Production and characterization of monoclonal antibodies against the two subunits proteins B1 and B2 of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.     

Anaerobic cytochrome b1 in Escherichia coli: association with and regulation of nitrate reductase.

Nitrate reductase solubilized from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit, not present in the purified enzyme used to prepare the antibody. This third subunit was identified as the cytochrome b1 apoprotein. This cytochrome is bound to nitrate reductase from wild-type E. coli in a ratio of 2 mol of cytochrome per mol of enzyme complex. In mutants unable to synthesize heme, this cytochrome b1 apoprotein is not bound to nitrate reductase. In these same mutants, the enzyme is overproduced and accumulates in the cytoplasm. The absence of cytochrome also affects the stability of the membrane-bound form of the enzyme.     

Activation of C1 by monoclonal antibodies directed against C1q

Eleven monoclonal antibodies directed against the subcomponent C1q of the first component of human complement, C1, were prepared and tested for binding to intact C1q and to the collagenous portion, the C1q stalks. All of the monoclonals bound well to the intact C1q. Eight out of the eleven exhibited strong binding to the collagenous stalks, while three bound very weakly, if at all, to the stalks and, thus, were presumed to bind to the pepsin-sensitive region which includes the C1q heads. For one of the latter monoclonals, this was confirmed by electron microscopy. Five of the monoclonals were purified by C1q affinity chromatography. When tested with C1 reassembled from its subunits, two of these purified monoclonal antibodies markedly enhanced the rate of spontaneous activation.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.     

A photoaffinity-labeled allosteric site in Escherichia coli ribonucleotide reductase

The B1 subunit of Escherichia coli ribonucleotide reductase is coded for by the nrdA gene, of determined structure. Protein B1 contains two types of allosteric binding sites. One type (h-sites) determines the substrate specificity while the other type (l sites) governs the overall activity. The effectors dGTP and dTTP bind only to the h-sites while dATP and ATP bind to both the h- and the l-sites. Protein B1 has been photoaffinity-labeled with radioactive dTTP and dATP using direct UV irradiation. Following tryptic digestion of labeled protein B1 only one peptide labeled with dTTP was found, while several peptides were labeled with dATP. One of the dATP-labeled peptides had chromatographic properties very similar to that labeled with dTTP and this peptide most likely forms part of the h-site of protein B1. Labeling of the l-site could not be conclusively shown since substantial non-specific labeling occurred with dATP. CNBr fragments of dTTP-labeled protein B1 were used to localize the region of nucleotide binding in the deduced primary structure of the nrdA gene. The dTTP label was further localized to a tryptic octapeptide with the sequence Ser-X-Ser-Gln-Gly-Gly-Val-Arg. The labeled amino acid was found at position 2, but the residue itself could not be directly identified. Unexpectedly, this sequence was not found in the earlier reported primary structure of the nrdA gene. However, a recent revised structure of the gene identifies the labeled residue as Cys-289 and fully confirms the rest of the peptide sequence. Thus the present result clearly defines one of the allosteric binding sites in ribonucleotide reductase.     

Immunochemical studies on blood groups. The combining site specificities of mouse monoclonal hybridoma anti-A and anti-B

Mouse monoclonal hybridomas, five anti-blood group A, three anti-B, and one anti-AB, produced by various methods of immunization, have been characterized by quantitative precipitin tests and the fine structures of their combining sites have been mapped by oligosaccharide inhibition assays. The combining sites of antibodies of each specificity differed among themselves. Three of the five monoclonals were specific for difucosyl and two for monofucosyl A determinants. All but the anti-AB were strictly specific for blood group A or blood group B erythrocytes; all of the anti-A monoclonals gave essentially equivalent titers in hemagglutination tests with A1 and A2 erythrocytes except for a monoclonal anti-A prepared by immunization with a human gastric cancer cell line. The data provide additional evidence for the heterogeneity of the antibody response to the different antigenic determinants present on blood A and B substances and emphasize the importance of difucosyl determinants which comprise most of the determinants on the water-soluble blood group substances.     

Characterization of monoclonal antibodies directed against pyruvate oxidase from Escherichia coli: modulation of antibody-induced inhibition by enzyme conformation

Monoclonal antibodies have been prepared against pyruvate oxidase, a flavoprotein dehydrogenase isolated from Escherichia coli. Six monoclonals were obtained, but only one was found to bind to the native form of the enzyme. This monoclonal, 1I1, was a potent inhibitor. Although this antibody inhibited the unactivated and lipid-activated forms of the enzyme, it had much less of an inhibitory effect on the protease-activated form of the enzyme, although the antibody still bound to this form. Hence, the coupling between antibody binding and the conformation at the active site can itself be modulated by the conformation of the protein.     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.     

Evidence for a new ribonucleotide reductase in anaerobic E. coli

E. coli conditional iron-containing ribonucleotide reductase (Fe-RR) mutant and wild type strains grew anaerobically under conditions when Fe-RR was absent or inhibited. Furthermore, a B12-independent, hydroxyurea-resistant RR activity, unaffected by monoclonal antibodies against either subunit B1 or B2 of Fe-RR, was partially purified from anaerobically grown mutant and wild-type E. coli. These findings indicate that E. coli has a second RR representative of a new class of RRs and that this is the first report where both in vivo and in vitro evidence is presented. It is probable that other facultative anaerobes also have two different RRs such that an optimal supply of deoxyribonucleotides is maintained under all growth conditions.     

Antibody against extracellular vaccinia virus (EV) protects mice through complement and Fc receptors

Protein-based subunit smallpox vaccines have shown their potential as effective alternatives to live virus vaccines in animal model challenge studies. We vaccinated mice with combinations of three different vaccinia virus (VACV) proteins (A33, B5, L1) and examined how the combined antibody responses to these proteins cooperate to effectively neutralize the extracellular virus (EV) infectious form of VACV. Antibodies against these targets were generated in the presence or absence of CpG adjuvant so that Th1-biased antibody responses could be compared to Th2-biased responses to the proteins with aluminum hydroxide alone, specifically with interest in looking at the ability of anti-B5 and anti-A33 polyclonal antibodies (pAb) to utilize complement-mediated neutralization in vitro. We found that neutralization of EV by anti-A33 or anti-B5 pAb can be enhanced in the presence of complement if Th1-biased antibody (IgG2a) is generated. Mechanistic differences found for complement-mediated neutralization showed that anti-A33 antibodies likely result in virolysis, while anti-B5 antibodies with complement can neutralize by opsonization (coating). In vivo studies found that mice lacking the C3 protein of complement were less protected than wild-type mice after passive transfer of anti-B5 pAb or vaccination with B5. Passive transfer of anti-B5 pAb or monoclonal antibody into mice lacking Fc receptors (FcRs) found that FcRs were also important in mediating protection. These results demonstrate that both complement and FcRs are important effector mechanisms for antibody-mediated protection from VACV challenge in mice.     

Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products

Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.     

The B cell surface molecule B1 is functionally linked with B cell activation and differentiation

The B1 molecule is a 32,000 m.w. phosphorylated cell surface protein expressed exclusively by B cells from the mid pre-B until the plasma cell stage of differentiation. Two monoclonal antibodies (gamma 2a and mu) reactive with this molecule were used to assess the role of B1 in B cell activation, proliferation, and differentiation. The anti-B1 antibodies at concentrations ranging from 0.1 to 100 micrograms/ml significantly inhibited B cell proliferation induced by anti-mu antibodies, Staphylococcus aureus Cowan strain 1, activated T cells, and Epstein Barr virus. Although capable of inhibiting proliferation, anti-B1 antibody in soluble form or coupled to beads did not activate B cells or induce proliferation. Antibodies of comparable isotypes or against other B cell-restricted antigens, including B2, B4, B5, and HB-5, did not inhibit activation. Pretreatment of B cells with anti-B1 antibody did not inhibit activation, indicating that B cells had to be cultured with anti-B1 antibody for anti-B1-mediated inhibition to occur. Maximum inhibition was obtained when anti-B1 antibody was added at the initiation of culture. In agreement with this, growth factor-dependent proliferation of preactivated B cells was not inhibited by anti-B1 antibodies. Comparable inhibition of B cell activation was noted with antibodies reactive with class II antigens of the major histocompatibility complex with the exception that anti-B1 antibody inhibited immunoglobulin secretion in pokeweed mitogen assays, whereas anti-DR antibody did not. These results suggest that the B1 molecule may serve a central role in the regulation of B cell activation and differentiation.     

Alternative model for the internal structure of laminin

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.     

Identification and characterization of the Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2).

In pursuing studies on the early events in the infection of human B cells by Epstein-Barr virus (EBV), we examined the host cell attachment phase with a panel of B-cell-specific monoclonal antibodies. One of the monoclonal antibodies, OKB7, directly blocked the attachment of purified EBV to B lymphocytes in the absence of a second anti-immunoglobulin antibody and thereby prevented EBV infection of tonsil and peripheral blood B cells. Although earlier studies have shown a close association of the EBV and complement receptor (CR2), an anti-CR2 monoclonal antibody, anti-B2, did not directly block the binding of EBV to B cells. A comparison of the structures recognized by these monoclonal antibodies on various cell types and their functional and physiochemical properties was undertaken. Flow cytometric analysis revealed that the molecules detected by OKB7 and anti-B2 were coexpressed to the same extent on B cells but were not expressed on T-cell lines. OKB7 and anti-B2 both immunoprecipitated a 145,000-molecular-weight membrane protein with an isoelectric point of 8.2 from membrane extracts of Raji lymphoblastoid cells. OKB7 and, to a lesser extent, anti-B2 directly blocked the attachment of C3d,g-coated fluorescent microspheres and sheep erythrocytes bearing C3d to B cells, indicating that these antibodies also react with CR2. These studies indicate that the EBV-CR2 receptor is a single membrane glycoprotein which possesses multiple antigenic and functional epitopes.     

Assignment of the structural gene for subunit M1 of human ribonucleotide reductase to the short arm of chromosome 11

By using a species-specific monoclonal antibody that recognizes subunit M1 of ribonucleotide reductase from human but not hamster origin, we have been able to assign the structural gene for the human protein M1 to the short arm of chromosome 11. Protein extracts from a panel of human-Chinese hamster somatic cell hybrids were subjected to electrophoresis in sodium dodecyl sulfate (SDS) denaturating polyacrylamide gels, and then transferred and coupled covalently to diazobenzyloxymethyl paper. These were screened for human protein M1 by incubation with the mouse monoclonal anti-M1 antibody AD 203, followed by rabbit anti-mouse IgG, 125I-labelled Staphylococcus protein A and finally autoradiography. In all tested hybrids the detection of human protein M1 was correlated with the presence of chromosome 11, specifically with the short arm of this chromosome. This region also contains the human genes for insulin, insulin-like growth factor II, and the c-Harvey-ras 1 oncogene.     

Ribonucleotide reductase from Escherichia coli: demonstration of a highly active form of the enzyme.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low. Enzyme activity is stimulated 5 to 10-fold by addition of an excess of either subunit. Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20-fold higher activity than extracts from mechanically disrupted cells. This activity was not further stimulated by either B1 or B2. The system is suitable for complementation tests for the analysis of temperature-sensitive mutants affecting the ribonucleotide reductase system. Concentrated high-speed supernatants from E. coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2. This active form of the enzyme was unstable and could not be purified. The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures.     

Study of the structural characteristics of the regulatory subunit of cAMP-dependent protein kinase type II using monoclonal antibodies

Monoclonal antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from porcine brain were used to study the antigenic properties of the enzyme regulatory subunit (RII). The monoclonal antibodies were bound to linear antigenic determinants on the protein molecule surface. The cAMP binding to RII interfered with the interaction between monoclonal antibodies and the protein. The use of different proteolytic fragments of RII allowed for the localization of antigenic determinants in the N-terminal moiety of RII.     

Ribonucleotide reductase from Escherichia coli. Identification of allosteric effector sites by chromatography on immobilized effectors.

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.