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.

Two B cell epitopes of HIV-1 Tat protein have limited antigenic polymorphism in geographically diverse HIV-1 strains.

HIV-1 Tat, a secreted virally encoded toxin, enhances chronic viral replication and induces immune suppression, activities blocked in vitro and in vivo by anti-Tat antibodies. We mapped HIV-1 Tat B cell epitopes, determined sequence variation within them in 350 Tat sequences in GenBank, and determined antigenic cross-reactions between significant amino acid polymorphs. Two of the four B cell epitope sequences identified had limited or no antigenic polymorphism within geographically diverse strains. For epitope 1 in primates, (V,I)4DP(R,K,S,N)7L(E,D)9PW(N,K)12, the most frequent antigenic polymorphs were VDPRLEPWK in B clades (75%) and VDPNLEPWN in non-B clades (64%), with five additional sequences occurring at lower incidence. Epitope 2 in primates, K41(G,A)42LGISYGRK50, had no antigenic polymorphism. These two epitopes have potential utility for the generation of universal vaccine immunogens and therapeutic antibodies.

Minimization of chronic plasma viremia in rhesus macaques immunized with synthetic HIV-1 Tat peptides and infected with a chimeric simian/human immunodeficiency virus (SHIV33).

HIV-1 Tat protein activates resting cells, rendering them permissive for viral replication. Replication of HIV-1 in vitro is enhanced by intercellular passage of Tat protein and inhibited by anti-Tat antibodies. Tat dependence of HIV-1 replication in vivo during acute, chronic asymptomatic and AIDS stages of infection was assessed by comparisons of plasma viremia in Tat-immunized or control monkeys challenged with SHIV(33) or SHIV(33A). Chronic plasma viremia became undetectable or minimized in Tat-immunized asymptomatic SHIV(33)-infected monkeys (p<0.008) while the high viral loads of acute infection or SHIV(33A)-induced simian AIDS were unaffected by Tat immunization. Active or passive immunotherapies targeting Tat provide potential approaches to controlling chronic HIV-1 viremia and preventing AIDS.

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30-positive cells appear in the 'apoptotic' subG1 peak. Tests with synthetic peptides define positions 387-396 of CK18, with a liberated C-terminus at the caspase cleavage site DALD-S, as the ten-residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo-epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin-fixed material.

Antibody-Defined Epitope Mapping Using the Multipin Method of Peptide Synthesis

The multipin method for peptide synthesis was developed for antibody epitope mapping. From its initial invention as a method for testing many short peptides on the solid surface used for their synthesis, it has evolved into a method for producing solutions of peptides of a wide range of lengths in many "formats," such as tagged (biotinylated) peptides for binding studies or affinity chromatography. The power of the multipin technique lies in the large number and variety of peptides that can be rapidly made and efficiently tested.

Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor.

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.

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.

Monoclonal anti-H1 histone autoantibodies from unimmunized Balb/c mice. Specificity and VH and VL domain sequences.

Examination of the nuclear reactivities of monoclonal IgM kappa autoantibodies, secreted by GFM-5 1B12 and NU-6 1F12 hybridomas derived from germ-free and nude mice, respectively, demonstrated homogeneous nuclear immunofluorescence staining patterns consistent with the recognition of histones. Under these conditions, GFM-5 1B12 and NU-6 1F12 mAbs produced species non-specific binding to components within the nuclei of mouse, human and Drosophila melanogaster cells. Immunoblotting confirmed the binding of these two autoantibodies to autologous H1 histones as well as bovine and insect H1 histones. Identification of the epitopes bound by GFM-5 1B12 and NU-6 1F12 mAbs within the D. melanogaster H1 histones was undertaken using 248 overlapping octapeptides encompassing the entire sequence of D. melanogaster H1 histones. GFM-5 1B12 mAbs bound several octapeptides derived from the amino- and carboxyl-terminal regions of D. melanogaster H1 histones with accessible KT, AT or VT amino acids. NU-6 1F12 mAbs, which stained nuclei within sections of D. melanogaster lavae, failed to bind to any of the 248 linear octapeptides, implying recognition of a conformational H1 histone epitope by this autoantibody. ELISA analysis of the polyspecific binding properties of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that both antibodies exhibited unique polyspecificity profiles. GFM-5 1B12 mAbs recognized bovine carbonic anhydrase and mouse IgG1, while NU-6 1F12 bound bovine cardiolipin, rat cytochrome c, Escherichia coli beta-galactosidase, toxoid from Clostridium tetani, mouse IgG1 and the haptens, DNP and FITC from the 24 antigen test panel. Comparison of the VH and VL domain sequences of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that the variations in autoreactivity and polyspecificity profiles resulted from amino acid variations in the CDRs of the VH and VL domains of these autoantibodies. Significantly, major differences in the VH domain sequences of the NU-5 1F12 and GFM-5 1B12 mAbs suggest that the VH domains may preferentially contribute to the unique specificities of the two anti-H1 histone autoantibodies.

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.

Epitope analysis using kinetic measurements of antibody binding to synthetic peptides presenting single amino acid substitutions.

The fine modulation of peptide-antibody interactions was investigated with anti-peptide monoclonal antibodies recognizing peptide 125-136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcore, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope resulted in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide-antibody interactions.

Differing epitope selection of experimentally-induced and natural antibodies to a disease-specific autoantigen, the E2 subunit of pyruvate dehydrogenase complex (PDC-E2).

Naturally-occurring autoantibodies to a family of mitochondrial enzymes, the 2-oxoacid dehydrogenase complexes (2-OADC), characterize the human liver disease primary biliary cirrhosis. The immunodominant epitope for these autoantibodies is associated with the lipoyl-binding domain of the E2 subunit of the enzymes. The reactivity of these disease-associated autoantibodies was compared with that of antibodies raised in rats and rabbits, by immunization with various preparations derived from the 2-OADC enzymes, using immunization protocols that have successfully induced various organ-specific autoimmune diseases in animals. The immunogens included the intact pyruvate dehydrogenase complex (PDC) from bovine heart, human recombinant PDC-E2, and short synthetic peptides representing the immunodominant lipoic acid binding sequences of the 2-OADC enzymes. The techniques for antibody analysis included immunofluorescence, immunoblotting on mitochondrial extracts, ELISAs using entire PDC, PDC-E2, or synthetic peptides, epitope mapping by peptide scanning on overlapping octameric peptides representing the human PDC-E2 sequence, affinity purification on PDC-E2, and inhibition in vitro by sera of the catalytic function of PDC. Experimental immunization did not elicit any evidence of autoimmune disease. Moreover, the experimentally-induced antibodies in striking contrast to the natural autoantibodies showed preferential reactivity with PDC-E2 rather than with intact PDC, failed to inhibit in vitro the catalytic function of PDC, and, on peptide scanning, reacted with discrete epitopes, but at sites other than the lipoyl-binding region of PDC-E2. Our data indicate that 'multisystem' autoimmune diseases including primary biliary cirrhosis may not be elicitable experimentally because a critical disease-relevant autoepitope is not engaged by the immune system.

Use of the multipin peptide synthesis technique for the generation of antipeptide sera.

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pin-synthesized peptides and beta-amide peptides and the protein antigen coated to microtiter plates.

Simultaneous multiple synthesis of peptide-carrier conjugates.

Recently, the multipin approach for simultaneous multiple peptide synthesis was applied to the analysis of T cell determinants by using a novel cleavage method (Maeji et al., 1990). A diketopiperazine forming linker allowed cleavage of peptides into aqueous buffer which, without further purification, could be used immediately in cell culture assays. Another potential application of the technique is the simultaneous cleavage and coupling of peptides to immunogenic carriers. Without further purification the resulting conjugates can be used for the production of antipeptide antisera. The choice of carrier and conjugation chemistry is not restricted as peptide/pin cleavage occurs in aqueous solution over a range of pH and ionic strength. The method was assessed using the 2,4-dinitrophenyl group as a model hapten, diphtheria toxoid as the carrier, and N-(epsilon-maleimidocaproyloxy)succinimide as the cross-linking reagent. The resulting DNP-DT conjugate was used to prepare high titered specific anti-DNP antisera in mice.

Extension of a minimal T cell determinant allows relaxation of the requirement for particular residues within the determinant.

The determinant recognized by a class II restricted helper T cell clone raised against a peptide corresponding to the C-terminal 24 residues of the heavy chain of influenza virus hemagglutinin (HA) was examined in detail. The sequence 309VKQNTLKL316 was identified as the minimal determinant for T cell activation but its stimulatory capacity was augmented by extension at either end. Sets of peptide analogs, in which each residue within the minimal determinant was replaced in turn by every one of the other naturally occurring amino acids, revealed either an absolute requirement for the native residue or a very limited degree of replaceability, at seven of the eight positions. Only the N-terminal residue 309V could be replaced with almost any other amino acid without loss of reactivity; in fact, substitution at this position with residues containing bulky side groups enhanced the response. The reactivity of the clone with analogs of the longer peptide 307KYVKQNTLKL316, which induces maximal levels of stimulation, revealed a very different pattern of replaceability for certain residues; in particular, the requirement for a lysine at position 310 was no longer apparent. This study presents a complete analysis of the importance of each individual residue to the integrity of a T cell determinant and provides evidence that the critical requirement for a particular amino acid at a given location may be overridden by N-terminal extension of the minimal determinant. These findings indicate that, within different homologs of the native sequence, particular residues may assume quite different roles.

Analysis of the molecular mimicry between HLA-B27 and a bacterial OmpA protein using synthetic peptides.

In spite of a lack of sequence 'homology' between HLA-B27 and the bacterial OmpA outer membrane proteins, they both react with the Ye-2 monoclonal anti-HLA-B27 antibody. The Ye-2 antibody also reacted positively in ELISA with a synthetic peptide derived from the segment spanning residues 63-84 of B*2705. The critical peptide residues were determined by testing first with overlapping peptides, followed by a replacement set made according to the determined epitope. The results were compared with those with overlapping eight mers made to span a carboxyl fragment of the Escherichia coli OmpA protein. They indicate the reason why Ye-2 reacts with both sets of peptides is because it has a preference for polymers of arginine.

Framework peptides from kappa IIIb rheumatoid factor light chains with binding activity for aggregated IgG.

Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly the kappa IIIb subfamily. Very few sequence differences are found between these light chains. Light chains with similar variable region framework sequences are also found in some mouse monoclonal RF derived from mice stimulated with lipopolysaccharide or secondarily immunized with protein antigens. There are two likely explanations for this restriction in framework sequences between the two species: (a) the sequences are important for the immunoregulation of RF production or (b) the sequences are concerned with the antibody binding specificity of the RF. We have examined overlapping octapeptides from the kappa IIIb light chain variable region and show that some framework peptides have the ability to bind aggregated IgG. Replacement of amino acids within the peak binding peptide have indicated the critical amino acids necessary for binding.

Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands.

The fractionation of polyclonal antibodies on multiple peptide ligands is described. The method is an application of a procedure for the synthesis of large numbers of peptides on individual polyethylene pins (Geysen et al., 1987). In this application, each pin-bound peptide is used as an affinity support. Antibodies bound to the peptides are then eluted, using buffers of either high or low pH. Each eluted antibody is then tested for specific binding to peptides or proteins, using ELISA procedures. A rabbit antiserum raised to gonococcal pilin was fractionated on a complete set of octapeptides homologous with the sequence of the pilin protein. Antibodies eluted from some of the peptides bound to pilin in solution. In a second example three hyperimmune sera raised to three different potyviruses were fractionated on their respective homologous peptide sequences. Testing the eluted antibodies on the three virus coat proteins revealed peptides which bound cross-reacting antibodies. Thus the method can be used to confirm direct peptide binding evidence for sequential epitopes. These peptides can then be used in affinity chromatography to increase the specificity of polyclonal sera. This can be achieved either by elution of the specific antibody from the peptide or by removal of cross-reacting antibodies from the whole serum by absorption on peptide.

Universal antibodies to human interferon-alpha subtypes--the production of antipeptide antibodies to conserved regions of interferon-alpha.

Antibodies to three conserved regions of all human interferon (IFN)-alpha 1 subtypes were raised by immunizing rabbits with short synthetic peptides coupled to a carrier. These peptides correspond to amino acid residues 29-36, 31-36, 126-131, 139-151, and 142-151 of the consensus sequence of IFN-alpha 1. The antibodies were tested for reactivity with IFN-alpha 1, -alpha 2a, -alpha 2b, -alpha 4a, and -alpha 14 and IFN-beta. Most antipeptide antibodies react only weakly with IFN-alpha subtypes; only the antibodies raised against the peptide corresponding to residues 142-151 conjugated to keyhole limpet hemocyanin using glutaraldehyde react appreciably with all IFN-alpha subtypes tested. These antipeptide antibodies are potentially universal antibodies to the human IFN-alpha 1 subtypes, since they exhibit similar binding affinity to all the IFN-alpha subtypes tested and do not react with IFN-beta.