Use Of Synthetic Peptides Research Articles

Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat-shock protein

Adjuvant arthritis in Lewis rats is a model of T cell-mediated autoimmune arthritis resembling human rheumatoid arthritis. A nonapeptide from the 65-kD heat-shock protein of Mycobacterium bovis BCG, amino acid sequence 180-188, has been described to carry the dominant immunogenic epitope(s) for both arthritis-protective and arthritogenic T cell clones. Here we demonstrate that immunizations with the synthetic nonapeptide completely protected rats against adjuvant arthritis induced by M. tuberculosis. Interestingly, deletion of the N-terminal threonine of the nonapeptide resulted in loss of the protective activity. Pretreatments with the nonapeptide resulted in an immune response to the nonapeptide and to M. tuberculosis. After immunizations with the synthetic nonapeptide, only low titres of nonapeptide-specific antibodies were produced, whereas a significant cellular immune response to the nonapeptide was observed. In addition, the protection was transferable to naive rats by spleen T cells. These findings document the requirement of a T cell-specific immune response to the dominant epitope of the 65-kD mycobacterial heat-shock protein for the protection against adjuvant arthritis and suggest the feasibility of immune intervention in autoimmune arthritis through the use of synthetic peptides.

Amine-reactive isobaric tagging reagents: Requirements for absolute quantification of proteins and peptides

Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented.

Fibrinogen decreases cardiomyocyte contractility through an ICAM-1-dependent mechanism

IntroductionCardiomyocytes exposed to inflammatory processes express intracellular adhesion molecule-1 (ICAM-1). We investigated whether fibrinogen and fibrinogen degradation products, including D-dimer, could alter cardiomyocyte contractile function through interaction with ICAM-1 found on inflamed cardiomyocytes.MethodsIn vivo, rats were injected with endotoxin to model systemic inflammation, whereas isolated rat cardiomyocytes were treated with tumor necrosis factor-alpha to model the inflammatory environment seen following exposure to bacterial products such as lipopolysaccharide.ResultsIn vivo, endotoxin administration profoundly decreased cardiac contractile function associated with a large increase in intracardiac ICAM-1 and perivascular fibrinogen. Confocal microscopy with double-staining of isolated rat cardiomyocytes demonstrated colocalization of ICAM-1 and fibrinogen. This interaction was disrupted through pre-treatment of the cells with an ICAM-1-blocking antibody. Functionally, isolated rat cardiomyocyte preparations exhibited decreased fractional shortening when incubated with fibrinogen, and through the use of synthetic peptides, we determined that residues 117–133 of the fibrinogen gamma chain are responsible for this interaction with ICAM-1. Despite having crosslinked gamma chains, D-dimer retained the ability to decrease cardiomyocyte contractility.ConclusionSite 117–133 of the fibrinogen gamma chain is able to depress cardiomyocyte contractility through binding ICAM-1.

Peptide‐based immunotherapy of experimental autoimmune encephalomyelitis without anaphylaxis

Administration of peptide antigens in tolerogenic form holds promise as a specific treatment for autoimmune and allergic disorders. However, experiments in rodent autoimmune models have highlighted the risk of anaphylaxis in response to systemic peptide application once the aberrant immune response is underway. Thus, mice with clinical signs of experimental autoimmune encephalomyelitis (EAE) or diabetes have been reported to suffer fatal anaphylaxis upon administration of native autoantigenic peptides. Clearly, this might represent a significant barrier to the use of synthetic peptides in the treatment of ongoing human autoimmune conditions. Here we describe the development of an altered peptide ligand (APL) engineered to prevent anaphylaxis (no antibody binding) whilst retaining the ability to silence pathogenic myelin-reactive T lymphocytes. Administration of the APL to mice with an ongoing anti-myelin immune response did not cause anaphylaxis, but led to complete protection from the subsequent induction of EAE and, when given during ongoing EAE, led to a rapid remission of clinical signs. The approach of removing antibody recognition whilst maintaining the desired functional effect (in this case T cell tolerance) may be of value in other situations in which there is a risk of triggering anaphylaxis with peptide-based drugs.

Qualitative and Quantitative Analyses of Protein Phosphorylation in Naive and Stimulated Mouse Synaptosomal Preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

Use of Intein-Mediated Phosphoprotein Arrays to Study Substrate Specificity of Protein Phosphatases

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.

Attachment Activity of RGD-Motif Peptides to Osteoblast-Like Cell Line MC3T3-E1

Enhancement of the adhesion and migration of osteoblastic cells on a titanium surface are believed to increase the successful rate of implant therapy. The molecular basis of cell adhesive and migratory interactions with extracellular matrix molecules is dependent on specific adhesive functional motif sequences. These short peptide sequences, known as RGD in adhesion molecules, are thought to serve as a binding site for plasma membrane integrins. In this study, to clarify the usefulness of synthetic peptides for osteoblast attachment, we designed several short RGD-motif peptides from adhesion molecules and examined the attachment level to the osteoblastic cell line MC3T3-E1. To examine how well those designed peptides attach to MC3T3-E1 cells, we performed a competition assay using soluble RGD-motif peptides. Among those peptides used, the GRGDSP peptide derived from fibronectin not only expressed the highest binding activity to MC3T3-E1, but also increased in a dose-dependent manner. These findings suggest that the GRGDSP peptide may be useful in the development of functional titanium implants.

T-cell-directed therapy in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with significant morbidity and mortality that commonly afflicts young women. It ranges from a mild disease with a predominance of joint and skin manifestations to a severe life-threatening disease that can have serious CNS or renal involvement. While there are several immune effector mechanisms that contribute to the disease, it is clear that T lymphocytes have a central role in the immune pathogenesis of SLE. There are five broad approaches to T-cell-directed therapy in SLE that will be reviewed, including the use of monoclonal antibodies or decoy receptors directed against cytokines or cell surface costimulatory molecules; the use of synthetic peptides to selectively modulate autoantigen reactive T cells; cell-based therapies; restoration of abnormal T-cell receptor signaling events; and the correction of epigenetic abnormalities found in SLE. The current and future feasibility of each of these approaches to treatment are discussed.

Recent Improvements in the Use of Synthetic Peptides for a Selective Photodynamic Therapy

Photodynamic therapy (PDT) is a relatively new cytotoxic treatment, predominantly used in anti-cancer approaches, that depends on the retention of photosensitizers in tumor and their activation after light exposure. Photosensitizers are photoactive compounds such as porphyrins and chlorins that upon photoactivation, effect strongly localized oxidative damage within the target cells. The ability to confine activation of the photosensitizer by restricting illumination to the tumor allows for a certain degree of selectivity. Nevertheless, the targeted delivery of photosensitizers to defined cells is a major problem in PDT of cancer, and one area of importance is photosensitizer targeting. Alterations or increased levels in receptor expression of specific cellular type occur in the diseased tissues. Therefore, photosensitizers can be covalently attached to molecules such as peptides, leading to a receptor-mediated targeting strategy. These active-targeting approaches may be particularly useful for anti-vascular PDT. Moreover, it has been shown that the photocytotoxicity of photodynamic drugs could be enhanced by delivering high amounts of a photosensitizer into subcellular organelles such as the nucleus where nucleic acids represent target molecules sensitive to photodamage. The recent progresses in the use of active-targeting strategy with synthetic peptides and the interest of using an active-targeting strategy in PDT, which could allow efficient cellular internalization of photosensitizers, are described in this review.

Synthetic peptides: managing lipid disorders

Recent publications related to the potential use of synthetic peptides for the management of lipid disorders and their vascular complications are reviewed. The potential use of synthetic peptides for the management of lipid disorders and their vascular complications has emerged in recent years. These peptides are models of apolipoproteins, but are much smaller in size than the apolipoproteins. Oral peptides that improve the antiinflammatory properties of HDLs have been shown to potently inhibit atherosclerosis in mouse models. Injection of a peptide with a class A amphipathic helix in a rat model of diabetes dramatically reduced endothelial sloughing and improved vasoreactivity. Injected synthetic peptides have also been described that dramatically lower plasma cholesterol and restore endothelial function in a rabbit model of familial hypercholesterolemia. These studies suggest the therapeutic potential for synthetic peptides in the management of lipid disorders and their vascular complications. Synthetic peptides much smaller than exchangeable human plasma apolipoproteins but with physical and chemical characteristics similar to the plasma apolipoproteins have shown promise in the management of lipid disorders and their vascular complications in animal models. The initial success of these animal studies suggests that synthetic peptides have the potential to emerge as a new therapeutic class of agents in the management of patients with lipid disorders.

Structural Changes in the Lectin Domain of CD23, the Low-Affinity IgE Receptor, upon Calcium Binding

CD23, the low-affinity receptor for IgE (Fc epsilonRII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca2+. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca2+ bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

Stabilization of the Biologically Active Conformation of the Principal Neutralizing Determinant of HIV-1IIIBContaining acis-Proline Surrogate: 1H NMR and Molecular Modeling Study

The V3 loop is part of the gp120 glycoprotein, an extracellular protein located on the membrane of the human immunodeficiency virus (HIV-1). This loop is significantly important in many biological processes of the virus and contains the principal neutralizing determinant (PND). The PND is one of the most variable regions of the envelope, and this is probably related to the ability of the HIV virus to escape the immunologic defenses of the target host. Particular attention has been paid to the central part of the V3 loop which contains a highly conserved GPGR/GPGQ sequence and represents the binding site for antibodies. Many attempts have been made to design synthetic peptides as mimics of the V3 loop capable of eliciting immune response. However, this strategy suffers from the great conformational flexibility small peptides have in solution, which together with bioavailability represents the most important limitation to the usefulness of synthetic peptides as drugs and as synthetic immunogens. The use of conformationally constrained peptides can alleviate this problem. Early works using NMR studies have shown that a V3(IIIB) loop-derived peptide is conformationally heterogeneous when free in water. Upon complexation with 0.5beta, a monoclonal neutralizing antibody specific for the HIV-1(IIIB) strain, it adopts a beta-hairpin conformation with the central proline forming a type VIb beta-turn. In this study, we report the design and characterization of a conformationally restricted peptide with a sequence identical to that previously described, but with thiazolidine derivatives replacing the proline. The affinity of the 2,2-dimethylthiazolidine derivative for 0.5beta demonstrates that this moiety can successfully be used to mimic the proline in a cis conformation. This peptide not only displays a high propensity to adopt a beta-hairpin conformation but also retains the type VIb RGPG beta-turn similar to that found in the native complex. These compounds could help in elaborating more efficient immunogens for HIV-1 synthetic vaccine development.

Use of Synthetic Peptides to Locate Novel Integrin α2β1-binding Motifs in Human Collagen III

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

CpG oligodeoxynucleotides activate dendritic cells in vivo and induce a functional and protective vaccine immunity against a TERT derived modified cryptic MHC class I-restricted epitope

The use of synthetic peptides derived from tumor-associated Ags is attractive for the development of antitumoral vaccines as far as strong adjuvants are found to render them immunogenic. Here, we investigated the possibility to enhance the CD8 response against the human and mouse shared TERT 572Y HLA-A*0201 restricted modified cryptic peptide by using ODN-CpG as adjuvant. Humanized transgenic mice were immunized with the TERT 572Y modified cryptic peptide in the presence of ODN-CpG and compared to mice immunized in IFA. By contrast with IFA, we first showed that, in vivo, ODN-CpG leads to the recruitment of dendritic cells in the lymph nodes draining the injection site. Those cells and especially the CD11c + CD11b − CD8a + lymphoid and the CD11c + B220 + plasmacytoid dendritic cells were activated as shown by up-regulation of CD40 at their cell surface. Immunization against TERT 572Y peptide in the presence of ODN-CpG rather than IFA led to a strong CD8 response and can delayed mortality in an induced tumor model. Study of the CD8 response obtained after antigenic challenge suggested that a functional memory response is induced upon vaccination with ODN-CpG. Thus, MHC class I-restricted epitope in combination of ODN-CpG is a promising and rather simple cancer vaccine formulation.

Comparison between RGD-peptide-modified titanium and borosilicate surfaces

The use of synthetic peptides containing adhesive sequences, such as the Arg-Gly-Asp (RGD) motif, represents a promising strategy to control biological interactions at the cell-material interface. These peptides are known to improve the tissue-material contact owing to highly specific binding to cellular membrane receptors known as integrins, thereby promoting the adhesion, migration and proliferation of cells. The peptides were coupled to borosilicate glass and titanium surfaces using silanisation chemistry. A tryptophan residue was incorporated into the amino acid sequences of selected peptides to facilitate the detection of the covalently bound peptides. Successful peptide immobilisation was proven by fluorimetric measurements. The confocal imaging analysis suggests a homogeneous distribution of the immobilised peptide across the biomaterial surface. In vitro cell proliferation assays were employed to compare the adhesion potentials of the well-known RGD-containing peptides GRGDSP, GRADSP and RGDS to the three peptides designed by our group. The results demonstrate that the RGD sequence is not necessarily required to enhance the adhesion of cells to non-biological surfaces. Moreover, it is shown that the number of adhering cells can be increased by changes in the peptide hydrophobicity. Changes in the cytoskeleton are observed depending on the type of RGD-peptide modification.

Use of Peptide Strategy for Study of Molecular Mechanisms of Hormonal Signal Transduction into Cell

At present the peptide strategy is used extensively to study molecular mechanisms of interaction between signal proteins, components of hormonal signal systems. The strategy is based on use of synthetic peptides as probes corresponding to functionally important sites of these proteins. This review summarizes and analyzes literature data and results of our own works on use of the peptide strategy for studying functional coupling of receptors of serpentine and tyrosine kinase types with heterotrimeric G-proteins. Alongside with peptides derived from the primary structure of cytoplasmic loops and transmembrane domains as well as from different sites of G-protein α, β, and γ-subunits, natural and synthetic peptides are considered which have no homology with receptors and G-proteins, but are able to affect effectively interaction between them.

Jararhagin and its multiple effects on hemostasis

Jararhagin is a 52 kDa hemorrhagic P-III metalloproteinase isolated from the venom of the medically important Brazilian pit-viper Bothrops jararaca. It is a member of the reprolysin family of zinc metalloproteinases containing a catalytic metalloproteinase domain followed by a disintegrin-like and a cysteine-rich domain. The impact of jararhagin on hemostasis has been extensively studied using in vitro and in vivo model systems as well as in clinical studies. Jararhagin-induced hemorrhage is the result of the degradation of sub-endothelial matrix proteins leading to the disruption of the blood vessel endothelium, with accompanying disturbances in platelet function. The versatility of jararhagin is further demonstrated by its direct action on von Willebrand factor, the degradation of fibrinogen, by its inhibition of platelet adhesion to collagen and by its inability to be affected by the plasma inhibitor α 2-macroglobulin. Collagen-induced platelet aggregation is inhibited by jararhagin though the binding of the molecule to the α 2 subunit I domain of the platelet surface α 2β 1 integrin (collagen receptor). Jararhagin also cleaves the β 1 subunit of the same integrin, inhibiting platelet interaction and ultimately causing impairment of signal transduction. The effect of jararhagin on cell systems other than platelets is evaluated; in fibroblasts, jararhagin functions as a collagen-mimetic substrate and, in endothelial cells, it causes apoptosis and indirectly inhibits cell proliferation by release of angiostatin-like compounds. Jararhagin induces a strong pro-inflammatory response characterized by intense leukocyte accumulation at the site of the injection. Although hemorrhage and edema are a response to the direct effect of jararhagin, jararhagin-induced inflammation and necrosis are dependent on macrophages and key pro-inflammatory cytokines or their receptors. Some data also indicate that the toxin possesses anti-tumorgenic properties. Methods for inhibiting jararhagin are reviewed; this encompasses the use of synthetic peptides to the isolation of naturally occurring mammalian peptides and the development of toxin-specific antibodies through DNA immunisation and monoclonal antibody technologies. The availability of jararhagin makes it an important tool for research into the mechanisms of action of similar toxins, for insights into cellular interactions and for clinical investigations into the treatment of envenomings from B. jararaca.

Coming of age: carbohydrates and immunity

Adaptive immune responses have long been considered the "territory" of antigenic proteins, whereas carbohydrates are characterized as T-cell-independent antigens that are not typically recognized by the complete adaptive machinery. The current modus operandi when searching for dominant epitopes is the use of synthetic peptides designed from the primary structure of interesting target proteins; however, there is growing evidence that sugars can also play a critical role in immune recognition. Findings reported in this issue of the European Journal of Immunology begin to shed light on the differences in protein glycosylation that can occur in association with disorders like rheumatoid arthritis and the effect these changes have on collagen recognition by the immune system. Other recent studies have shown that immunodominant glycopeptide "remnant" epitopes as well as glycosylation changes on self-proteins can generate autoimmunity. Finally, some types of carbohydrates are now known to be processed and presented to T cells by class II MHC. Taken together, these advances illustrate a clear importance for carbohydrate recognition in foreign and self antigens by the adaptive immune system. With the common presence of carbohydrate molecules on eukaryotic, prokaryotic, and viral surfaces, the impact of carbohydrates on adaptive immunity is now indisputable.

Assignment of the Binding Site for Haptoglobin on Apolipoprotein A-I

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Inhibition of <I>Porphyromonas gingivalis</I> Hemagglutinating Activity by Synthetic Peptides Derived from Phage Display Selection Using MAb Against the Recombinant Outer Membrane Protein

Porphyromonas gingivalis has been implicated as an pathogen in the development of periodontitis, and hemagglutinins have been identified as an important adhesion onto the gingival tissue cells, and to attach and lyse erythrocytes to uptake Fe ion as an essential nutriant. The 40-kDa outer membrane protein (OMP) has been moleculary cloned from P. gingivalis 381. Since the antibody against recombinant (r) 40-kDa OMP inhibited the hemagglutinating activity, and the polymeric form of r40-kDa OMP itself expressed hemagglutinating activity, the 40-kDa OMP is thought to be one of the hemagglutinins. Moreover, we established MAbs against r40-kDa OMP which were capable of inhibiting hemagglutinating activity of P. gingivalis vesicles. In the present study, a phage-displayed epitope mapping system was used to identify the functional domain expressing hemagglutinating activity by biopanning using the neutralizing mAb, Pg-ompA1. The minimal epitope requirements of the MAb and the predicted amino acid sequences were identified in the region of 96IALDQTLGIP105 in 40-kDa OMP. Synthetic peptide, 87WPRVGQLFIALDQTLGIPTFSVCRME116, mapped the relevant molecule within a short stretch and is corresponding to residues of 40-kDa OMP. Chemically synthesized peptide was used to determine its inhibitory activity against hemagglutinating activity. The synthetic peptide significantly abolished hemagglutinating activity in a dose-dependent manner. These findings suggest that the synthetic peptide is an effective antagonist of erythrocyte binding, and this peptide may be a potent inhibitor of hemagglutination of P. gingivalis cells. The use of synthetic peptide neutralizing hemagglutinating activity of P. gingivalis represents a possible new therapeutic approach to P. gingivalis infected periodontitis.