Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.     

Selection of phage-displayed peptides mimicking an extracellular epitope of human MDR1-P-glycoprotein

To study the structural conformation of the MM4.17 monoclonal antibody (mAb) epitope, twenty-six mAb MM4.17-specific phage clones were affinity-isolated and their inserts characterized for amino acid composition and homology with MDR1 gene product (MDR1-P-glycoprotein). The resulting sequence alignment shows that a unique consensus sequence, which corresponds to the previously mapped TRIDDPET linear peptide identified through synthetic peptide scanning, could not be identified. However, similarities between the inserts of positive phage clones and P-glycoprotein primary structure, consisting in two or three amino acid-long sequences, were observed. An analysis of the over-represented amino acid residues in the inserts of positive clones, and their comparison with the sequence of the antigen was also performed. The two different procedures led to the identification of four regions in which these similarities are clustered, indicating that four different antigen regions, one of which includes the TRIDDPET linear amino acid sequence, might participate in forming the structure of monoclonal antibody MM4.17 epitope.     

Development of a polymeric surgical paste formulation for taxol

The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RTlu haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184-198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine-specific, but cross-reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence was also autoantigenic. Use of independently synthesized and purified 184-198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA: this was found to delay the onset of disease, reduce mean disease severity, shift the anti-CII antibody response to favour antibodies of the IgG1, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184-198 peptide. The dominant 184-198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre-treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen.     

Activation of a murine autoreactive B cell by immunization with human recombinant autoantigen La/SS-B: characterization of the autoepitope

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.     

Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein

The reactions of a panel of 34 mouse monoclonal antibodies (MAbs) specific for the dengue-2 virus nonstructural-1 glycoprotein (NS1), were analysed using 174 overlapping synthetic nonameric peptides covering the entire sequence. Using this methodology, four epitopes were identified. One pair of MAbs, which defined a dengue-2/4 virus subcomplex epitope (24C: amino acids 299-309) using native NS1 proteins, showed the same reaction pattern with synthetic peptides containing the corresponding NS1 sequences of each virus serotype. One amino acid substitution, present in the sequences from the dengue-1/3 virus subcomplex abrogated almost all reaction by these MAbs. A dengue complex epitope (LX1: amino acids 111-121) was also located and peptides containing the sequences of each serotype were shown to contain only antigenically silent amino-acid substitutions. In contrast, MAbs which defined a dengue type-specific epitope (LD2: amino acids 25-33) and another dengue subcomplex epitope (24A: amino acids 61-69) failed to show the same reaction profiles using peptides of each serotype, suggesting that these determinants were partially dependent upon conformation. The LX1 epitope is a good candidate for further trials aimed at generating cross-protective immune responses to these viruses without the risk of antibody-dependent enhancement.     

Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage

The structural requirements for peptide binding to an antibody may be elucidated by probing it with a variety of peptides having different constraints. To this end, we have constructed and screened a panel of peptide libraries displayed by filamentous bacteriophage. The peptides in most of the libraries have the potential for constraint by fixed Cys residues, which have been placed at different sites within a randomized amino acid sequence of varying length. When taken together, the binding data obtained from screening the panel with a given antibody allow one to determine the types of constraints that promote binding, as well as the residues that are critical for binding. We describe the construction of 11, pVIII-displayed, peptide libraries, whose sizes range from 150 million to 10 billion clones. The libraries were screened with a number of polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates. Cross-reactivity with peptides was always found for antibodies produced against peptides, linear epitopes on folded proteins and, surprisingly, carbohydrates, whereas antibodies against discontinuous epitopes on proteins were found less frequently. The implications of these results are discussed in terms of the structural basis for cross-reactivity with peptides.     

Characteristics and determinants of sumatriptan-associated chest pain

Analysis of epitopes recognized by therapeutic monoclonal antibodies (mAb) is critical in clinical applications and in structure/function studies of target antigen. mAb MGr6 recognizes the extracellular domain of the p185HER2 oncoprotein and is a promising candidate for cancer immunodiagnosis and immunotherapy. Thus, epitope location and structure on p185HER2 need to be investigated. The use of MGr6-selected phage-displayed peptides for epitope analysis served to dissect the MGr6 epitope into at least two subregions, mimicked by CHSDC- and (L)P-(L)K(L) phage displayed peptides, respectively. Comparison of amino acid sequences of CHSDC peptides with the p185HER2 protein sequence and analysis of MGr6 reactivity with p185HER2 deletion mutants identified the linear subregion CCHEQCAAG of the MGr6 epitope, corresponding to amino acids 235-243 of the p185HER2 protein. This continuous subregion is part of a larger conformational epitope, and other amino acids, including a proline, a lysine and leucine residues contained in (L)P-(L)K(L) phage-displayed peptides appear to contribute to the formation of the MGr6 epitope surface.     

Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI

Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with factor XIIa.     

Variant antibody identification by peptide mapping

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A-purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H-chain) band with a molecular weight slightly lower than that of the principal H-chain band on SDS-PAGE. The N-terminal amino acid sequence of this minor H-chain species indicated that at least the first 30 amino acids were identical to those of the antibody light-chain (L-chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg108 of the L chain and Ala124 of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful "first-line" analytical tool in the characterization of the quality of the monoclonal antibody.     

Definition of essential amino acid residues in the recognition of a peptide by a mouse monoclonal antibody

A mouse monoclonal antibody reacting in ELISA with a synthetic peptide representing a linear amino acid stretch of the protein antigen was tested on all overlapping 5-mer to 9-mer fragments of the peptide, as prepared by multi-pin synthesis. Analysis of the binding data suggests that several residues in the peptide might be relatively unrelevant for recognition, while few others seem to play a critical role as key residues. On the basis of such observations, we attempted to reconstruct an alternative essential epitope by introducing multiple amino acid substitutions in the 9-mer peptide exhibiting the best binding activity, and then tested its ability to be recognized by the monoclonal antibody.     

The role of structure in antibody cross-reactivity between peptides and folded proteins

Peptides have the potential for targeting vaccines against pre-specified epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced fit of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with sub-micromolar affinities. The strong preference for a type II beta-turn predicted for one peptide was confirmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.     

Mapping and characterization of a sequential epitope on the rabies virus glycoprotein which is recognized by a neutralizing monoclonal antibody, RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Analysis of a kanamycin resistance gene (kmr) from Streptomyces kanamyceticus and a mutant with increased aminoglycoside resistance

The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes. The dominant epitope, VIVMLTPLVEDGVKQC (aa 805-820), which elicited the highest T-cell responses in all at-risk relatives, has 56% identity and 100% similarity over 9 amino acids (aa) with a sequence in VP7, a major immunogenic protein of human rotavirus. Both peptides bind to HLA-DR4(*0401) and are deduced to present identical aa to the T-cell receptor. The contiguous sequence of VP7 has 75% identity and 92% similarity over 12 aa with a known T-cell epitope in glutamic acid decarboxylase (GAD), another autoantigen in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity over 8-14 aa to sequences in Dengue, cytomegalovirus, measles, hepatitis C, and canine distemper viruses, and the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 aa to herpes, rhino-, hanta- and flaviviruses. Two others are 80-82% similar over 10-11 aa to sequences in milk, wheat, and bean proteins. Further studies should now be carried out to directly test the hypothesis that T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and (for rotavirus) GAD.     

Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.     

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.     

Primary structure of a cadmium-induced metallothionein from the insect Orchesella cincta (Collembola)

The induction of metallothionein was studied in the springtail Orchesella cincta (Collembola), a species of insect living in forest soils. Upon dietary exposure to Cd, two Cd-binding, cysteine-rich peptides were isolated from whole-body homogenates, using gel filtration and reversed-phase FPLC. Mass spectrometric analysis revealed that the molecular masses of these peptides were 2989 Da and 4139 Da, respectively. Amino acid sequencing of the 2989-Da peptide resulted in a sequence typical for a metallothionein. Sequencing of the 4139-Da protein was unsuccessful, probably due to N-terminal blockage. Using different PCR techniques (3' and 5' RACE) with (degenerate) primers based on the identified amino acid sequence of the 2989 Da peptide, a metallothionein cDNA was isolated. The sequence of this cDNA potentially codes for a protein of 77 amino acids. The 2989 Da peptide corresponds to the C-terminal part of this protein. The 4139-Da protein is probably encoded by the N-terminal part of this protein. These results suggest that the identified peptides are products of one gene, and that the primary gene product is subject to post-translational processing. The deduced amino acid sequence of the O. cincta metallothionein shows low sequence similarity with metallothioneins from Drosophila. The similarity between O. cincta MT and MTs of invertebrates is not higher than that between O. cincta and vertebrates.     

Fine epitope specificity of antibodies to region II of the Plasmodium vivax circumsporozoite protein correlates with ability to bind recombinant protein and sporozoites

Recent work has suggested that important B- and T-cell epitopes on the circumsporozoite protein (CSP) of Plasmodium vivax lie external to the major repeat regions of the protein. We have studied two naturally exposed human populations (Caucasian and Papua New Guineans) and determined the antibody response to yeast-derived recombinant CSPs, overlapping synthetic peptides spanning amino acids 76 348 of the Belem P. vivax CSP and overlapping peptides representing the variant repeats of the VK247 strain of P. vivax. We have demonstrated that the P. vivax CSP-specific antibody response is directed towards areas within the repeat region as well as areas external to this; but the dominant epitopes recognized by the two populations studied, were distinct. One epitope, lying external to the repeats and recognized by both populations, partially overlaps an area of the protein referred to as region II-plus. Sera from malaria-exposed Papua New Guineans and Thais contained antibodies to this epitope (V22, single letter amino acid sequence TCGVGVRVRRRVNAANKKPE) which were capable of recognizing sporozoites, as determined by quantitative inhibition IFA. Seventeen percent of PNG sera had antibodies to this peptide compared with 33% who had antibodies to the central repeats of the protein. Immunization of mice with recombinant CSP did not induce antibodies to V22. However, immunization with overlapping peptide epitopes representing this region (V21 or V22) induced specific antibodies but only two sera recognized both V21 and V22 and, by inference, the overlapping peptide sequence (TCGVGVRVRR). Antibodies in these two sera could bind recombinant CSP in ELISA; however, in contrast, nine sera which recognized either V21 or V22 alone did not bind CSP. Only one of two sera containing antibodies recognizing CSP stained P. vivax sporozoites. This serum also recognized an epitope dependent upon two amino acids aminoterminal to V22. These data suggest that the fine specificity of antibodies is a critical determinant for binding to both recCSP and sporozoites.     

Monoclonal antibody against bovine Lactoferricin and its epitopic site

Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".