Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

An nmr conformational analysis of a synthetic peptide Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from the New World Centruroides noxius 2 (Cn2) scorpion toxin: comparison of the structure with those of the Centruroides scorpion toxins

The solution structure of a synthetic peptide, Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from toxin 2 (Cn2) of the New World scorpion Centruroides noxius was determined using nmr and molecular dynamics calculations. The peptide has no significant secondary structure such as an alpha-helix or a beta-sheet, yet it has a fixed conformation for the first chain. The backbone secondary structure involving residues 6-12 in this peptide shows an excellent overlap with the structures of natural neurotoxins from Centruroides sculpturatus Ewing. Residues 6-9 form a distorted type I beta-turn and residues 10-12 form a gamma-turn. As residues 7-10 in the Centruroides toxins correspond to one of the regions of highest sequence variability, it may account for the species specificity and/or selectivity of toxic action. The conformation of this region evidently plays an important role in receptor recognition and in binding to the neutralizing monoclonal antibody BCF2 raised against the intact toxin.     

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom.

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether-a-go-go-related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long-QT syndrome and Drosophila 'seizure' mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50=16+/-1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure-function relationships and novel pharmacological tools with potential therapeutic efficacy.     

Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.     

Directed evolution, phage display and combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.     

Mapping and regulation of the tumor-associated epitope recognized by monoclonal antibody RS-11

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the alpha-helix 2B domain of keratin 18 in which two amino acids (Leu(366) and Lys(370)) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.     

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.     

Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na⁺ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.     

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.     

Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody

Mouse monoclonal IgG2b antibodies to leukotriene B4 bind [3H]leukotriene B4 with an affinity one-thirtieth to one-third that of different rabbit antibodies to leukotriene B4. The concentrations of related ligands required to inhibit by 50% the binding of [3H]leukotriene B4 define cross-reactivities of approximately 100% for carboxyl-derivatives of leukotriene B4, 10% for 12(S)-leukotriene B4 and 8 cis-leukotriene B4, which were not distinguished from leukotriene B4 by polyclonal antibodies, 3-5% for the two isomers of 6 trans-leukotriene B4, 5% for 20-OH-leukotriene B4 and 20-COOH-leukotriene B4, and less than 1% for other leukotrienes, mono-hydroxy-eicosatetraenoic acids, and the two leukotriene B4-like isomers of 8, 15-di-hydroxy-eicosatetraenoic acid. Thus the monoclonal combining site is highly specific for the di-hydroxy-triene portion of leukotriene B4.     

Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.

A monoclonal antibody, 5D2, which inhibits human lipoprotein lipase (hLPL) activity has been widely used for assessment of LPL immunoreactive mass in the clinical evaluation of patients [1] and for analysis of structure-function relationships of LPL [2,3]. We have mapped the epitope on LPL, recognized by the 5D2 antibody, within residues 396-405. Ala400 is the critical amino acid residue conferring epitope specificity. This knowledge confirms that the C-terminal domain of LPL plays a critical role in LPL activity and also provides important information for studies exploring the structure-function relationship of LPL using this antibody.     

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann.

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II-9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A-D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.     

Identification and characterization of a neutralizing monoclonal antibody for the epitope on HM-1 killer toxin

Killer toxin-neutralizing monoclonal antibody (nmAb-KT) against HM-1 killer toxin (HM-1) produced by yeast Williopsis saturnus var. mrakii IFO 0895 reduces both the killing and glucan synthase inhibitory activity of HM-1. nmAb-KT is classified as IgG1kappa and has been shown to be ineffective against HYI killer toxin produced by the related yeast W. saturnus var. saturnus IFO 0117. To determine the epitope for nmAb-KT, overlapping peptides were synthesized from the primary structure of HM-1. nmAb-KT reacted with peptides P5 (33NVHWMVTGGST43), P6 (39TGGSTDGKQG48) and P7 (44DGKQGCATIWEGS56), which represent the middle region of the HM-1 sequence. P6 reacted most strongly with nmAb-KT. Combined analysis by immunoblotting, surface plasmon resonance (SPR) analysis and yeast growth inhibition assay showed that nmAb-KT recognizes a specific epitope within peptide P6. The K(d) value of nmAb-KT against HM-1 and P6 were determined to be 5.48 x 10(-9) M and 1.47 x 10(-6) M by SPR analysis, respectively. These results strongly indicate that nmAb-KT binds to HM-1 at the sequence 41GSTDGK46, and not to HYI at the same position. The potential active site of HM-1 involved in the killing activity against sensitive yeast is discussed.     

Development of a new epitope tag recognized by a monoclonal antibody to Rickettsia typhi

The epitope recognized by a mouse monoclonal antibody (MAb) to the crystalline surface layer protein of Rickettsia typhi, SRT10, was mapped to 10 amino acid residues (SRTag TFIGAIATDT). The oligonucleotide sequence covering the epitope recognized by SRT10 was inserted into a mammalian expression vector together with multiple cloning sites. When the SRTag was fused in frame to the coding region of the NCC27/CLIC1 gene and expressed in mammalian cells, the MAb SRT10 could detect the tagged protein by immunoblotting, immunocytochemistry, and immunoprecipitation. In addition to the SRT-NCC27/CLIC1, SRT10 could detect N-terminal-tagged MEF2D and C-terminal-tagged CD4 by immunocytochemistry. We suggest that this specific recognition of the SRTag by SRT10 is generally applicable to cellular and molecular biology research that requires the expression and detection of fusion proteins.     

Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope.

A monoclonal antibody was produced to the exterior envelope glycoprotein (gp120) of the human T-cell lymphotropic virus (HTLV)-IIIB isolate of the human immunodeficiency virus (HIV). This antibody binds to gp120 of HTLV-IIIB and lymphadenopathy-associated virus type 1 (LAV-1) and to the surface of HTLV-IIIB- and LAV-1-infected cells, neutralizes infection by cell-free virus, and prevents fusion of virus-infected cells. In contrast, it does not bind, or weakly binds, the envelope of four heterologous HIV isolates and does not neutralize heterologous isolates HTLV-IIIRF and HTLV-IIIMN. The antibody-binding site was mapped to a 24-amino-acid segment, using recombinant and synthetic segments of HTLV-IIIB gp120. This site is within a segment of amino acid variability known to contain the major neutralizing epitopes (S. D. Putney, T. J. Matthews, W. G. Robey, D. L. Lynn, M. Robert-Guroff, W. T. Mueller, A. J. Langlois, J. Ghrayeb, S. R. Petteway, K. J. Weinhold, P. J. Fischinger, F. Wong-Staal, R. C. Gallo, and D. P. Bolognesi, Science 234:1392-1395, 1986). These results localize an epitope of HIV type-specific neutralization and suggest that neutralizing antibodies may be effective in controlling cell-associated, as well as cell-free, virus infection.     

Delineation of the epitope recognized by an antibody specific for N- glycolylneuraminic acid-containing gangliosides

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc- containing gangliosides. It also reacts with antigens expressed in human breast tumors (Vazquez et al. (1995) Hybridoma , 14, 551-556). In this work, the binding specificity of P3 has been characterized in more detail using a panel of glycolipids that included several disialylated gangliosides and several chemical derivatives of NeuGc-GM3. The carboxyl group and the nitrogen function of sialic acid were found to play important roles in the antibody binding, whereas the glycerol tail appears to be nonrelevant. Molecular modeling was used to analyze the binding data, including the finding that P3 selectively recognizes the internal NeuGc in GD3. For this purpose, conformational studies of GD3 were performed using molecular dynamics. It was concluded that sialic acid binds the P3 antibody through its upper face (the one on which the carboxyl group is exposed) and the C4-C5 side of the sugar ring, whereas none or very little contact between the galactose residue and the protein is evident. Conformational analysis of GD3 revealed that, despite the large flexibility of the NeuGcalpha8NeuGc linkage, the P3 binding epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the internal sialic acid presents the epitope in a proper way for several of these conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained.      

Neoantigens in complement component C3 as detected by monoclonal antibodies. Mapping of the recognized epitopes by synthetic peptides.

The different fragments of the third complement component, C3, generated upon complement activation/inactivation have the ability to bind to several other complement components and receptors as well as to proteins of foreign origin. These multiple reactivities of C3 fragments are associated with a series of conformational changes occurring in the C3 molecule during its degradation. The conformations acquired by the different C3 fragments are also associated with the exposure of neoantigenic epitopes that are specific for (a) particular fragment(s). In order to study these epitopes and thus the conformational changes occurring in C3, monoclonal antibodies (mAbs) recognizing such epitopes were produced in Balb/c mice after immunization with denatured human C3. Two of the three antibodies (7D84.1 and 7D264.6) presented in this study recognized predominantly surface-bound iC3b, and one mAb (7D323.1) recognized both surface-bound and fluid-phase iC3b. Although none of the mAbs recognized any other fluid-phase C3 fragment, all three antibodies detected micro-titre-plate-fixed C3b and iC3b, but not C3c or C3d. In addition to the reaction with human C3, mAb 7D323.1 also bound to micro-titre-plate-fixed rabbit C3. The epitopes recognized by the three mAbs were further localized by using synthetic peptides that were designed on the basis of the differential binding of the mAbs to the C3 fragments. All three antibodies reacted with C3-(924-965)-peptide, which represents the region of C3 between the kallikrein-cleavage site (923-924) and the elastase-cleavage site (965-966). On the basis of the binding of the mAbs to five different overlapping peptides spanning the region between residues 924 and 965 of the human C3 sequence, and the sequence similarity between human C3 and rabbit C3 within this area, the epitopes recognized by these antibodies are mapped. The contribution of the individual amino acid residues in the formation of the epitopes is discussed.     

Mass spectrometric characterization of a discontinuous epitope of the HIV envelope protein HIV-gp120 recognized by the human monoclonal antibody 1331A

The characterization of a discontinuous epitope in the C5 region of the HIV envelope protein HIV-gp120, recognized by 1331A, a human mAb, is reported. Regions involved in affinity binding in the HIV-gp120 molecule were identified by epitope excision/extraction methods followed by matrix assisted laser desorption-time of flight mass spectrometry. In epitope excision, the protein is bound in its native conformation to an immobilized Ab and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the Ab. A series of proteolytic digestions of the 1331A/HIV-gp120 complex allowed the identification of protected amino acids in two noncontinuous regions of the C5 region of HIV-gp120. Interaction of the Ab with amino acids I487 and E507 of HIV-gp120 is essential for efficient binding. This is the first application of this approach for the identification and characterization of a discontinuous epitope. The results are consistent with molecular modeling results, indicating that these amino acids are located on opposite sides of a hydrophobic pocket. This pocket is thought to be of importance for the interaction of HIV-gp120 with the transmembrane protein HIV-gp41.     

Localization on the mitochondrial F1 ATPase alpha subunit of an epitope masked in the membrane-bound enzyme using a monoclonal antibody and synthetic peptides.

The epitope of the monoclonal antibody 20D6 was localized by N-terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1 alpha subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven-amino-acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the alpha subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti-F1 antisera showed that this region is highly immunodominant. In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the alpha subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane.