Peptide A2 Research Articles

Binding epitope for recognition of human TRPM4 channel by monoclonal antibody M4M

Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4.

Neuron-recognizable characteristics of peptides recombined using a neuronal binding domain of botulinum neurotoxin

Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2ʹ and EGFP-RBDʹ, exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBDʹ to SV2C-LD was determined to be 5.45 μM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2ʹ, respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBDʹ to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2ʹ, respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBDʹ. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.

Characterization of T cell phenotype and function in a double transgenic (collagen-specific TCR/HLA-DR1) humanized model of arthritis

IntroductionT cells orchestrate joint inflammation in rheumatoid arthritis (RA), yet they are difficult to study due to the small numbers of antigen-specific cells. The goal of this study was to characterize a new humanized model of autoimmune arthritis and to describe the phenotypic and functional changes that occur in autoimmune T cells following the induction of pathological events.MethodsWe developed a double transgenic mouse containing both the HLA-DR1 transgene and an HLA-DR1-restricted collagen-specific TCR in order to obtain large numbers of antigen-specific T cells that can be used for immunologic studies.ResultsIn vitro, CII-specific T cells from this mouse proliferated vigorously in response to the CII immunodominant peptide A2 and the cells altered their phenotype to become predominately CD62Llow and CD44high “activated” T cells. The response was accompanied by the production of Th1, Th2, and Th17-type cytokines. Following immunization with bovine CII/CFA, these mice develop an accelerated arthritis compared to single transgenic HLA-DR1 mice. On the other hand, when the mice were treated orally with the analog peptide A12, (a suppressive analog of collagen we have previously described), arthritis was significantly suppressed, despite the fact that >90% of the CD4+ T cells express the TCR Tg. In GALT tissues taken from the A12-treated mice, IL-2, IFN-γ, and IL-17 production to the autoimmune collagen determinant dropped while high levels of IL-10 and IL-4 were produced.ConclusionsWe have developed a humanized model of autoimmune arthritis that will be useful for the study of T cell directed therapies as well as T cell mediated mechanisms of autoimmune diseases.

Identification of an Epitope within Human Integrin α6 Subunit for the Binding of Autoantibody and Its Role in Basement Membrane Separation in Oral Pemphigoid

Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin alpha6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin alpha6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin alpha6. Control sera did not bind to the full-length integrin alpha6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin alpha6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.

Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2

Antigenic domain A is a highly conserved unit on envelope protein E2 of classical swine fever virus (CSFV). It was found that mutant E2 containing only unit A, with the unit BC deleted, provided immunized pigs with complete protection against the lethal challenge. In this study, six overlapping peptides (A1–A6) covering this unit were synthesized and conjugated to bovine serum albumin (BSA). Two candidate multi-peptide-vaccines (MPVs) using aluminum adjuvant successfully induced potent immunity against CSFV in pigs. Although both candidate MPVs failed to provide complete protection, they showed better protective activity than that induced by C-strain. Subsequently, neutralizing epitopes in unit A were identified using a panel of peptide-vaccines (PVs). Six candidate peptide-vaccines (PV-An, n = 1–6) were separately given to six groups of pigs. Among these candidates, PV-A2 and PV-A6 exhibited the most potent protective activity, while the other four showed weaker or almost no effects. Moreover, the polyclonal antibodies induced by PV-A2 and PV-A6 were capable of neutralizing C-strain virus at the dilution 1:16 in vitro. Thus, two principal sequential neutralizing determinants covered by peptide A2 (aa792–814) and A6 (aa844–865) were demonstrated to exist in the antigenic domain A, and can be recruited in developing new effective “marker vaccine” against CSFV.

Correlation of Three-dimensional Structures with the Antibacterial Activity of a Group of Peptides Designed Based on a Nontoxic Bacterial Membrane Anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.

CDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.

Implications of HLA-E allele expression and different HLA-E ligand diversity for the regulation of NK cells

The interaction of HLA-E with CD94/NKG2A is dependant on the binding of HLA class I signal sequence derived peptides to HLA-E. In the caucasoid population two HLA-E alleles are observed at equal frequencies. Here we study the functional differences between the two HLA-E molecules with regard to cell surface expression, peptide binding, and potential to inhibit lytic activity of a CD94/NKG2A + NK cell line. In contrast to the HLA-E R allele, the HLA-E G allele shows considerable cell surface expression even in the absence of endogenous HLA class I signal sequence derived HLA-E ligands. Eighteen HLA-E allele/HLA-E ligand combinations were analyzed. No correlation between cell surface expression of HLA-E and NK cell inhibition was observed. The peptides present in the signal sequences of HLA-B15, -Cw0402, and -Cw7 bound to both HLA-E alleles but did not lead to an inhibition of NK cell lysis. In our experimental system the peptides A2 and G were not effective with regard to NK cell inhibition when bound to the HLA-E R allele. These results may be of functional significance particularly in the placenta where the only HLA-E ligands are derived from HLA-G and -C.

Characterization of a Peptide Analog of a Determinant of Type II Collagen That Suppresses Collagen-Induced Arthritis

Immunization of susceptible strains of mice with type II collagen (CII) elicits an autoimmune arthritis known as collagen-induced arthritis (CIA). One analogue peptide of the immunodominant T cell determinant, A9 (CII245-270 (I260-->A, A261-->B, F263-->N)), was previously shown to induce a profound suppression of CIA when coadministered at the time of immunization with CII. In the present study, A9 peptide was administered i.p., orally, intranasally, or i.v. 2 to 4 wk following CII immunization. We found that arthritis was significantly suppressed even when A9 was administered after disease was induced. To determine the mechanism of action of A9, cytokine responses to A9 and wild-type peptide A2 by CII-sensitized spleen cells were compared. An increase in IL-4 and IL-10, but not in IFN-gamma, was found in A9 culture supernatants. Additionally, cells obtained from A9-immunized mice produced higher amounts of IL-4 and IL-10 when cultured with CII compared with cells obtained from mice immunized with A2, which produced predominantly IFN-gamma. Suppression of arthritis could be transferred to naive mice using A9-immune splenocytes. Lastly, phosphorylation of TCRzeta was not altered in the immunoprecipitates from the lysates of cells exposed to analogue peptides (A9 and A10) together with wild-type A2 in a T cell line and two I-Aq-restricted, CII-specific T hybridomas. We conclude that analogue peptide A9 is effective in suppressing established CIA by inducing T cells to produce a Th2 cytokine pattern in response to CII.

The class II MHC protein HLA-DR1 in complex with an endogenous peptide: implications for the structural basis of the specificity of peptide binding.

Class II major histocompatibility complex (MHC) proteins are cell surface glycoproteins that bind peptides and present them to T cells as part of the mechanism for detecting and responding to foreign material in the body. The peptide-binding activity exhibits allele-specific preferences for particular sidechains at some positions, although the structural basis of these preferences is not understood in detail. We have determined the 2.45 A crystal structure of the human class II MHC protein HLA-DR1 in complex with the tight binding endogenous peptide A2 (103-117) in order to discover peptide-MHC interactions that are important in determining the binding motif and to investigate conformational constraints on the bound peptide. The bound peptide adopts a polyproline II-like conformation and places several sidechains within pockets in the binding site. Bound water molecules mediate MHC-peptide contacts at several sites. A tryptophan residue from the beta 2 'lower' domain of HLA-DR1 was found to project into a pocket underneath the peptide-binding domain and may be important in modulating interdomain interactions in MHC proteins. The peptide-binding motif of HLA-DR1 includes an aromatic residue at position +1, an arginine residue at position +2, and a small residue at position +6 (where the numbering refers to the normal MHC class II convention); these preferences can be understood in light of interactions observed in the peptide-MHC complex. Comparison of the structure with that of another MHC-peptide complex shows that completely different peptide sequences bind in essentially the same conformation and are accommodated with only minimal rearrangement of HLA-DR1 residues. Small conformational differences that are observed appear to be important in interactions with other proteins.

Determination of HLA-B7 T-cell epitopes

Abstract A series of peptides de rived from the αl and α2 regions of the HLA-B7 and HLA-A2 mole cules was synthesized with an auto matic peptide synthesizer using the FMOC (9–fluorenylmethoxycarbon-yl) technique. Peptides were ana lyzed and purified by reversed-phase high pressure liquid chroma-tography (HPLC). Peptide purity was > 96%. The effect of B7 pep tides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cy-tolysis. The data show that the pep tides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62–70 in HLA-B7) and peptide A2 (residues 62–70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7–specific CTLs were incubated with these peptides at 37 °C for 90 min and cell fluorescence measured by flow cy-tometry. The B7 peptide was bound by 60 % of the CTLs whereas the A2 peptide, as a negative control, bound only 10 %. The molecular size of the ligands to which the peptides bind are being characterized by im-munoprecipitation.

Determination of HLA-B7 T-cell epitopes.

A series of peptides derived from the alpha 1 and alpha 2 regions of the HLA-B7 and HLA-A2 molecules was synthesized with an automatic peptide synthesizer using the FMOC (9-fluorenylmethoxycarbonyl) technique. Peptides were analyzed and purified by reversed-phase high pressure liquid chromatography (HPLC). Peptide purity was > 96%. The effect of B7 peptides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cytolysis. The data show that the peptides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62-70 in HLA-B7) and peptide A2 (residues 62-70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7-specific CTLs were incubated with these peptides at 37 degrees C for 90 min and cell fluorescence measured by flow cytometry. The B7 peptide was bound by 60% of the CTLs whereas the A2 peptide, as a negative control, bound only 10%. The molecular size of the ligands to which the peptides bind are being characterized by immunoprecipitation.

Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus. Involvement of conserved residues in ligand binding.

The nucleotide sequence of two genes encoding fibronectin (Fn) receptors FnBA and FnBB of Streptococcus dysgalactiae S2 revealed the presence of repeated motifs (called RA1-A3 and RB1-B3, respectively) which encode Fn binding activity (Lindgren, P.-E., McGavin, M. J., Signäs, C., Guss, B., Gurusiddappa, S., Höök, M., and Lindberg, M. (1993) Eur. J. Biochem. 214, 819-827). Synthetic peptides of 32-37 amino acids, corresponding to individual repeated motifs, were assayed for the ability to inhibit Fn binding to cells of S. dysgalactiae. Within the RA motifs, peptide A2 was 10-fold more active than either A1 or A3, while in the RB motifs, only B3 was active. The same level of activity is observed when these synthetic peptides were assayed for inhibition of Fn binding to cells of Staphylococcus aureus. Likewise, synthetic peptides corresponding to the RD1-D3 motifs, which comprise a ligand binding domain in a Fn receptor from S. aureus, inhibit binding of Fn to both S. aureus and S. dysgalactiae. Assays of chemically modified peptides and peptide fragments derived from chemical or proteolytic cleavage suggest that a conserved core sequence, defined as ED(T/S) (X9,10)GG(X3,4)(I/V)DF, within a 30-amino acid-long segment is present in the active RA and RD motifs. Analyses of the importance of individual residues of this core sequence indicate that the ED(T/S) motif is nonessential, whereas the GG and the (I/V)DF together with additional acidic residues in the C-terminal half of the peptide are required for activity.

Structural characterization of the phosphorylation sites of human erythrocyte spectrin.

The phosphorylation sites of spectrin dimer were characterized before and after incubation of intact human red cells with [32P]orthophosphate. Phosphate measurements of unlabeled spectrin dimer show it contains 4.0 +/- 0.3 covalent protein phosphates, all located on band 2. Quantitation of the number of exchangeable phosphorylation sites in erythrocytes labeled with [32P]orthophosphate yields 3.9 +/- 0.3 phosphates/spectrin dimer, indicating that all four spectrin phosphorylation sites are metabolically active. Tryptic and chymotryptic peptide mapping reveals that the dimer contains three unique 32P-labeled tryptic peptides of approximately 4,600 (A1), 3,500 (A2), and 2,400 (B) daltons. Peptide A1 contains both phosphoserine and phosphothreonine while peptides A2 and B possess only phosphoserine. Peptides A1 and A2 remain associated after trypsinization of spectrin dimer and are only separable in detergents. All three 32P-labeled tryptic peptides are contained within a 20,000 dalton cyanogen bromide fragment which is within a 60,000 dalton staphylococcal protease phosphopeptide. All these fragments are found within a 90,000 dalton nitrothiocyanobenzoic acid phosphopeptide. The purified 20,000 dalton fragment contains no homoserine or homoserine lactone and is the COOH-terminal cyanogen bromide peptide of band 2. The isolated tryptic peptide B possesses no lysine or arginine and is presumably the COOH-terminal tryptic peptide of band 2 and the most distal phosphopeptide. Thus, the four phosphorylation sites of spectrin dimer are clustered at the extreme COOH-terminal end of band 2.

CHOLERA TOXIN

CHOLERA TOXIN