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BACKGROUND: Pemphigus vulgaris (PV) is a blistering skin disease caused by IgG autoantibodies against desmoglein 3 (Dsg3). We have recently developed an active disease mouse model for PV by adoptive transfer of splenocytes from immunized or naive Dsg3-/- mice into Rag2-/- recipient mice. OBJECTIVE: In this study, we characterized the conformational epitopes of anti-Dsg3 IgG antibodies and their pathogenic activities in the PV model mice. METHODS: The binding regions of anti-Dsg3 IgG antibodies were assessed by competition ELISAs with domain-swapped mouse Dsg1/Dsg3 molecules in PV model mice receiving immunized (n = 53) or naive (n = 56) splenocytes. To compare the pathogenic activity of antibodies against N-terminal versus C-terminal extracellular domains, Dsg3-/- mice were immunized with the residues 1-162 or the residues 403-565 of mouse Dsg3, and the splenocytes were adoptively transferred into Rag2-/- mice. RESULTS: The middle to C-terminal extracellular domains of Dsg3 (residues 195-565) showed >50% competition in 51/53 (96.2%) and 45/56 (80.4%) while the N-terminal domain (residues 1-162) showed >50% competition only in 3/53 (5.7%) and 8/56 (14.3%) in mice receiving immunized and naive splenocytes, respectively. The mice receiving Dsg3-/- splenocytes immunized with the residues 403-565 developed the PV phenotype as early as and as severely as the mice receiving splenocytes immunized with the residues 1-162. CONCLUSIONS: In PV model mice the antibodies were dominantly raised against the middle to C-terminal extracellular domains of mouse Dsg3 where amino acid sequences are less conserved among desmoglein isoforms and that those antibodies may also be involved in the blister formation.
RhD is a highly immunogenic erythrocyte membrane protein, implicated in hemolytic disease of the newborn and other hemolytic disorders. Anti-RhD antibodies are used in the treatment of such disease states. Six mutant forms of recombinant RhD were stably expressed in K562 cells, and these cells were used to investigate epitope specificities of four anti-RhD monoclonal antibodies (mAbs). Amino acid substitutions were made in the exofacial loops of RhD to the corresponding residues found in the related RhCE polypeptide; M169L/M170R and I172F in the third loop, F223V and E233Q in the fourth loop, and D350H and G353W/A354N in the sixth loop. Each mAb was found to have a unique fine specificity and recognized multiple distant sites within RhD. The mAbs also differed in how they recognized individual amino acids in the exofacial loops of RhD.
The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies (mAbs) raised against synapsins I and II and have characterized their ability to interfere with the interactions of the synapsins with protein kinases, actin and Src homology-3 (SH3) domains. The epitopes of the six mAbs were found to be concentrated in the N-terminal region within domains A and B for the synapsin II-reactive mAbs 19.4, 19.11, 19.51 and 19.21, and in two C-terminal clusters in the proline-rich domains D for synapsin I (mAbs 10.22, 19.51, 19.11 and 19.8) and G for synapsin II (mAb 19.8). The synapsin II-specific mAbs 19.4 and 19.21, whose overlapping epitopes are adjacent to phosphorylation site 1, specifically inhibited synapsin II phosphorylation by endogenous or exogenous cAMP-dependent protein kinase. While all the anti-synapsin I mAbs were unable to affect the interactions of synapsin I both with Ca2+/calmodulin-dependent protein kinase II and with actin monomers and filaments, mAbs 19.8 and 19.51 were found to inhibit the binding of Grb2 SH3 domains to the proline-rich C-terminal region of synapsin I.
A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was located in the Asp77 to Gly103 sequence. This antibody could be useful for the characterization of CAP and would be a valuable probe for the detection of CAP antigen in the sera of patients with invasive candidiasis.
A common meningococcal antigen designated P64k has been identified, cloned and expressed in Escherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B-cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre-immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti-P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp(524)-Gly(533). Consistently, in this protein segment there was an amino acid stretch located in a beta-hairpin loop, which is exposed to the solvent in the previously determined three-dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 A.
Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50-66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.
The pan-leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4-8-thymocytes and the functionally mature single-positive subset, but is largely absent from the intermediate CD4+8+ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX-79 and OX-80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4-8-thymocytes, mouse CD53 is clearly detectable on the earliest CD44high25- subset, but down-regulated on the later CD44high25+, CD44low25+ and CD44low25- stages. Also, the subsequent transient TcR-/low CD4-8+ cells and most CD4+8+ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4+8+ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the re-expression of CD53 on CD4+8+ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H-2b/influenza nucleoprotein-peptide-specific TcR, back-crossed onto an H-2q or H-2b background of RAG-2-deficient mice. CD4+8+ thymocytes from non-selecting H-2q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4+8+ thymocytes from positively selecting H-2b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histocompatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53-negative, non-selected CD4+8+ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation.
Monoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1-32, 1-32, and 33-59 of CEA, respectively, and that two discrete regions 1-32 and 60-93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27-29), eight residues (21, 27-29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA.
Epitopes recognized by nine monoclonal antibodies (mAbs) on the envelope protein, gp53, of two strains of bovine viral diarrhoea virus (NADL and Oregon C24V) were mapped by competitive binding assays and by the characterization and sequence analyses of mAb neutralization escape mutants. This defined an antigenic domain on gp53 that was shared by many BVDV strains, while other less conserved epitopes were possibly distinct. Sequencing of escape mutant viruses revealed that a cluster of three amino acids in the N-terminal half of gp53 were involved in the main antigenic domain shared by both NADL and Oregon C24V viruses, while an amino acid 31 residues further toward the N-terminus was involved in a second site present only on the NADL strain. Since other amino acids defining these epitopes were located at distant positions within the gp53 protein, it is likely that a major domain [corrected] on gp53 consist of composite, conformation-dependent epitopes.
Previously we reported that the N-terminal sequence 120/123-129 of the preS2 region of the hepatitis B virus surface antigen plays an important role on peptide antigenicity against a monoclonal antibody H8 (H8 mAb) by affecting the B cell epitope conformation of a peptide existing within the sequence 130-145 (Lee et al., Biochem. Mol. Biol. Int., 34, 159-168, 1994). In this study, we try to map the H8 mAb binding site using a series of substituted peptides in the sequence 131-143 by competitive ELISA. Peptide antigenicities were greatly reduced when the residues 131 (L), 137 (R), 140 (Y), 141 (F) and 142 (P) were substituted. The residues 133 (D), 134 (P) and 136 (V) had a slight affect on the mAb binding, whereas the residues 135 (R) and 139 (L) had no effects on the mAb binding. In contrast to H8 mAb, however, three anti-HBsAg polyclonal antisera showed the lowest bindings to the peptide substituted at position 135. These results suggest that the epitope against H8 mAb is discontinuously conformational.
Despite extensive experimental and clinical evidence demonstrating the critical role of flavocytochrome b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct structural data defining its topology in the phagocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 binds exclusively to an extracellular domain, and identification of its epitope should provide novel insight into the membrane topology of Cyt b. To that end, we examined biochemical features of 7D5-Cyt b binding and used the J404 phage display nonapeptide library to identify the bound epitope. 7D5 precipitated only heterodimeric gp91-p22phox and not individual or denatured Cyt b subunits from detergent extracts of human neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p22phox complexes from detergent extracts of the biosynthetically active gp91-PLB cells, demonstrating that complex carbohydrates were not required for epitope recognition. Epitope mimetics selected from the J404 phage display library by 7D5 demonstrated that (226)RIVRG(230) and (160)IKNP(163) regions of gp91phox were both bound by 7D5. These studies reveal specific information about Cyt b membrane topology and structure, namely that gp91phox residues (226)RIVRG(230) and (160)IKNP(163) are closely juxtaposed on extracytoplasmic domains and that predicted helices containing residues Gly(165)-Ile(190) and Ser(200)-Glu(225) are adjacent to each other in the membrane.
The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341-1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.
A complex between sialyl Lewisx (alpha-D-Neu5Ac-[2-->3]- beta-D-Gal-[1-->4]-[alpha-L-Fuc-(1-->3)]-beta-D-GlcNAc-O-[CH2]8 COOMe) and E-selectin was studied using saturation transfer difference (STD) nuclear magnetic resonance (NMR) experiments. These experiments allow the identification of the binding epitope of a ligand at atomic resolution. A semiquantitative analysis of STD total correlation spectroscopy spectra provides clear evidence that the galactose residue receives the largest saturation transfer. The protons H4 and H6 of the galactose residue are in especially close contact to the amino acids of the E-selectin binding pocket. The fucose residue also receives a significant saturation transfer. The GlcNAc and Neu5Ac residues, with the exception of H3 and H3' of Neu5Ac, were found to interact weakly with the protein surface. These findings are in excellent agreement with a recently published X-ray structure and with the earlier findings from syntheses and activity assays. To further characterize the binding pocket of E-selectin, an inhibitory peptide, Ac-TWDQLWDLMK-CONH2, was synthesized and the binding to E-selectin studied utilizing transfer nuclear Overhauser effect spectroscopy (trNOESY) experiments. Finally, competitive trNOESY experiments were performed, showing that the synthetic peptide is a competitive inhibitor of sialyl Lewisx.
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The reactivity of a panel of 12 monoclonal antibodies raised against the human respiratory syncytial virus 22 kDa (22K) protein was tested by Western blotting with a set of 22K deletion mutants. The results obtained identified sequences in the C-terminal half of the 22K polypeptide required for integrity of most antibody epitopes, except for epitope 112, which was lost in mutants with short N-terminal deletions. This antibody, in contrast to the others, failed to immunoprecipitate the native 22K protein, indicating that the N terminus of this protein is buried in the native molecule and exposed only under the denaturing conditions of Western blotting. In addition, N-terminal deletions that abolished reactivity with monoclonal antibody 112 also inhibited phosphorylation of the 22K protein previously identified at Ser-58 and Ser-61, suggesting that the N terminus is important in regulating the 22K protein phosphorylation status, most likely as a result of its requirement for protein folding.
Ole e I is the major allergen derived from olive tree pollen (Olea europaea) and it is composed of two polypeptides with molecular weights (MWs) of 18 and 20 kD. A panel of six monoclonal antibodies (mAbs) has been prepared and used to map antigenic determinants on this molecule. Four epitope determinants have been identified on Ole e I. Using the purified mAbs produced against Ole e I, we have analyzed the common epitope determinants in olive (O. europaea) and different Oleaceae pollens: ash (Fraxinus excelsior); privet (Ligustrum vulgare); lilac (Syringa vulgaris), and forsythia (Forsythia suspensa). ELISA showed three reactivity groups depending on the recognition of monoclonal antibodies: (1) olive and ash; (2) olive, ash, privet and lilac; and (3) olive, ash, privet, lilac and forsythia. Immunoblotting studies on Oleaceae pollen extracts with these mAbs showed a very similar cross-reactivity pattern. The 18- and 20-kD MW proteins were present in each pollen, except in the case of forsythia. In this case the reactivity pattern was associated with 50- to 55-kD protein bands. This band was recognized by a pool of sera from olive-allergic patients. Finally, ultrastructural localization of Ole e I antigen was performed on the mature olive pollen grain. Ole e I was located in association with dilated endoplasmic reticulum cisternae. Pollen grain walls, nuclei and cytoplasmic organelles were totally devoid of the allergen.
We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.