Delineation and conformational analysis of two synthetic peptide models of antigenic sites on rodent cytochrome c

Two regions of rodent cytochrome c, one within the first four residues of the molecule, which is N-acetylated, and one at a beta bend around residue 44, are known to be immunogenic and antigenic in rabbits. Using sequential peptide synthesis, we have determined the residues required for linear synthetic peptides within these sequences to bind to antibody raised in rabbits to intact rat cytochrome c. The residues that were important in binding the N-terminal peptides were N-acetylglycine at position 1 and valine at position 3. The smallest peptide sequence around residue 44 that would bind to antibodies was Gln-Ala-Ala-Gly-Phe. A theoretical conformational analysis of these peptides showed that the amino-terminal tetrapeptide adopts a wide statistical ensemble of conformational states and that the addition of residues beyond 41 and 45 in the other sequence does not appear to stabilize longer peptides in the native beta-bend conformation. Thus, the antigenicity conferred by Phe-46 and Gln-42 in this peptide is most likely due to the direct interaction of the side chains of these residues with the antibody binding site. The demonstration here that native conformation is not essential for antigenic peptides to bind to antibodies raised against the whole protein indicates that the association energy between antigen and antibody can be sufficient to induce conformation in conformationally flexible peptides. This supports the concept that anti-protein and anti-peptide antibodies may invoke conformational changes in cross-reactive protein antigens and may explain why longer peptides, which may adopt stable nonnative secondary structure, often do not bind to antibodies raised to the whole molecule.     

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.     

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.     

Self-assembly of trimethyl chitosan and poly(anionic amino acid)-peptide antigen conjugate to produce a potent self-adjuvanting nanovaccine delivery system

Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.     

Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.     

Location of the actual signal in the negatively charged leader sequence involved in the import into the mitochondrial matrix space

Proteins destined for the mitochondrial matrix space have leader sequences that are typically present at the most N-terminal end of the nuclear-encoded precursor protein. The leaders are rich in positive charges and usually deficient of negative charges. This observation led to the acid-chain hypothesis to explain how the leader sequences interact with negatively charged receptor proteins. Here we show using both chimeric leaders and one from isopropyl malate synthase that possesses a negative charge that the leader need not be at the very N terminus of the precursor. Experiments were performed with modified non-functioning leader sequences fused to either the native or a non-functioning leader of aldehyde dehydrogenase so that an internal leader sequence could exist. The internal leader is sufficient for the import of the modified precursor protein. It appears that this leader still needs to form an amphipathic helix just like the normal N-terminal leaders do. This internal leader could function even if the most N-terminal portion contained negative charges in the first 7-11 residues. If the first 11 residues were deleted from isopropyl malate synthase, the resulting protein was imported more successfully than the native protein. It appears that precursors that carry negatively charged leaders use an internal signal sequence to compensate for the non-functional segment at the most N-terminal portion of the protein.     

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.     

Identification of MHC class II-associated peptides that promote the presentation of toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.     

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.     

Structural analysis of adult and larval isozymes of sn-glycerol-3-phosphate dehydrogenase of Drosophila melanogaster

Compositional analysis of the soluble tryptic peptides representing about 70% of the 293 residues of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster reveals a single peptide difference between the sn-glycerol-3-phosphate dehydrogenase adult (GPDHF-1) and larval (GPDHF-3) isozymes. This peptide was shown to be the carboxyl terminus by sequence determination and by carboxypeptidase A digestion of the native protein. For GPDHF-1, the sequence of the COOH-terminal tryptic peptide is Asn-His-Pro-Glu-His-Met-Gln-Asn-Leu-COOH, while that of GPDHF-3 is Asn-His-Pro-Glu-His-Met-COOH.     

Allergic encephalomyelitis: characterization of the determinants for delayed type hypersensitivity

Three non-encephalitogenic peptides derived from the encephalitogenic myelin basic protein of the central nervous system, produce delayed type hypersensitivity responses and elicit delayed skin reaction in guinea pigs sensitized with either peptide, the encephalitogenic tryptophan region (peptide E) or the basic protein. The amino acid sequence of the peptides is N-Acetyl-Ala-Ser-Ala-Gln-Lys-OH, forming the N-terminal region of the basic protein molecule, H-Gly-Ser-Leu-Pro-Gln-Lys-OH and H-Gly-Ala-Glu-Gly-Gln-Lys-OH representing residues number 69–74 and 117–122 of the basic protein respectively.     

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.     

Shortening and modifying the 1513 MSP-1 peptide's alpha-helical region induces protection against malaria

Immunogenic and protective peptide sequences are of prime importance in the search for an anti-malarial vaccine. The MSP-1 conserved and semi-conserved sequences have been shown to contain red blood cell (RBC) membrane high affinity binding peptides (HABP). HABP 1513 sequence ((42)GYSLFQKEKMVLNEGTSGTA(61)), from this protein's N-terminal, has been shown to possess a T-epitope; however, it did not induce a humoral immune response or complete protection when evaluated in Aotus monkeys. Analogue peptides with critical binding residues replaced by amino acids with similar mass but different charge were synthesised and tested for immunogenicity and protectivity in monkey. NMR studies correlated structural behaviour with biological function. Non-immunogenic and non-protective 1513 native peptide presented a helical fragment between residues L(4) and E(14). C-terminal, 5-residue-shorter, non-immunogenic, non-protective peptide 17894 contained an alpha-helix from Q(6) to L(12) residues. Immunogenic and protective peptide 13946 presented a shorter alpha-helix between K(7) to N(13) residues. These data suggest that changing certain residues permits better peptide fit within the MHC class II-peptide-TCR complex, thus activating the immune system and inducing a protective immune response.     

Membrane interaction of Escherichia coli hemolysin: flotation and insertion-dependent labeling by phospholipid vesicles

The 1,024-amino-acid acylated hemolysin of Escherichia coli subverts host cell functions and causes cell lysis. Both activities require insertion of the toxin into target mammalian cell membranes. To identify directly the principal toxin sequences dictating membrane binding and insertion, we assayed the lipid bilayer interaction of native protoxin, stably active toxin, and recombinant peptides. Binding was assessed by flotation of protein-liposome mixtures through density gradients, and insertion was assessed by labeling with a photoactivatable probe incorporated into the target lipid bilayer. Both the active acylated hemolysin and the inactive unacylated protoxin were able to bind and also insert. Ca(2+) binding, which is required for toxin activity, did not influence the in vitro interaction with liposomes. Three overlapping large peptides were expressed separately. A C-terminal peptide including residues 601 to 1024 did not interact in either assay. An internal peptide spanning residues 496 to 831, including the two acylation sites, bound to phospholipid vesicles and showed a low level of insertion-dependent labeling. In vitro acylation had no effect on the bilayer interaction of either this peptide or the full-length protoxin. An N-terminal peptide comprising residues 1 to 520 also bound to phospholipid vesicles and showed strong insertion-dependent labeling, ca. 5- to 25-fold that of the internal peptide. Generation of five smaller peptides from the N-terminal region identified the principal determinant of lipid insertion as the hydrophobic sequence encompassing residues 177 to 411, which is conserved among hemolysin-related toxins.     

The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1

The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.     

Analysis of Heparin-Binding Sites in Human Lipoprotein Lipase Using Synthetic Peptides

Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388–395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261–287) and cluster 3 (residues 147–151) peptides followed by cluster 2 (residues 290–302) peptide. A peptide corresponding to LPL cluster 4 (residues 405–414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications.