Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structure.

Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.

Ratio encoding combinatorial libraries with stable isotopes and their utility in pharmaceutical research.

Combinatorial libraries are an important tool for lead discovery in the pharmaceutical industry. Advances in high throughput screening coupled with combinatorial chemistry can significantly reduce the time to find lead compounds. A major difficulty in developing large combinatorial libraries is the ability to identify active compounds. This paper describes a rapid and sensitive encoding/decoding methodology that utilizes stable isotopes and mass spectrometry. The ability of mass spectrometry to precisely determine the intensity of isotopic abundances provides a unique encoding strategy employing synthetically generated ratios of stable isotopes in a compound as the code. The application of ratio encoding is demonstrated using peptoid and imidazole chemistries. Supporting data demonstrate that the incorporation of one or more stable isotopes using unique-predetermined ratios can encode chemical libraries. In addition, the presence of a unique isotopic pattern in a ligand can facilitate the pharmacokinetic analysis. Isotope incorporation into a compound and subsequently into its metabolites reliably distinguishes products from other molecules in the mass spectrum. This is illustrated by metabolic analyses of peptoid and imidazole compounds.

Isotope or mass encoding of combinatorial libraries.

BACKGROUND: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds. RESULTS: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution. CONCLUSIONS: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.

High-throughput purity estimation and characterisation of synthetic peptides by electrospray mass spectrometry.

High-throughput analysis for purity and molecular weight determination of synthetic peptides including characterisation of any peptidic byproducts arising from synthesis is described. The data from electrospray mass spectrometry are processed with an algorithm that calculates the contribution of the target peptide and each of the identifiable contaminants to the total ionizable material in a sample of synthetic peptide. All essential data are obtained by one instrumental technique in < 3 min per sample. The technique has distinct advantages in the rapid analysis of the many hundreds of peptides/peptidomimetics required in systematic quantitative structure-activity relationship and other investigations.

Analysis of the requirements for class II-restricted T cell recognition of a single determinant reveals considerable diversity in the T cell response and degeneracy of peptide binding to I-Ed.

The amino acid sequences recognized by five I-E(d)-restricted and one E alpha A beta d-restricted murine T cell clones were determined. The clones had been raised to a synthetic peptide representing amino acids 305-328 of influenza virus hemagglutinin. It was found that although all of the T cell clones recognized a single 10-residue region of the peptide, 307KYVKQNTLKL316, different clones could recognize minimal ("core") determinants spanning 8, 9, or 10 of these amino acids. To see whether particular amino acids within the sequence 307-316 were universally important for T cell recognition, the six clones were assayed for their ability to tolerate single amino acid substitutions of the 10 residue peptide. In all, 190 analogues of the peptide in which each amino acid in the sequence was replaced, in turn, by each of the other 19 naturally occurring amino acids were tested. It was shown that 1) the six T cell clones had very different requirements for recognition of the peptide, 2) substitutions at every single position within the peptide could be shown to affect recognition in a T cell-specific manner, and 3) every single position within the peptide could be replaced by a large number of amino acids and still be recognized by at least one T cell clone. These results demonstrate the great diversity exhibited by the T cell repertoire in recognizing a 10-amino acid determinant, as well as the degeneracy of peptide binding to I-E(d).

A single-bead decode strategy using electrospray ionization mass spectrometry and a new photolabile linker: 3-amino-3-(2-nitrophenyl)propionic acid.

A new linker that employs a photosensitive 3-amino-3-(2-nitrophenyl)propionyl functionality (ANP-resin) has been developed for the preparation of C-terminal carboxamides. A wide range of carboxamides were prepared and identified using the ANP-resin and electrospray ionization mass spectrometry. A single bead containing tripeptide Fmoc-Asp-Arg(Tos)-Val-NH2 was isolated, photocleaved and the peptide was characterized by tandem mass spectrometry, thereby verifying a library decode strategy that avoids complex tagging procedures.

Recognition of multiple peptide cores by a single T cell receptor.

We present evidence that a single T cell clone can recognize at least five different overlapping peptides, each with its distinct core structure, in the context of the same major histocompatibility complex (MHC) molecule. Distinct core residues are crucial for triggering the T cell receptor (TCR) in each case. These results suggest that the TCR (a) has multiple sets of contact residues for alternative peptide-MHC ligands, the binding to any one of which can trigger the cell; and/or (b) is able to attach to the peptide-MHC complex in more than one orientation. In this sense, the TCR is a multisubsite structure capable of being stimulated by a variety of peptide ligands associated with the same MHC molecules.

Immunodominant framework region 3 peptide from TCR V beta 8.2 chain controls murine experimental autoimmune encephalomyelitis.

Previous work has demonstrated the existence of regulatory circuitry that controls response to the dominant determinant Ac1-9 of myelin basic protein (MBP) which is highly restricted in TCR V gene usage to V beta 8.2 and V alpha 2.3. In particular, a CD4+ V beta 14+ regulatory T cell was shown to be a vital component of this circuit. In our work presented here, the peptide specificity of the response to V beta 8.2 peptides was examined. Five overlapping peptides, B1 through B5, were studied for their ability to induce a proliferative response: B2 (21-50), B4 (61-90), and B5 (76-101) each had this capacity in the B10.PL or (SJL x B10.PL)F1 mice. The determinant within the TCR peptide B5 appears dominant, whereas determinants within the B2 and B4 peptides are physiologically cryptic. Furthermore, only B5 could down-regulate the response to MBP Ac1-9 and significantly protect mice from MBP- or Ac1-9-induced EAE, whereas B2 or B4 treatment had no significant effect. Treatment of mice with B5 did not result in generalized deletion or inactivation of V beta 8.2+ T cells. The core residues of the B5 determinant lie within framework region 3 of the V beta 8.2 chain and do not include residues from the joining CDR3 region. Response to B5 was restricted by the I-Au MHC molecule. Furthermore, B5 only induced responses in mice with certain MHC alleles. It is evident that by specifically down-regulating the initial dominant response to Ac1-9, Ag-induced disease can be prevented. These data have implications for understanding induction of TCR-based regulation, as well as relevance to possible therapeutic approaches for oligoclonal responses in human autoimmune diseases.

Limited T cell response to donor MHC peptides during allograft rejection. Implications for selective immune therapy in transplantation.

Previously, we have demonstrated that during allograft rejection, MHC molecules of the donor are processed and presented to alloreactive CD4+ T lymphocytes in the form of peptides associated with the MHC class II molecules of the recipient. There is an increasing body of evidence that this indirect pathway of allorecognition may play a major role in allograft rejection. Herein, we have used a series of overlapping MHC peptides progressing along the sequence of the donor MHC molecule in single residue steps. We have mapped all potential MHC Ag determinants to which T cell responses could be generated after s.c. injection of allogeneic splenocytes. We have shown that splenic T cell proliferative responses to the beta 1 domains of donor Ak, Ad, and A(s) mouse MHC class II molecules were directed toward a single immunodominant determinant in each of three donor/recipient combinations. Interestingly, after allogeneic spleen cell transplantation, an additional determinant on donor MHC could be detected in the draining lymph nodes. This result shows that the fine specificity of T cell response to donor transplantation Ags can differ between lymphoid organs. Then, we investigated whether limitation of T cell response to donor MHC peptides applies to the clinical situation of a graft. We have shown that after an allogeneic skin graft, self-restricted alloreactive T cells proliferated to the same determinant on the donor MHC molecule. These results indicate that immune intervention, such as tolerance induction to the dominant T cell determinant on donor MHC molecules, may be developed for the prevention of allograft rejection.

Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides.

Major T helper epitopes of medically important antigens can be located by measuring the proliferative responses of human peripheral blood mononuclear cells (PBMC) to pools of short synthetic peptides. The length and endings of the peptides used were shown to be critical for success in identifying Th cell epitopes. Many epitopes would be missed if either long (31mers) or short (less than 12mers) peptides were used. Pools of 14 and 16mers were more efficient than 12mers spanning the same region, however, for a promiscuous Th cell epitope of tetanus toxin (tt 947-967), two of three donors tested did not respond to 18mers or shorter peptides spanning this region. Although peptides with either unblocked or blocked ends were stimulatory, peptides with blocked ends were generally more efficient. The peptide concentration and number of available APC were also found affect the efficiency of the proliferation assay as a measure of peptide recognition by Th cells. Two screenings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulatory regions. Thus, PBMC and peptide pools can be used for the reproducible identification of Th cell epitopes. After immunization with tetanus toxoid (TT), peptide-responsive cells increased in frequency in parallel to the increase in TT responsive cells, indicating that the peptide-responsive cells were primed by TT.

T cell determinant structure of myelin basic protein in B10.PL, SJL/J, and their F1S.

Recent experiments have shown that during the course of chronic experimental allergic encephalomyelitis, there is a shift in the determinant hierarchy away from the dominant to other subdominant and cryptic self determinants. It was therefore of interest to define the pattern of dominance for mouse myelin basic protein in the three commonly used experimental allergic encephalomyelitis model strains of mice, i.e., B10.PL, SJL/J, and (SJL x B10.PL)F1. Our studies indicate that many cryptic determinants are demonstrable, which only activate T cells on injection as individual peptides and not with the native protein. The core amino acid residues of the various determinants are defined and range in size between 5 and 10 amino acids. Interestingly, there is a bias toward H-2u-restricted response vis-a-vis the H-2s-restricted response in the (SJL x B10.PL)F1 strain. The TCR V beta 8.2 gene segment was not predominantly used for responses to other determinants, although some B10.PL and (SJL x B10.PL)F1 cell lines expressed V beta 8.2 more than others. This study represents the most comprehensive analysis so far of the pattern of dominant and cryptic proliferative T cell determinants and their core sequences for mouse myelin basic protein.

Disruption of the determinant hierarchy on a self-MHC peptide: concomitant tolerance induction to the dominant determinant and priming to the cryptic self-determinant.

The presentation of self-peptides in a self-restricted manner plays a critical role in the complex positive and negative selective process of T cell recognition of self-determinants. The population of determinants comprises (i) a dominant set which is efficiently presented and induces clonal elimination or inactivation of the corresponding autoreactive T cells, and (ii) a cryptic set which is not processed efficiently enough to reach the threshold of presentation to make an impact on the T cell repertoire during thymic selection. Here we have studied a self-MHC peptide, Ld 61-85, which as shown in earlier work was able to induce vigorous T cell proliferation in syngeneic animals. Despite the fact that this peptide as a whole is 'cryptic', the fine specificity of the class II restricted response was complex, in that there were three distinct and overlapping T cell determinants: the dominant determinant, Ld 65-80, flanked by two cryptic determinants, Ld 61-75 and Ld 73-85, all of which compete for stimulating in vivo autoreactive T cell proliferative responses. The hierarchy of these determinants bears an interesting relationship to tolerance. Ld 61-85 or Ld 61-80 priming induces proliferation only to Ld 65-80; likewise, tolerance induction to Ld 61-80 prevents elicitation of a subsequent response to Ld 61-80 or Ld 65-80 in the local lymph nodes. However, in the Ld 61-80 tolerant mice, in vivo challenge with Ld 61-80 induces a strong T cell proliferative response directed towards cryptic Ld 61-75.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping the major human T helper epitopes of tetanus toxin. The emerging picture.

Progress on the mapping of Th epitopes of tetanus toxin (tt) has been slow due to reliance on studies of clones. In this paper, human Th cell epitopes of tt were mapped using proliferation tests on PBMC in response to synthetic peptides. PBMC from nine donors were tested over the entire set of tt homologous overlapping dodecapeptides. The 1304 peptides were initially tested as 66 pools, each containing an average of 20 peptides. PBMC from individual donors responded to as few as 1 and as many as 17 of the 66 peptide pools. The sequences responsible for proliferation were identified for the two most frequently recognized pools, and for another two pools within a major immunodominant region. Three new epitope sequences were mapped in detail and based on their recognition by most individuals are likely to be promiscuous. A cocktail of peptides including the newly identified Th cell epitopes was able to induce proliferation in PBMC from 24 of 31 tetanus toxoid (TT)-responsive donors. This cocktail is a chemically defined reagent that can be used to quantitate in vitro Ag-specific Th cells in PBMC from most subjects, and may thus be useful for serial measurements of specific immunity such as in subjects undergoing immunotherapy or immunosuppressive treatment.

Degenerate recognition of a dissimilar antigenic peptide by myelin basic protein-reactive T cells. Implications for thymic education and autoimmunity.

The key event in the induction of an immune response is the recognition by a T lymphocyte of an antigenic peptide bound to a MHC molecule. In the absence of structural coordinates of the TCR and class II MHC molecules, various models of T cell recognition have been proposed, with the emerging dogma that T cell recognition is exquisitely specific. We show here that T cell clones specific for the N-terminal fragment of myelin basic protein, acetylated Ac1-11, recognize a set of unrelated peptides in the context of the same I-Au molecule. Moreover, immunization with the peptide mimic is sufficiently cross-reactive with Ac1-9 to either induce the Ac1-9-reactive clones or to induce tolerance as well as protection against Ac1-9-induced EAE. This observed degeneracy in T cell recognition has important implications for thymic selection and induction of autoimmune disease states by "molecular mimicry."

Systematic study of substance P analogs. I. Evaluation of peptides synthesized by the multipin method for quantitative receptor binding assay.

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Systematic study of substance P analogs. II. Rapid screening of 512 substance P stereoisomers for binding to NK1 receptor.

Recent developments in multiple peptide synthesis have made it feasible to synthesize and screen large numbers of peptide analogs simultaneously. We report here a model study of large scale screening of stereoisomers of substance P with systematic D-amino acid replacements. Such studies are useful in exploring conformational requirements of peptide-receptor interaction and to provide empirical information for peptide drug design. 512 stereoisomers of SP were prepared by the multipin peptide synthesis method. Receptor binding affinities of these analogs were estimated by an iterative competition assay. Results obtained form a comprehensive database on the stereochemical requirement of SP binding to central NK1 receptor. Data from analogs with single D-amino acid replacement are consistent with those previously reported showing that SP binding is highly sensitive to the chirality change of the C-terminal residues (Gln6-Leu10), but less sensitive to the chirality change of the N-terminal residues (Arg1-Gln5). A qualitative analysis of the database by comparison of series of peptide pairs revealed a repeated pattern of affinity change with D-amino acid replacement, suggesting a largely additive binding activity of SP from each residue. On the other hand, possible intramolecular interactions between some N-terminal and C-terminal residues to form an optimal binding conformation were also found. A set of 189 peptides with IC50 values less than 5 microM was subjected to an empirical QSAR analysis using a linear additive model. The relative contribution coefficients obtained agreed with the observation that the predominant contribution comes from the C-terminal residues, suggesting considerable independency of each residue on binding to NK1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports.

The multipin peptide synthesis procedure has been adapted to allow the synthesis of peptides at micromole loadings. The original solid pin support was replaced with a detachable crown-shaped polyethylene support with an increased surface area. In addition, the polyethylene crowns were radiation-grafted with 2-hydroxyethyl methacrylate monomer instead of acrylic acid to yield hydroxy functionalized supports with a larger concentration of polymer and hence a larger peptide capacity. Fmoc-beta-Alanine was directly esterified to the HEMA hydroxy groups with subsequent addition of a diketopiperazine-forming handle for peptide attachment. Peptides varying in length from 10 to 25 residues were assembled at a number of loadings from 1.0 to 2.2 mumol. Purity of peptides at all loadings was equal to, and in some instances superior to, that achieved on conventional solid-phase supports.

Principles and pitfalls in designing site-directed peptide ligands.

An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallographically refined structures of 14 peptide-protein complexes at 2.7 A resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change in conformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program an end-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligands were diffused into preexisting crystals. In such a large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints.

Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.