells showed an increase in the level of cellular LBP. The VEE virus replication in transformed cells (9S2) was more than 2000 times higher compared to 293 cells. The results obtained demonstrate a principal role of cellular LBP in VEE virus entry into mammalian cells. It can be proposed that LBP is a key cellular Protein at the early stage of VEE virus replication in cells. So, LBP might be a target Protein for development of some new generation of antiviral drugs that would be able to inhibit (enhance) the alphavirus replication in human cells.
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inhibition of replication of venezuelan equine encephalomyelitis with polyclonal antibodies to Laminin Binding Protein
Voprosy virusologii, 2004Co-Authors: E I Bondarenko, E V Protopopova, Alexander N Shvalov, V. B. LoktevAbstract:A study of temporal and quantitative characteristics of inhibition of replication of Venezuelan equine encephalomyelitis (VEE) virus, strain TC-83, in Vero and CPE on PK cells showed purified polyclonal rabbit antibodies to human recombinant Laminin-Binding Protein (LBP) to be able to block completely the development of cytopathic effect (CPE) in such cells, when infected with 10(7) CPE60. The extent of VEE infection inhibition in Vero was in direct proportion to a concentration of specific antibodies within a range of 0.44-3 microg/100 microl. When antibodies were added to Vero cells after they were infected, there was a gradual attenuation of the inhibition effect, which stopped almost completely 9 hours after the antibodies were placed. Inhibition was effective at 4 degrees C and 37 degrees C. A lack of synthesis of viral glycoProtein E2 in Vero cells infected in the presence of antibodies to LBP is an extra argument proving that the VEE replication is inhibited at early infection stages. The data obtained demonstrated the general LBP significance for the penetration of VEE into mammalian cells and the related importance of designing new antiviral drugs against alpha-viral infection, which are based on blocking the mechanism of receptor penetration of the virus into the cell.
Pijush K Das - One of the best experts on this subject based on the ideXlab platform.
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membrane orientation of Laminin Binding Protein
FEBS Journal, 2003Co-Authors: Keya Bandyopadhyay, Sudipan Karmakar, Aruna Biswas, Pijush K DasAbstract:Earlier we presented several lines of evidence that a 67-kDa Laminin Binding Protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa Laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania Laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface Proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane Protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using Laminin as primary probe revealed that a major part of this Protein harbouring the Laminin Binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact Protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane Protein, having significant portion of N-terminal end as well as the Laminin Binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.
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high affinity Binding between Laminin and Laminin Binding Protein of leishmania is stimulated by zinc and may involve Laminin zinc finger like sequences
FEBS Journal, 2002Co-Authors: Keya Bandyopadhyay, Sudipan Karmakar, Abhijit Ghosh, Pijush K DasAbstract:In the course of trying to understand the pathogenesis of leishmaniasis in relation to extracellular matrix (ECM) elements, Laminin, a major ECM Protein, has been found to bind saturably and with high affinity to a 67-kDa cell surface Protein of Leishmania donovani. This interaction involves a single class of Binding sites, which are ionic in nature, conformation-dependent and possibly involves sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on Laminin. Inhibition studies with monoclonals against polypeptide chains and specific peptides with adhesive properties revealed that the Binding site was localized in one of the nested zinc finger consensus sequences of B1 chain containing the specific pentapeptide sequence, YIGSR. Furthermore, incubation of L. donovani promastigotes with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67-kDa Laminin-Binding Protein (LBP) induced tyrosine phosphorylation of Proteins with a molecular mass ranging from 115 to 130 kDa. These studies suggest a role for LBP in the interaction of parasites with ECM elements, which may mediate one or more downstream signalling events necessary for establishment of infection.
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role of 67 kda cell surface Laminin Binding Protein of leishmania donovani in pathogenesis
Journal of Biochemistry, 2001Co-Authors: Keya Bandyopadhyay, Sudipan Karmakar, Abhijit Ghosh, Pijush K DasAbstract:The role that interaction with lnminin may play in Leishmania donovani infection was investigated. Binding of 12SI-radiolabeled lnminin, in a liquid-phase assay, by the parasite was rapid, saturable, specific, reversible, and of high affinity. Using a Western blotting procedure, a 67 kDa Laminin-Binding Protein (LBP) was identified from the membrane of both the promastigote and amastigote forms of L. donovani. Subsequently, the Protein was purified by affinity chromatography. Immunofluorescence with a polyclonal antibody against LBP as well as flow cytometric analysis demonstrated its presence at the parasite surface. After stimulation with phorbol-12-myristate-13-acetate (PMA), U937 cells exhibited the ability to adhere to lnminin and LBP specifically inhibited this adhesion. The reduced parasite adhesion after tunicamycin treatment suggested the importance of sugar residues in cell adhesion. Although co-administration of either Laminin or LBP or anti LBP antibody reduced parasite virulence, resulting in a lower level of infection in the BALB/c mouse model, an in vitro macrophage culture-enhanced level of infection was observed in the case of Laminin-coated parasites. The results collectively suggest a role for LBP in the interaction of the parasite with extracellular matrix elements, which may constitute a basis for the homing of the parasite to its physiological address. ,i
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isolation of a Laminin Binding Protein from the protozoan parasite leishmania donovani that may mediate cell adhesion
Biochemical Journal, 1999Co-Authors: Abhijit Ghosh, Keya Bandyopadhyay, Labanyamoy Kole, Pijush K DasAbstract:Extracellular matrix (ECM)-Binding Proteins on the surface of Leishmania are thought to play a crucial role in the onset of leishmaniasis, as these parasites invade mononuclear phagocytes in various organs after migrating through the ECM. In a previous report, we presented several lines of evidence suggesting that Leishmania has a speci®c receptor for Laminin, a major ECM Protein, with a Kd in the nanomolar range. Here we describe the identi®cation, puri®cation and biochemical characterization of the Leishmania Laminin receptor. When the outer membrane Proteins of L. dono�ani were blotted on to nitrocellulose paper and probed with Laminin, a prominent Laminin-Binding Protein of 67 kDa was identi®ed. The puri®ed Protein was isolated by a three-step process involving DEAE±cellulose, Con A (concanavalin A)±Sepharose and Laminin±Sepharose affinity chromatography and was used to raise a monospeci®c antibody. The same Protein was obtained when parasite membrane extracts were adsorbed to antibody affinity matrix and eluted with glycine. The affinity-puri®ed Protein bound to Laminin in a detergentsolubilized form as well as after integration into arti®cial bilayers, and was subsequently characterized as an integral membrane Protein. Metaperiodate oxidation and metabolic inhibition of glycosylation studies indicate the Binding Protein to be glycoProtein in nature and that N-linked oligosaccharides play a part in the interaction of Laminin with the Binding Protein. Surface-labelled parasites attached to microtitre wells coated with Laminin and the 67 kDa Protein blocked the adhesion to Laminin substrate. We propose that the 67 kDa Protein is an adhesin involved in the attachment of Leishmania to host tissues.
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evidence of a Laminin Binding Protein on the surface of leishmania donovani
Biochemical and Biophysical Research Communications, 1996Co-Authors: Abhijit Ghosh, Keya Bandyopadhyay, Labanyamoy Kole, Kakali Sarkar, Pijush K DasAbstract:Abstract Both the promastigote and amastigote forms of the intracellular parasite,Leishmania donovanibind the basement membrane glycoProtein Laminin with high affinity (Kd= 3.56 × 10−9M and 3.98 × 10−9M respectively) with ∼9000 and ∼800 sites per cell. Bound Laminin was identified by direct autoradiography and the Binding Protein through analysis of the parasite extract by SDS-PAGE and immunoblotting. A major component of 67 kDa was detected. The same Protein was obtained when parasite outer membrane Proteins were adsorbed to Laminin-sepharose affinity matrix and subsequently eluted with SDS. The Binding affinity of the isolated receptor was similar to that of the whole cells. Such a receptor isolated inLeishmaniafor the first time, may function as one of the bridging molecules for extracellular matrix recognition.
Jean R. Starkey - One of the best experts on this subject based on the ideXlab platform.
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tumor shedding of Laminin Binding Protein modulates angiostatin production in vitro and interferes with plasmin derived inhibition of angiogenesis in aortic ring cultures
International Journal of Cancer, 2006Co-Authors: Britney L Moss, Dmitri Kazmin, Lara Taubner, Yekaterina K Sample, Valerie Copie, Jean R. StarkeyAbstract:The growth of solid tumors is largely controlled by the process of angiogenesis. A 67 kDa Protein, the Laminin Binding Protein (LBP), is shed from malignant cells in significant amounts and binds to Laminin-1 (Starkey et al., Cytometry 1999;35:37–47; Karpatova et al., J Cell Biochem 1996;60:226–34). However, the functions of shed LBP are not fully understood. We hypothesize that matrix-bound LBP could modulate local tumor angiogenesis. In support of this hypothesis, we demonstrate that shed LBP exhibits sulfhydryl oxidase-like activities, and modifies the production of angiostatins from plasmin in vitro. The molecular weights of the autocatalytic products of lys-plasmin incubated with LBP in vitro suggest that PMDs (plasmin A chains attached to degraded B chains) (Ohyama et al., Eur J Biochem 2004;271:809–20) are preferentially generated. Using rat aortic ring assays, we also show that shed LBP reverses plasmin-dependent inhibition of vascular outgrowth. To elucidate which LBP region(s) are active in modulating angiogenesis, limited proteolysis experiments were conducted to determine stable rLBP domains likely to fold correctly, and these were cloned, expressed and purified. The stable LBP fragments were tested for Binding to Laminin-1 and for competition with shed LBP activity in the aortic ring assay. Results of these studies suggest that the active LBP domains lie within the 137–230 amino acid sequence, a region known to contain 2 bioactive sequences. Since this fragment binds to Laminin-1 and modulates angiogenesis, it appears likely that Binding of shed LBP to matrix Laminin-1 is related to its functions in tumor angiogenesis. The findings presented in this manuscript suggest that LBP shedding could provide a useful therapeutic target. © 2005 Wiley-Liss, Inc.
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Comparative modeling of the N-terminal domain of the 67kDa Laminin-Binding Protein: implications for putative ribosomal function.
Biochemical and Biophysical Research Communications, 2003Co-Authors: Dmitri Kazmin, Yurii Chinenov, Eric T. Larson, Jean R. StarkeyAbstract:Abstract Laminin-Binding Protein/p40 (LBP/p40) precursor appears to be involved in two seemingly unrelated activities—cell adhesion and ribosomal biogenesis. Analysis of primary structure revealed a two-domain organization of the LBP/p40. The N-terminal portion of LBP is similar to the S2 family of prokaryotic ribosomal Proteins, while the C-terminus is unique for Metazoa and is involved in extraribosomal functions. To gain insight into putative ribosomal functions of LBP we performed comparative modeling of the N-terminal domain using crystal structures of S2p from Thermus thermophilus . The LBP model assumes an α–β sandwich fold similar to that of S2. Modeling revealed the loss of a significant portion of ribosomal RNA (rRNA) interaction domain, lack of conservation of many residues involved in interactions with rRNA, and a major shift in surface charge distribution (compared to the S2 Protein). The overall stability of the fold argues against a proposed transmembrane domain in the central part of the Protein. Partial overlap in S2 and Laminin-Binding domains suggests that ribosomal and surface receptor functions would be mutually exclusive. The possible biological role of LBP/p40 bifunctionality is discussed.
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cell surface and substrate distribution of the 67 kda Laminin Binding Protein determined by using a ligand photoaffinity probe
Cytometry, 1999Co-Authors: Jean R. Starkey, Selvanayagam Uthayakumar, Deborah L BerglundAbstract:Background: Peptide 11, a nine–amino acid sequence from the β1 chain of Laminin-1, has been reported to inhibit tumor cell invasion of basement membranes, and to reduce tumor lung colonization (Iwamoto et al.: Science 238:1132–1134, 1987; Landowski et al.: Clin Exp Metastasis 13:357–372, 1995). The peptide is a ligand for the 32/67-kDa Laminin-Binding Protein (LBP); however, the mechanism by which the 67-kDa LBP promotes invasion is unknown. Methods: We have synthesized a highly specific probe for the 67-kDa LBP by adding a biotinylated residue, and replacing the required tyrosine in peptide 11 with the photoactivatable bezophenone crosslinker, 4-benzoyl-l-phenylalanine. This probe was used to follow the distribution of the 67-kDa LBP by gel electrophoresis, fluorescence-activated cell scanning, and confocal microscopy techniques. Results: A single crosslinked Protein, consistent with the high molecular weight form of the LBP, was found on Western blots of membrane detergent extracts from cells treated with the ligand probe. A CHO cell line, manipulated to overexpress the Laminin-specific α6β1 integrin, exhibited increased invasiveness, and expressed more cell surface 67-kDa LBP. Membrane-associated 67-kDa LBP was found in the vicinity of focal adhesion plaques and also associated with the matrix substrate. Studies on conditioned medium indicated that the matrix-associated LBP derived from material that was shed from the cells, with more being shed from the more invasive CHO variants. Conclusions: These results demonstrate the utility of this novel probe in diverse experimental protocols, and suggest that shedding of the 67-kDa LBP may have a role in promoting tumor cell invasion. Cytometry 35:37-47, 1999. © 1999 Wiley-Liss, Inc.
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studies of the structure of the metastasis associated 67 kda Laminin Binding Protein fatty acid acylation and evidence supporting dimerization of the 32 kda gene product to form the mature Protein
Biochemistry, 1995Co-Authors: T H Landowski, Edward A Dratz, Jean R. StarkeyAbstract:The level of expression of the 67 kDa high-affinity Laminin Binding Protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the Protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC-MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the Protein to be 66.7 kDa, compatible with the SDS-PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O-glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the Protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated Proteins. Reduction with dithiothreitol or beta-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity Laminin Binding characteristic of the Protein may be modulated by an, as yet, unidentified membrane accessory molecule.
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control pathways of the 67 kda Laminin Binding Protein surface expression and activity of a new ligand Binding domain
Clinical & Experimental Metastasis, 1995Co-Authors: Terry H Landowski, Selvanayagam Uthayakumar, Jean R. StarkeyAbstract:A number of papers have been published on the clinical correlation of the expression of the 67 kDa Laminin Binding Protein (LBP) with the metastatic potential of solid tumors. Both mRNA and Protein expression levels have been reported, but both the relationship between them and the molecular nature of the 67 kDa surface product remain unclear. We have utilized a homotypic overexpression system to investigate the cell surface presentation of the 67 kDa LBP and the contribution of this Protein to the invasive phenotype of cultured cell lines. We report here that the cellular mRNA levels do not directly reflect the levels of the 67 kDa LBP observed on the cell surface in this overexpression system. Methotrexate amplification of transfected plasmids expressing the 67 kDa LBP leads to an initial elevation of both the LBP mRNA and surface Protein levels. This is accompanied by an altered, more flattened, cell morphology. Later, apparent adaptation of the cells to methotrexate is accompanied by a down-regulation of the surface expression of the Protein. mRNA levels, however, remain elevated. A nine amino acid sequence, CDPGYIGSR (peptide 11), within the β chain of Laminin 1 has been identified as a probable Binding domain for the 67 kDa LBP. Previous studies have identified a region of the 67 kDa LBP which may be involved in Laminin interaction, although not necessarily via the peptide 11 domain. We have identified a second site within the amino acid coding sequence of the 67 kDa LBP which also shows biological activity both in vitro and in vivo. A peptide with this sequence, LBP residues 205-229, binds Laminin-1 in a peptide 11 inhibitable manner. The receptor-derived peptide modulates invasion of basement membrane matrix in vitro and inhibits experimental lung colony formation when injected along with B16BL6 mouse melanoma cells. However, pretreatment of the melanoma cells with the peptide enhances lung colony formation. Thus, the interaction of the 67 kDa LBP with basement membrane matrix appears to involve a complex series of events including multiple adhesive sites and tight regulation of cell surface expression.
Franco D Menozzi - One of the best experts on this subject based on the ideXlab platform.
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mycobacterial heparin Binding hemagglutinin and Laminin Binding Protein share antigenic methyllysines that confer resistance to proteolysis
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Kevin Pethe, Pablo Bifani, Herve Drobecq, Christian Sergheraert, Anne-sophie Debrie, Camille Locht, Franco D MenozziAbstract:Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette–Guerin produce a heparin-Binding hemagglutinin adhesin (HBHA) required for extrapulmonary dissemination and a Laminin-Binding Protein (LBP) involved in cytoadherence through Laminin recognition. These adhesins bear posttranslational modifications that are not present when the Proteins are produced in a recombinant (r) form in Escherichia coli. Mass spectrometry analysis of HBHA revealed that the posttranslational modifications are borne by the C-terminal moiety, which comprises the heparin-Binding domain made of repeated lysine-rich motifs. Amino acid sequencing showed that these modifications consist of mono- and dimethyllysines within these motifs. The methyllysine-containing repeats were recognized by mAb 4057D2 and were also detected in LBP, which is equally recognized by mAb 4057D2. This Ab does not recognize the recombinant forms of these Proteins. However, when rHBHA and rLBP were subjected to NaBH4 and formalin treatment to induce lysine methylation, reactivity with mAb 4057D2 was recovered. Methylated rHBHA displayed enhanced resistance to proteolysis compared with rHBHA, as previously observed for native HBHA. S-adenosylmethionine-dependent HBHA methyltransferase activity was detected in the cell-wall fractions of M. bovis bacillus Calmette–Guerin and of Mycobacterium smegmatis, a species that produces LBP but naturally lacks hbhA, suggesting that the same enzyme(s) methylate(s) both LBP and HBHA. This hypothesis was confirmed by the fact that HBHA produced by recombinant M. smegmatis was also methylated. These results show that mycobacteria use enzymatic methylation of lysines to ensure greater stability of their adhesins.
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Mycobacterial heparin-Binding hemagglutinin and Laminin-Binding Protein share antigenic methyllysines that confer resistance to proteolysis
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Kevin Pethe, Pablo Bifani, Herve Drobecq, Christian Sergheraert, Anne-sophie Debrie, Camille Locht, Franco D MenozziAbstract:Abstract Mycobacterium tuberculosis and Mycobacteriumbovis bacillus Calmette–Guerin produce a heparin-Binding hemagglutinin adhesin (HBHA) required for extrapulmonary dissemination and a Laminin-Binding Protein (LBP) involved in cytoadherence through Laminin recognition. These adhesins bear posttranslational modifications that are not present when the Proteins are produced in a recombinant (r) form in Escherichia coli. Mass spectrometry analysis of HBHA revealed that the posttranslational modifications are borne by the C-terminal moiety, which comprises the heparin-Binding domain made of repeated lysine-rich motifs. Amino acid sequencing showed that these modifications consist of mono- and dimethyllysines within these motifs. The methyllysine-containing repeats were recognized by mAb 4057D2 and were also detected in LBP, which is equally recognized by mAb 4057D2. This Ab does not recognize the recombinant forms of these Proteins. However, when rHBHA and rLBP were subjected to NaBH4 and formalin treatment to induce lysine methylation, reactivity with mAb 4057D2 was recovered. Methylated rHBHA displayed enhanced resistance to proteolysis compared with rHBHA, as previously observed for native HBHA. S-adenosylmethionine-dependent HBHA methyltransferase activity was detected in the cell-wall fractions of M. bovis bacillus Calmette–Guerin and of Mycobacteriumsmegmatis, a species that produces LBP but naturally lacks hbhA, suggesting that the same enzyme(s) methylate(s) both LBP and HBHA. This hypothesis was confirmed by the fact that HBHA produced by recombinant M. smegmatis was also methylated. These results show that mycobacteria use enzymatic methylation of lysines to ensure greater stability of their adhesins.
