M467: a murine IgA myeloma protein that binds a bacterial protein. I. Recognition of common antigenic determinants on Salmonella flagellins.

We have studied the binding of M467, an IgA murine myeloma protein, to flagellin from seven species of Salmonella. It was found that M467 was reacting with antigenic determinants that were common to all the flagellins studied. These determinants were not related to serotypic antigens. Electronmicrographs of unreduced M467 showed a variety of polymeric species bound to flagella in a manner that could produce immobilization as well as agglutination and precipitation through cross-linking of antigenic determinants. Immunodiffusion in agar gel revealed that M467 was recognizing more than one group of peptide determinants on the flagellins studied. Passive hemagglutination inhibition and a solid phase radioimmunoassay provided evidence that there were differences in binding avidities between M467 and the various Salmonella flagellins studied. It was concluded that M467 is binding more than one specific group of antigenic peptide determinants on flagellin molecules. Flagellin from four of the seven species of Salmonella studied were deficient in one or more of these determinants.     

Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface

PhoE protein is an abundant outer membrane protein of theEscherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foreign antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein ofMycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuatedSalmonella strains, which can be used as live oral vaccines, was shown.Paper awarded the Kluyver Prize 1990 by the Netherlands Society of Microbiology to Dr. M.C. Agterberg     

A semi-empirical method for prediction of antigenic determinants on protein antigens.

Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi-empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that our method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.     

Recombinant plasmids containing hybrid protein genes with antigenic determinants of the foot and mouth virus

Plasmids have been constructed which contain genes coding for fused proteins including beta-galactosidase or human leukocyte interferon alpha 2 and monomeric or pentameric form of the main antigenic determinant of the foot-and-mouth disease virus (FMDV) serotype 01K. Expression of the hybrid genes has been studied. It is shown that fused proteins, containing beta-galactosidase and the antigenic determinant (monomer or pentamer), interact specifically with anti-FMDV anti-sera and with antibodies against peptide 141-160 of FMDV VP1 coat protein.     

Genetic mapping of antigenic determinants on a membrane protein

The antigenic determinants recognized by two monoclonal antibodies were mapped on LamB, an outer membrane protein of Escherichia coli. The procedure consisted of performing immunoprecipitation experiments with extracts of strains which produced truncated fragments of LamB, either in a free form (deletion and nonsense mutants) or fused to another polypeptide (malK-lamB and lamB-lacZ fusion strains). The conclusion is that the two antigenic determinants are located within 70 residues from the COOH-terminal end of LamB, which contains a total of 421 amino acids. Since these two antigenic sites were previously demonstrated to be exposed at the cell surface, it follows that a COOH-terminal portion of LamB must be located on the outer surface of the outer membrane.     

Expression of foreign antigenic determinants on bacterial envelope proteins

Conclusions and perspectives We have demonstrated that the LamB153 vector was expressed in attenuated Salmonella typhi and typhimurium (Charbit et al., unpublished) so that the development of live oral vaccines based on this vector can be envisaged.Using the same procedure for detecting permissive sites, we have constructed a number of other expression vectors based on other sites in LamB (Charbit et al., in preparation), but also based on other envelope proteins of the system for maltose and maltodextrins transport (Duplay et al., 1987; Martineau, unpublished results; Dassa, unpublished results). This provides a useful material to study the influence on the immune response of the site of insertion of the foreign epitope into a protein and of the location of the protein within the bacterial cell.     

PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.     

ADEPT: a computer program for prediction of protein antigenic determinants

ADEPT, a program that can be used for prediction of proteinantigenic determinants from the amino acid sequence alone forthe cases of humoral and cellular immune response, is described.Most methods presently utilized for this purpose are implementedin the program along with some original parameters for the caseof humoral immune response. There is also a possibility to combineseveral methods for the case of humoral immune response, whichprovides a means to create new predictive methods. ADEPT alsoincludes a literature-derived database of proteins in the SWISS-PROTstandard with experimentally determined antigenic determinants,so the predictive ability of new methods can be assessed withinthe program and corresponding statistical information is calculated.ADEPT may be useful for solving vamious tasks involving thedelineation of antigenic regions on proteins or characterizationof peptide fragments, arising, for examnple, in protein engineering(e.g. selection of protein portions most suitable for insertionof peptide sequences that are antigenically active or possessingother desired characteristics), and in particular for constructionof vaccines containing B- and/or T-cell epitopes, as well asfor other problems of medico-biological research.     

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.     

Construction of a fusion protein carrying antigenic determinants of enteric clostridial toxins

Clostridium difficile and Clostridium perfringens type A are infectious agents of enteric diseases. The main virulence factors of these microorganisms include toxins A and B of C. difficile (ToxA and ToxB) and enterotoxin of C. perfringens (Cpe). In this study genetic constructions have been created for the expression of ToxA, ToxB and Cpe fragments either as individual components or as a hybrid multidomain (ToxA-ToxB-Cpe) protein. Rabbit monospecific sera raised against individual peptides reacted with the chimeric product indicating that the corresponding antigenic determinants were correctly expressed on the hybrid molecule. Furthermore, mice immunized with the fusion protein produced antibodies specific to each of the three separate components. These data suggest that the constructed three-domain molecule could be used in future studies for development of a vaccine against enteric clostridial diseases.     

C4b-binding protein and a 60,000-Dalton plasma protein share antigenic determinants with membrane cofactor protein of complement.

Membrane cofactor protein (MCP) of the C system is a widely distributed regulatory molecule with C3b/C4b binding and factor I-dependent cofactor activity. A rabbit polyclonal antibody was raised against purified human MCP, and it was found to also immunoprecipitate C4b-binding protein (C4bp). Other related complement regulatory proteins, factor H, C3b/C4b receptor, and decay-accelerating factor, were not recognized by this polyclonal antibody to MCP. The cross-reactive epitope was sensitive to reduction with 2-ME and about 3% of the anti-MCP antibody reacted with C4bp. The amino-terminal 48,000-Da, chymotryptic fragment of C4bp was recognized by the antibody to MCP. This fragment of C4bp contains a seven-amino acid peptide that is identical, in its sequence and its location in the third short consensus repeat, to one found in MCP. Two polyclonal antibodies to C4bp, one raised to native and the other to reduced C4bp, did not cross-react with MCP. In addition to this one-way cross-reaction with C4bp, a protein with a m.w. of approximately 60,000 (p60) was found in two of three C4bp preparations that also cross-reacted with antiserum to MCP. p60 was present in trace quantities in the C4bp preparation and was successfully isolated from plasma by C3b affinity chromatography. Its Mr was distinct from that of MCP and other known C3b/C4b binding proteins. Furthermore, p60 was isolated by two different procedures and such material possessed no detectable cofactor activity. Based on these results, p60 is a plasma C3b-binding protein that shares epitopes with C4bp and MCP, and is probably not a soluble form of MCP.     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.     

Steric hindrance as a factor in the reaction of labeled antibody with cell surface antigenic determinants.

The binding of rabbit anti-human IgG labeled with 125I, shellfish glycogen or ferritin to human IgG attached to the surface of rabbit RBC with chromic chloride was studied. Maximum binding was noted with 125I labeled antibody. Slightly but consistently less binding was found with shellfish glycogen labeled antibody. The binding of ferritin labeled antibody was strikingly reduced--usually one-third or less of that found with 125I labeled antibody alone. This suggests that under the conditions of these experiments, the attachment of large labels to antibody molecules results in reduced antibody binding to surface antigen. Steric hindrance is probably at least in part responsible for this reduced binding.     

Mutations affecting antigenic determinants of an outer membrane protein of Escherichia coli.

The Escherichia coli LamB protein is located in the outer membrane. It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10. It is a trimer composed of three identical polypeptide chains, each containing 421 residues. Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface. Some of the mutations also altered the binding site for phage lambda. All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide. This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane. This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.     

Construction and analysis of recombinant DNAs containing a structural gene for rat prolactin.

Poly A-containing RNA enriched in prolactin-coding sequences was isolated from female rate pituitaries after induction with diethylstilbesterol. Double stranded cDNA was synthesized from this RNA and inserted into plasmid pBR322 at the Pst I site via the poly(dG):polyy(dC) tailing method. E. coli was transformed with this DNA and the recombinant plasmid in one of the transformants characterized in detail. About half of its 900 base pair cDNA insert was sequenced. The DNA sequence is consistent with most of the reported amino acid sequence of rat preprolactin. In addition, the recombinant plasmids in two of the other transformants appear to contain growth hormone coding sequences.     

Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction.

The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs.     

Monoclonal antibody studies of the antigenic determinants of human plasma retinol-binding protein

A battery of monoclonal antibodies (MoAbs) against human retinol-binding protein (RBP) was produced to obtain useful probes for the study of the antigenic determinants of RBP. The 12 antibodies all reacted with human RBP by immunoblotting. Based on antibody cross-competition radioimmunoassays, four distinct and different groups of antibodies were identified: group I, 1A4 and 2F4; group II, 1G10, 5C5, 6F4, and 7G3; group III, 5H6, 6C7, 10G5, and 14E3; and group IV, 5H9 and 13A1. Information about the epitopes of RBP recognized by these MoAbs was obtained by testing the reactivity of each antibody with human, rabbit, and rat RBPs by immunoblotting. Group I and group IV antibodies reacted to a similar extent with human, rabbit, and rat RBPs. Group II antibodies reacted strongly with human and rabbit RBPs, but reacted very weakly with rat RBP. Group III antibodies reacted strongly with human RBP, but did not react with rabbit or rat RBP. Thus, the epitopes for group I and group IV antibodies appear to be regions of the RBP molecule that are conserved across the three species, whereas group III antibodies recognized only human RBP. In a preliminary study, the reactivity of each antibody with purified cyanogen bromide fragments of RBP was tested by slot immunoblotting. None of the MoAbs reacted with any of the cyanogen bromide fragments. This study shows that MoAbs specific for at least four different regions of the RBP molecule can be produced; hence, RBP contains at least four major antigenic domains.     

Application of a novel radioimmunoassay to identify baculovirus structural proteins that share interspecies antigenic determinants

Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120, 1979) and Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with ¹²⁵I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin (the occlusion body protein from NPVs) recognized antigenic determinants on all the polyhedrins and granulins (occlusion body protein from GVs) that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures.