Mapping of antigenic domains on phytochrome from etiolated Avena sativa L. by immunoblot analysis of proteolytically derived peptides

Several monoclonal antibodies to phytochrome that interact with putative functionally important domains have been previously identified. The locations of some of these domains are determined here by epitope mapping experiments that utilize immunoblot analyses of proteolytically degraded phytochrome. Seven independent epitopes are identified. An epitope that is recognized by monoclonal antibody Oat-25 is confirmed to be wholly located near the N terminus of phytochrome. This domain undergoes a conformational change when phytochrome is interconverted between its red- and far-red-absorbing forms and is recognized by Oat-25 better in the red-absorbing form. A second domain that also undergoes a photointerconvertible conformation change and that contains the epitope for Oat-16 is localized near the site of chromophore attachment, which is about 36 kDa from the N terminus. A third domain, which contains the most highly conserved epitope on phytochrome that has so far been identified, is recognized by Pea-25 and is located about 85 kDa from the N terminus. Other epitopes and their approximate distances from the N terminus are those recognized by Oat-22 (36 kDa), Oat-13 (65 kDa), and Oat-8 and Oat-28 (70-75 kDa). Even though epitopes for Oat-16 and Oat-22, as well as for Oat-8 and Oat-28, are close together, competitive binding assays indicate that they are different. Immunoblot analyses also indicate that the epitope for Oat-28 is further from the N terminus of phytochrome than is that for Oat-8.     

Identification with Monoclonal Antibodies of a Second Antigenic Domain on Avena Phytochrome that Changes upon Its Photoconversion

An enzyme-linked immunosorbent assay that revealed an antigenic difference between the red-absorbing and far-red-absorbing forms of phytochrome (Pr and Pfr, respectively) near its amino terminus (Cordonnier M-M, H Greppin, LH Pratt 1985 Biochemistry 24: 3246-3253) was used to screen eight additional monoclonal antibodies directed to phytochrome from etiolated oats. While six of these antibodies detected Pr and Pfr with equal affinity, two of them, designated Oat-9 and Oat-16, bound to Pfr 1.6 to 2.3 times better than to Pr. Competitive enzyme-linked immunosorbent assays indicate (a) that Oat-9 and Oat-16 probably bind to the same domain on phytochrome and (b) that this domain is at least 3.5 nanometers away from the epitope near its amino terminus that was shown earlier to change upon phototransformation. Neither the absorbance spectra of Pr and Pfr, nor the rate of dark reversion of Pfr to Pr, was influenced by the presence of Oat-9. Immunoblotting of sodium dodecyl sulfate polyacrylamide gels after electrophoretic separation of phytochrome fragments obtained by endogenous proteolytic digestion indicates that Oat-16 binds to an epitope located on the chromophore half of this chromoprotein. The observation that the epitope recognized by Oat-9 and Oat-16 is also present on at least some of the immunochemically distinct phytochrome that is obtained from green oat shoots (Shimazaki Y, LH Pratt 1985 Planta 164: 333-344), together with the evidence that this epitope undergoes a change upon photoransformation, indicates that it may play an important role in phytochrome function.     

A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification

Accompanying the phototransformation of native 124-kilodalton (kDa) oat phytochrome from red-absorbing form (Pr) to far-red-absorbing form (Pfr), there is a photoreversible change in circular dichroism (CD) in the far-UV region indicative of a 3% increase in alpha-helical folding of apoprotein. To elucidate the conformational change involved in the phytochrome phototransformation, several monoclonal antibodies have been used as epitope-specific probes. Monoclonal antibody oat-25 suppressed the photoreversible CD spectral change using phytochrome with an A666/A280 as Pr of 1.13. Monoclonal antibodies oat-22, oat-13, and oat-31 did not significantly affect the CD spectral change of phytochrome. Oat-25 requires an epitope near the N-terminus of phytochrome. Oat-22, oat-13, and oat-31 recognize epitopes on the N-terminus, chromophore-containing half of phytochrome, albeit further removed from the N-terminus than that recognized by oat-25. Interestingly, oat-13 and oat-31 did, however, induce a time-dependent decrease in the far-UV CD, apparently due to aggregation of phytochrome (both Pr and Pfr forms). Monoclonal antibodies oat-26 and oat-28, which recognize epitopes on the C-terminus half of phytochrome, also did not suppress the photoreversible CD change, although oat-26 and oat-28 slightly inhibited it. The photoreversible CD spectral change can also be inhibited by sodium borohydride, which bleaches the chromophore by reducing it, and by tetranitromethane, which oxidizes the chromophore of phytochrome. Although explanations of these results based on indirect interactions between the chromophore and the N-terminus segment are possible, we propose that an additional alpha-helical folding of the Pfr form of the phytochrome may result from a photoreversible interaction between the Pfr form of the chromophore and the N-terminus segment.     

Identification of a highly conserved domain on phytochrome from angiosperms to algae

A monoclonal antibody (Pea-25) directed to phytochrome from etiolated peas (Pisum sativum L., cv Alaska) binds to an antigenic domain that has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sample buffer extracts prepared from lyophilized tissue samples or freshly harvested algae. Pea-25 immunostained an approximately 120-kilodalton polypeptide from a variety of etiolated and green plant tissues, including both monocotyledons and dicotyledons. Moreover, Pea-25 immunostained a similarly sized polypeptide from the moss Physcomitrella, and from the algae Mougeotia, Mesotaenium, and Chlamydomonas. Because Pea-25 is directed to phytochrome, and because it stains a polypeptide about the size of oat phytochrome, it is likely that Pea-25 is detecting phytochrome in each case. The conserved domain that is recognized by Pea-25 is on the nonchromophore bearing, carboxyl half of phytochrome from etiolated oats. Identification of this highly conserved antigenic domain creates the potential to expand investigations of phytochrome at a cellular and molecular level to organisms, such as Chlamydomonas, that offer unique experimental advantages.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.     

Mapping of a putative surface-binding site of human coagulation factor XII

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.     

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.     

Immunoinformatics analysis of H5N1 proteome for designing an epitope-derived vaccine and predicting the prevalence of pre-existing cellular-mediated immunity toward bird flu virus in Indonesian population

As an essential step of adaptive immune response, the recognition between antigen and antibody triggers a series of self-protection mechanisms. Therefore, the prediction of antibody-binding sites (B-cell epitope) for protein antigens is an important field in immunology research. The performance of current prediction methods is far from satisfying, especially for conformational epitope prediction. Here a multi-perspective analysis was carried on with a comprehensive B-cell conformational epitope dataset, which contains 161 immunoglobulin complex structures collected from PDB, corresponding to 166 unique computationally defined epitopes. These conformational epitopes were described with parameters from different perspectives, including characteristics of epitope itself, comparison to non-epitope surface areas, and interaction pattern with antibody. According to the analysis results, B-cell conformational epitopes were relatively constant both in the number of composing residues and the accessible surface area. Though composed of spatially clustering residues, there were sequentially linear segments exist in these epitopes. Besides, statistical differences were found between epitope and non-epitope surface residues with parameters in residual and structural levels. Compared to non-epitope surface residues, epitope ones were more accessible. Amino acid enrichment and preference for specific types of residue-pair set on epitope areas have also been observed. Several amino acid properties from AAindex have been proven to distinguish epitope residues from non-epitope surface ones. Additionally, epitope residues tended to be less conservative under the environmental pressure. Measured by topological parameters, epitope residues were surrounded with fewer residues but in a more compact way. The occurrences of residue-pair sets between epitope and paratope also showed some patterns. Results indicate that, certain rules do exist in conformational epitopes in terms of size and sequential continuity. Statistical differences have been found between epitope and non-epitope surface residues in residual and structural levels. Such differences indicate the existence of distinctiveness for conformation epitopes. On the other hand, there was no accordant estimation for higher or lower values derived from any parameter for epitope residues compared with non-epitope surface residues. This observation further confirms the complicacy of characteristics for conformational epitope. Under such circumstance, it will be a more effective and accurate approach to combine several parameters to predict the conformation epitope. Finding conformational epitopes and analysing their properties is an important step to identify internal formation mechanism of conformational epitopes and this study will help future development of new prediction tools.     

Genetic Regulation of Development in Sorghum bicolor: VII. ma(3) Flowering Mutant Lacks a Phytochrome that Predominates in Green Tissue

Phytochrome content of three near-isogenic genotypes of Sorghum bicolor was analyzed using immunological and spectrophotometric means. Seedlings of the photoperiodically sensitive genotypes 90M (Ma₁Ma₁, Ma₂Ma₂, ma₃ma₃) and 100M (Ma₁Ma₁, Ma₂Ma₂, Ma₃Ma₃) contain 126- and 123-kilodalton phytochromes. The 126-kilodalton protein is immunostained by antibodies Oat-16 and Pea-25. The 123-kilodalton phytochrome is immunostained by antibodies Pea-25 and Green-Oat-7. Seedlings of the photoperiodically insensitive genotype 58M (Ma₁Ma₁, Ma₂Ma₂, ma₃^rma₃^r) contain only the 126-kilodalton phytochrome. In 58M seedlings, 123-kilodalton phytochrome is not detected by either Pea-25 or Green-Oat-7. Deetiolation by white light causes the 126-kilodalton phytochrome to disappear but does not greatly affect the abundance of the 123-kilodalton phytochrome. In 58M, 90M, and 100M seedlings, the 126-kilodalton phytochrome is the most abundant in etiolated tissue, whereas the 123-kilodalton phytochrome of 90M and 100M seedlings predominates in green tissue. Spectrophotometric assays show that the bulk phytochrome of etiolated tissues of all three genotypes degrades similarly upon exposure to light. At least two phytochromes are detected in sorghum: a light-labile 126-kilodalton phytochrome that predominates in etiolated tissue and a 123-kilodalton phytochrome that predominates in green tissue. Photoperiodic control of flowering in sorghum is correlated with the presence of the 123-kilodalton phytochrome.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Determination of protein-derived epitopes by mass spectrometry

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified. To obtain information on linear, conformational and/or discontinuous epitopes, various approaches have been developed in conjunction with mass spectrometry. These methods include limited proteolysis and epitope footprinting, epitope excision and epitope extraction for linear epitopes and probing the surface accessibility of residues by differential chemical modifications of specific amino acid side chains or by differential hydrogen/deuterium exchange of the protein backbone amides for conformational and discontinuous epitopes. Epitope mapping by mass spectrometry is applicable in basic biochemical research and, with increasing robustness, should soon find its implementation in routine clinical diagnosis.     

Determination of protein-derived epitopes by mass spectrometry

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified. To obtain information on linear, conformational and/or discontinuous epitopes, various approaches have been developed in conjunction with mass spectrometry. These methods include limited proteolysis and epitope footprinting, epitope excision and epitope extraction for linear epitopes and probing the surface accessibility of residues by differential chemical modifications of specific amino acid side chains or by differential hydrogen/deuterium exchange of the protein backbone amides for conformational and discontinuous epitopes. Epitope mapping by mass spectrometry is applicable in basic biochemical research and, with increasing robustness, should soon find its implementation in routine clinical diagnosis.     

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol^-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol^-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol^-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.Abbreviations ELISA enzyme-linked immunosorbent assay - McAb monoclonal antibody - PBS phosphate-buffered saline - Pfr (Pr) far-red-absorbing (red-absorbing) form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

金黄色葡萄球菌肠毒素A蛋白的B细胞表位的预测

目的预测金黄色葡萄球菌肠毒素A蛋白（SEA）的B细胞表位。方法以金黄色葡萄球菌合肥乳源分离株M3基因组DNA为模板,PCR扩增SEA基因并进行序列测定与分析。应用DNAstar protean软件对SEA蛋白的二级结构、柔性、亲水性、表面可能性和抗原指数等多参数进行综合分析,预测其B细胞表位。结果M3分离株的SEA基因全长774bp,编码由257个氨基酸组成的相对分子量为29．67kDa的SEA蛋白,M3分离株SEA基因与标准株的核苷酸序列与氨基酸序列同源性分别为98．7％和98．4％。SEA蛋白的优势B细胞表位位于肽链的第64—68、100～107、138—141、156—160、166～173、213～217和237～244区段。结论预测出SEA蛋白的7个优势B细胞表位,为进而克隆表达表位蛋白,制备针对SEA表位的单克隆抗体奠定了基础。     

Mapping of antibody epitopes based on docking and homology modeling

Antibodies are key proteins produced by the immune system to target pathogen proteins termed antigens via specific binding to surface regions called epitopes. Given an antigen and the sequence of an antibody the knowledge of the epitope is critical for the discovery and development of antibody based therapeutics. In this work, we present a computational protocol that uses template-based modeling and docking to predict epitope residues. This protocol is implemented in three major steps. First, a template-based modeling approach is used to build the antibody structures. We tested several options, including generation of models using AlphaFold2. Second, each antibody model is docked to the antigen using the fast Fourier transform (FFT) based docking program PIPER. Attention is given to optimally selecting the docking energy parameters depending on the input data. In particular, the van der Waals energy terms are reduced for modeled antibodies relative to x-ray structures. Finally, ranking of antigen surface residues is produced. The ranking relies on the docking results, that is, how often the residue appears in the docking poses' interface, and also on the energy favorability of the docking pose in question. The method, called PIPER-Map, has been tested on a widely used antibody–antigen docking benchmark. The results show that PIPER-Map improves upon the existing epitope prediction methods. An interesting observation is that epitope prediction accuracy starting from antibody sequence alone does not significantly differ from that of starting from unbound (i.e., separately crystallized) antibody structure.     

A meta-analysis of the existing knowledge of immunoreactivity against hepatitis C virus (HCV)

Approximately 3% of the world population is infected by HCV, which represents a major global health challenge. Almost 400 different scientific reports present immunological data related to T cell and antibody epitopes derived from HCV literature. Analysis of all HCV-related epitope hosted in the Immune Epitope Database (IEDB), a repository of freely accessible immune epitope data, revealed more than 1500 and 1900 distinct T cell and antibody epitopes, respectively. The inventory of all data revealed specific trends in terms of the host and the HCV genotypes from which sequences were derived. Upon further analysis we found that this large number of epitopes reflects overlapping structures, and homologous sequences derived from different HCV isolates. To access and visualize this information we developed a novel strategy that assembles large sets of epitope data, maps them onto reference genomes and displays the frequency of positive responses. Compilation of the HCV immune reactivity from hundreds of different studies, revealed a complex and thorough picture of HCV immune epitope data to date. The results pinpoint areas of more intense reactivity or research activities at the level of antibody, CD4 and CD8 responses for each of the individual HCV proteins. In general, the areas targeted by the different effector immune functions were distinct and antibody reactivity was positively correlated with hydrophilicity, while T cell reactivity correlated with hydrophobicity. At the sequence level, epitopes frequently recognized by both T cell and B cell correlated with low variability, and our analysis thus highlighted areas of potential interest for practical applications. The human reactivity was further analyzed to pinpoint differential patterns of reactivity associated with acute versus chronic infection, to reveal the apparent impact of glycosylation on T cell, but not antibody responses, and to highlight a paucity of studies involved antibody epitopes associated with virus neutralization.     

Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments.

The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120].     

Localization of epitopes for antibodies that differentially label sodium channels in skeletal muscle surface and T-tubular membranes

Summary We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1–30 (I^1–30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to II^1–30. L/D3 binding was quantitatively inhibited by oligopeptides 1–30, 7–30, 13–30, and 19–30 but not 25–30, while binding of A/B2 was blocked only by the intact I^1–30 peptide. This data implies that the epitope for L/D3 lies within residues 19–25 while the epitope for A/B2 is contained within residues 1–6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I^1–30 and immunoreactivity of a peptide corresponding to residues 1–12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.We wish to thank Dr. John Lambris for helpful discussions. We also thank Ms. Candace Mello and Mr. James Hills for their expert technical assistance. This work was supported in part by NIH Grant NS 18013 (RLB) and by a grant from the W.W. Smith Charitable Trust (SAC). SAC is a Scholar of the Pfizer Scholar's Program for New Faculty.