Topography of N-CAM structural and functional determinants. I. Classification of monoclonal antibody epitopes

12 distinct neural cell adhesion molecule (N-CAM) epitopes, each recognized by a different monoclonal antibody (mAb), have been characterized in terms of the major structural and functional features of the molecule. Seven antibodies, each recognizing the amino-terminal region of the molecule, altered the rate of N-CAM-mediated adhesion. Four of these were inhibitors, two of which also recognized a heparin-binding N-CAM fragment. The other three antibodies specifically enhanced the rate of N-CAM-mediated adhesion. Three epitopes, one polypeptide- and two carbohydrate-dependent, were associated with the sialic acid-rich central portion of the molecule. The remaining two antibodies were found to react with intracellular determinants, and are specific for the largest of the three major N-CAM polypeptide forms. Studies on the ability of one antibody to hinder recognition of native N-CAM by another antibody suggested that the epitopes associated with N-CAM binding functions are in close proximity compared with the other determinants. The classification of these mAb epitopes has allowed the topographical placement of key N-CAM features, as described in the following paper, and provides valuable probes for analysis of both the structure and function of N-CAM.     

Monoclonal antibody mapping of structural and functional plectin epitopes

To map structural and functional epitopes of the cytomatrix protein plectin, a set of mAbs was prepared by immunization of mice. Using immunoblot analysis of plectin fragments obtained after limited digestion with various proteases, two groups of mAbs were distinguished. The epitopes of one group (1) were located on a 130-kD terminal segment of the plectin 300-kD polypeptide chain, whereas those of the other group (2) bound within a 40kD segment confined to a central domain of the polypeptide chain. Domains containing the epitopes of group 2 mAbs were shown to include in vitro phosphorylation sites for kinase A, whereas kinase C phosphorylation sites were found on the same terminal segment that contained group 1 mAb epitopes. Rotary shadowing EM of mAb (Fab fragment) -decorated plectin molecules at various states of aggregation, ranging from characteristic dumbbell-shaped single molecules to highly complex multimeric structures, revealed that the epitopes of group 1 as well as those of group 2 mAbs were located on plectin's roughly 200-nm long rod domain interlinking its two globular end domains. Epitopes of group 1 mAbs were localized within a region near the center of the rod, those of group 2 in more peripheral sections near the globular end domains. Solid-phase binding assays carried out in the presence of Fab fragments of mAbs demonstrated an interference of certain group 1 mAbs in the interactions of plectin with vimentin and lamin B. On the other hand, plectin's self-interaction was inhibited mainly by Fab fragments with epitopes in the peripheral rod domain (group 2 mAbs). Together, these results suggested that the molecular binding sites of plectin for vimentin and lamin B, as well as the phosphorylation sites for kinase C, were confined to a defined central section of plectin's rod domain. In addition, they suggest an involvement of peripheral rod sections in plectin self-association.     

Localization of monoclonal antibody epitopes and functional domains in the hemagglutinin protein of measles virus.

The expression of chimeric proteins was performed for the localization of monoclonal antibody (MAb) epitopes and functional domains in the hemagglutinin (H) protein of measles virus. The fusion helper function of the H protein was ablated by a single amino acid substitution at residue 98. Loss of reactivity to MAb 79-XV-V17 and to MAbs 16-CD-11 and 80-II-B2 was attributed to substitutions between residues 211 and 291 and between 451 and 505, respectively. The 80-II-B2 MAb epitope also seemed to be within a domain required for hemadsorption and hemagglutination activities.     

Antigenic determinants on human choriogonadotropin alpha-subunit. II. Immunochemical mapping by a monoclonal antipeptide antibody

In order to study antigenic site(s) present in the carboxyl-terminal part of the alpha-subunit of human choriogonadotropin (hCG-alpha), we attempted to produce site-specific antibodies directed against a 34-residue synthetic peptide analogous to region 59-92 of hCG-alpha. From a fusion experiment performed with a mouse injected with hCG-alpha-(59-92)-peptide conjugated to tetanus toxoid as immunogen, we selected a monoclonal antipeptide antibody (designated FA36) which has high binding activity for 125I-hCG-alpha but not for 125I-hCG in a radioimmunoassay. This antibody is of the IgG1 subclass and displays an affinity constant for 125I-hCG-alpha of 3.1 x 10(8) M-1. Hapten inhibition experiments performed by either radioimmunoassay or enzyme-linked immunosorbent assay with synthetic peptides spanning different portions of the region (59-92) demonstrated that the binding site of FA36 resides on (minimally) the six COOH-terminal amino acids of hCG-alpha, namely Cys-Tyr-Tyr-His-Lys-Ser, and that FA36 binds preferentially to peptides containing a carboxyl group on the COOH-terminal residue. Monoclonal immunoradiometric assays were established to determine the location of antigenic regions recognized by FA36, by antibody AHT20 (which binds only to hCG-alpha), and by antibody HT13 (which binds to both hCG and hCG-alpha). FA36 has the capacity to bind to hCG-alpha bound to either AHT20 or HT13, demonstrating that both AHT20 and HT13 antibodies are directed against antigenic regions distinct from the epitope of FA36. Monoclonal immunoradiometric assays were also carried out to study the binding of FA36 to hCG, the ovine and equine lutropin alpha-subunit, or hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core). Whereas significant binding of 125I-FA36 was observed with the ovine lutropin alpha-subunit, no binding was found with the equine lutropin alpha-subunit. As expected, FA36 did not bind to hCG-alpha core. Binding was also not detected with hCG, confirming that FA36 is specific for free hCG-alpha and that the COOH-terminal part of hCG-alpha is either weakly or (more likely) not at all accessible in the alpha/beta-dimer for antibody binding. Finally, immunoblots performed on hCG-alpha-(59-62)-peptide and various denatured alpha-subunits indicated that, with the exception of the equine lutropin alpha-subunit, FA36 detected various denatured alpha-subunits and particularly the alpha-subunit of carp gonadotropin-thyrotropin. This latter observation suggests a high degree of homology between the COOH-terminal regions of the alpha-subunits of fish gonadotropin and analogous mammalian hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.     

A monoclonal antibody detecting unusual Thy-1 determinants

20-10-5S is a monoclonal antibody produced by the fusion of C3H anti-C3H.SW splenocytes with the SP2/0 cell line. The antibody appears to react with Thy-1 determinants by several criteria including cytotoxicity patterns, functional assays, genetic analyses, and competitive binding experiments. However, the antibody and the determinants it detects are unusual in that: 1) 20-10-5S is autoreactive; 2) the antibody shows allospecificity for Thy-1.2 vs Thy-1.1 antigens only on peripheral lymphocytes and not on thymocytes; and 3) the antibody reacts only with determinants on murine T cells and not with antigens on brain tissue or on rat thymocytes. It therefore seems that 20-10-5S reacts with murine T cell-specific Thy-1 determinants that are lost or modified during maturation of the cells on which they are expressed.     

Analysis of structural properties and cellular distribution of avian Ia antigen by using monoclonal antibody to monomorphic determinants

In this report, we describe the analysis of Ia-like antigens in the chicken by using a monoclonal antibody (CIa-1) reactive with monomorphic determinants of the Ia-like (B-L) antigens. This antibody reacts with determinants on B cells in all avian species tested, but does not detect antigens on lymphocytes of representative mammals, reptiles, and amphibians. In addition to B cells, this antibody defines a subpopulation of the monocyte-macrophage series and reacts with mitogen-activated T cells. Immunochemical analysis indicates that the CIa-1 reactive antigen is a 65,000-dalton glycoprotein consisting of an alpha-chain of 32,000 daltons noncovalently bound to a beta-chain of 27,000 daltons. Under nonreducing conditions, the beta-chain migrates with slightly faster mobility. Two-dimensional gel analysis indicates that the beta-chain is the more heterogeneous of the two chains. Thus, the antigen detected by CIa-1 antibody is similar in cell distribution and structure to the murine Ia antigens and human DR antigens. During in ovo development, Ia+Ig- cells were not found in the yolk sac but were detected in the spleen, mesonephros, and bursa of 9-day embryos. Two populations of Ia+Ig- cells were identified in the bursa: 40 to 60% of the bursacytes, mostly larger cells, exhibited brighter immunofluorescence reactivity than the smaller bursacytes.     

Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system

To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.     

Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains.

It has been proposed that scaffold-associated regions are DNA elements that form the bases of chromatin loops in eukaryotic cells. Recent evidence supports a role for these elements as cis-acting 'handlers' of both structural and functional chromatin domains.     

Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21-25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh 29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.     

Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences.

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence-specific reagents, along with the previously described anti-OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD-dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit alpha9 integrin-dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell-binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration.     

Use of a monoclonal antibody in structural investigations of the 49-kDa polypeptide of photosystem II

A monoclonal antibody, FAC2, was isolated by immunization of mice with a Photosystem II core preparation followed by splenic fusion and standard monoclonal antibody screening and production techniques. This antibody recognizes the 49-kDa polypeptide of Photosystem II which is the apoprotein of CPal. The antigenic determinant recognized by this antibody lies on a cyanogen bromide fragment which appears as a doublet with an apparent molecular mass of 14.5 kDa. FAC2 was used to follow the effects of trypsin on the 49-kDa polypeptide in a membrane environment. Our results indicate that the extrinsic polypeptides of Photosystem II which are known to be involved in oxygen evolution protect the 49-kDa polypeptide from tryptic attack. Additionally, Photosystem II membranes which are treated with alkaline Tris exhibit a large increase in the ability to bind FAC2. This increase is not observed with membranes treated with calcium chloride or sodium chloride. These results indicate that the 49-kDa polypeptide may be at least structurally associated with the component(s) responsible for oxygen evolution.     

A monoclonal antibody (ER-HR3) against murine macrophages. II. Biochemical and functional aspects of the ER-HR3 antigen

We describe the purification and intracellular distribution of an antigen present on a subpopulation of murine macrophages and recognized by monoclonal antibody ER-HR3 against bone marrow-derived haemopoietic reticulum cells. Using the ER-HR3 antibody as an immobilizing ligand, two proteins were isolated as determined by SDS polyacrylamide gel electrophoresis. Under non-reducing conditions, there was a major band with an apparent molecular mass of 69 kDa and a minor band of 55 kDa. Under reducing conditions, the apparent molecular mass of each band was estimated as 76 kDa and 67 kDa, respectively. Intracellularly, these proteins occurred in close association with membranous structures, as demonstrated with gold-labelled protein A in an electron-microscopic study of the ER-HR3-positive cell line AP284. Some of the antigen was present in vesicles To gain further insight into the possible function of the ER-HR3 antigen, its tissue distribution was investigated under distinct experimental conditions. In mice infected with Bacillus Calmette Gurèrin, ER-HR3-positive cells were observed in many, but not all, granulomata of the spleen, the lung and the liver. The ER-HR3 reactivity in these mice clearly differed from that of other antimacrophage monoclonal antibodies, such as F4/80, M5/114 and M1/70. Furthermore, phenylhydrazine-induced extramedullary erythropoiesis in the liver was accompanied by ER-HR3 expression on a subpopulation of macrophages. Finally, the addition of ER-HR3 to an antigen-specific T cell proliferation assay did not inhibit T cell proliferation.     

A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins.

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.     

Characterization of structural features and diversity of variable-region determinants of related quaternary epitopes recognized by human and rhesus macaque monoclonal antibodies possessing unusually potent neutralizing activities

A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes.     

Anti-bilirubin monoclonal antibody. II. Enzyme-linked immunosorbent assay for bilirubin fractions by combination of two monoclonal antibodies

Among 16 monoclonal antibodies raised against covalently coupled bilirubin-bovine serum albumin, we selected two antibodies: one (designated 24G7) reacted with unconjugated and conjugated bilirubin and the other (designated 25H17) reacted only with unconjugated bilirubin. Combination of these two antibodies enabled us to determine extremely low concentrations of unconjugated and conjugated bilirubin independently by enzyme-linked immunosorbent assay (ELISA). In the assay, samples were incubated with each anti-bilirubin IgG, and then free remaining IgG was allowed to bind to the immunotiter plates coated with bilirubin-bovine serum albumin. The bound fraction of the IgG was visualized with horseradish peroxidase-conjugated rabbit anti-mouse IgG and substrate. Bilirubin concentration was determined from the absorbance at 425 nm. In this system, we could measure 10(-7)-10(-5) M unconjugated and conjugated bilirubin in samples, which is 100-fold more sensitive than Micha?lsson's diazocoupling method. The assay results gave a good correlation coefficient (0.86) compared with those determined with high performance liquid chromatography.