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Biomedical subjects

M Hofnung

Publications and source records attributed to M Hofnung.

At least 91 records · Page 5Linked to original sources
PubMed1991

A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis.

We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis. It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins. One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised. The two "recipient" proteins used are the MalE and the LamB proteins from E. coli. The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent. We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus. With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice. The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen.

Animals↗
PubMed1991

Permissive sites and topology of an outer membrane protein with a reporter epitope.

We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12. This approach consists of inserting the same reporter foreign antigenic determinant (the C3 epitope from poliovirus) at different sites of LamB so that the resulting hybrid proteins have essentially kept the in vivo biological properties of LamB and therefore its cellular location and structure; the corresponding sites are called permissive sites. A specific monoclonal antibody can then be used to examine the position of the reporter epitope with respect to the protein and the membrane. We present an improved and efficient procedure that led us to identify eight new permissive sites in LamB. These sites appear to be distributed on both sides of the membrane. At one of them (after residue 253), the C3 epitope was detected on intact bacteria, providing the first direct argument for exposure of the corresponding LamB region at the cell surface. At this site as well as at four others (after residues 183, 219, 236, and 352), the C3 epitope could be detected with the C3 monoclonal antibody at the surface of the extracted trimeric LamB-C3 hybrid proteins. We provide a number of convergent arguments showing that the hybrid proteins are not strongly distorted with respect to the wild-type protein so that the conclusions drawn are also valid for this protein. These conclusions are essentially in agreement with the proposed folding model for the LamB protein. They agree, in particular, with the idea that regions 183 and 352 are exposed to the periplasm. In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.

Amino Acid Sequence↗
PubMed1991

Bacterial vectors to target and/or purify polypeptides: their use in immunological studies.

The construction of recombinant proteins by genetic engineering has opened new avenues in basic research (studies on protein organization, protein folding, immunogenicity of proteins, ...) and many different applications. Recombinant proteins which keep properties of both parental proteins are especially interesting. For example, if one protein--the vector protein--is targeted to a given cellular compartment, the other protein--the passenger--may be identically targeted. Also, if the vector protein can be purified by a simple affinity chromatographic procedure, this property may be extended to the passenger. The authors have developed a genetic procedure to detect "permissive" sites within potential vector proteins so that genetic fusion to these sites keep most or all biological properties of the vector. When they used LamB, an outer membrane protein from E. coli, foreign sequences could be expressed at the bacterial cell surface. This may lead to several types of applications: live bacterial vaccines, simple diagnostic tests, selection procedures for peptides with biological activity. When they used the MalE protein, a periplasmic maltose binding protein from E. coli, the passengers could be exported and purified in one-step high affinity chromatography in mild non-denaturing conditions. This led us to a simple preparation and purification scheme for the soluble part of the CD4 receptor for the Human Immunodeficiency Virus (HIV).

Bacterial Outer Membrane Proteins↗
PubMed1990

DNA polymerase I and a protein complex bind specifically to E. coli palindromic unit highly repetitive DNA: implications for bacterial chromosome organization.

Starting from a crude E. coli extract, two activities which specifically protect highly repetitive bacterial DNA sequences (called PU for Palindromic Unit or REP for Repetitive Extragenic Palindromic sequence) against a digestion with Exonuclease III have been purified. We show that one of these activities is due to the DNA polymerase I (Pol I). This constitutes the first indication for a specific interaction between Pol I and a duplex DNA. This interaction requires the presence of PU. It was confirmed and analyzed by native gel electrophoresis and DNase I footprinting experiments. The other activity contained at least five polypeptides. Its binding to PU DNA sequences was confirmed by native gel electrophoresis. Implications for the possible origin and functions of PU are discussed.

Bacterial Proteins↗
PubMed1990

Genetic approach to the role of tryptophan residues in the activities and fluorescence of a bacterial periplasmic maltose-binding protein.

The periplasmic maltose-binding protein (MBP or MalE protein) of Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins. Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3 A three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in Kd values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in Kd (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in Kd value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations. The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1990

Crystallization of genetically engineered active maltose-binding proteins, including an immunogenic viral epitope insertion.

Three mutants of the maltose- or maltodextrin-binding protein encoded by the malE gene of Escherichia coli, with extensive genetic changes, have been purified and crystallized in different crystal forms. Two of these mutant proteins, MalE178 and MalE341, carry net deletions of seven and 13 residues, respectively, near the surface of the molecule. These mutations have very little effect on either the transport activity of the mutant strains or the sugar-binding activity of the purified mutant proteins. The third mutant protein involves the insertion of an 11-residue peptide of the C3 epitope from type 1 poliovirus VP1 protein into the MalE178 deletion mutant, with retention of essentially all the biological properties of the wild-type and the immunological properties of the C3 epitope. We are undertaking three-dimensional structure analysis in order to understand how the protein accommodates these large changes in its surface structure and how the C3 epitope retains its immunological properties in this new environment. The same system could be used to determine easily the structures of other peptide epitopes, especially those in proteins with unknown structures.

ATP-Binding Cassette Transporters↗
PubMed1990

Induction of virus-neutralizing antibodies by bacteria expressing the C3 poliovirus epitope in the periplasm. The route of immunization influences the isotypic distribution and the biologic activity of the antipoliovirus antibodies.

Two viral epitopes (C3 neutralization epitope from poliovirus type 1 and the 132-145 peptide from the PreS2 region from hepatitis B virus) have been expressed in the Escherichia coli periplasm as protein fusion with the maltose binding protein (MalE protein). Immunization of mice with live bacteria expressing the foreign viral epitopes in their periplasm elicited high antibody titers against the viral peptide as well as against the corresponding virus. This demonstrates for the first time in the case of defined epitopes that, when live bacteria are used as immunogens, presentation at the cell surface is not a prerequisite to obtain an antibody response. On the other hand, the induction of antiviral antibody responses by these recombinant bacteria depended dramatically on the route of immunization: a response was induced by live bacteria through the i.v. route but not through the s.c. route. However, when bacteria were heat killed or when the MalE hybrid protein was released under a soluble form from the cell, a response was induced even upon s.c. immunization. From these results, we suggest that in order to induce high levels of antibodies by the s.c. route, a major parameter for bacterial Ag would be their capacity to be released into a soluble form before the interaction of the bacteria with the APC. This would permit the presentation by B cells rather than by phagocytic cells. Finally, we demonstrate that the route of immunization influences the isotypic distribution and the neutralizing activity of the antipoliovirus antibodies. Such results may have major implications for the development of bacterial vaccines based on fusion proteins.

ATP-Binding Cassette Transporters↗
PubMed1990

Critical role of neighbouring sequences on the immunogenicity of the C3 poliovirus neutralization epitope expressed at the surface of recombinant bacteria.

The C3 neutralization epitope of poliovirus type 1 (PV-1) is a continuous epitope comprised within residues 93-103 of capsid protein VP1. These residues form a loop at the surface of the virus particle. The authors compared the immunogenicity of two peptides which contain this epitope, when presented at the surface of Escherichia coli by genetic insertion in the outer membrane protein LamB. One peptide was 13 residues long (VP1:93-103) and the other one contained flanking sequences increasing its size to 35 residues (VP1:86-115). Mice and rabbits were immunized with recombinant bacteria expressing the corresponding LamB-VP1 hybrid proteins. Antibodies against synthetic peptides, against native and heat denatured viral particles, as well as neutralizing antibodies were monitored. In this mode of presentation the shortest form of the epitope was more immunogenic. We provide evidence that the conformation of the epitope is different in the two hybrid LamB proteins and discuss possible consequences for immunogenicity.

Amino Acid Sequence↗
PubMed1990

Immunogenicity and antigenicity of conserved peptides from the envelope of HIV-1 expressed at the surface of recombinant bacteria.

We expressed peptides from the HIV-1 envelope protein at the surface of Escherichia coli by genetic insertions into an exposed loop of the outer membrane protein LamB. Recombinant bacteria expressing eight peptides from gp110 (pep1-pep8), conserved between HIV-1 and HIV-2, were used as live immunogens in rabbits by the intravenous route. The eight constructions elicited anti-LamB antibodies, showing that the hybrid proteins were immunogenic. One of them, LamB-pep8, gave rise to antibodies able to react with gp160 and to neutralize HIV-1 in vitro. We also show that this type of recombinant E. coli can provide a convenient reagent to monitor and characterize specific antibodies. Recombinant clones were used to test sera of seropositive individuals, as well as to narrow down the monoclonal antibody 110-1 recognition site to a cluster of eight residues at the carboxy-terminal end of gp110.

Amino Acid Sequence↗
PubMed1990

Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12.

Osmotic stress produced by high concentrations of sucrose stimulated the high-affinity transport of glutamine in Escherichia coli cells. Glutamine transport via a low-affinity system was not affected. Osmotic stress produced by NaCl, in contrast, inhibited the transport of glutamine and some other amino acids. Maltose transport was strongly inhibited by osmotic stress.

Amino Acids↗
PubMed1990

Export and one-step purification from Escherichia coli of a MalE-CD4 hybrid protein that neutralizes HIV in vitro.

The 177 N-terminal amino acids of CD4, the receptor of the human immunodeficiency virus (HIV), have been expressed in Escherichia coli as genetic fusions to the periplasmic maltose-binding protein (MalE) from this organism. A large fraction of the hybrid proteins can be released from the periplasm by osmotic shock and purified in one step on a cross-linked amylose column eluted with maltose under mild conditions. One hybrid protein binds HIV envelope protein gp160 and neutralizes the virus in vitro. This provides the first example of the production and one-step purification of an active form of an eukaryotic protein by fusion to MalE. The use of this system for mass screening of CD4 mutants, high-scale production of the hybrid protein for structural studies on CD4, testing antiviral compounds, and therapeutic assays is discussed.

ATP-Binding Cassette Transporters↗
PubMed1989

Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12.

LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages. A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm. This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein. In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences. We chose four regions. Three of them [aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)] are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)] is predicted to be periplasmic. By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions. In some cases, the recognition sites for antibodies were narrowed down to parts of a region. In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4. Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: a potential new regulatory site in the interoperonic region.

The malE and malK genes from Salmonella typhimurium, and the malEFG operon and a portion of malK from Enterobacter aerogenes were cloned and sequenced. Plasmid-borne malE genes from both species and the malF and malG genes from E. aerogenes were expressed normally in Escherichia coli, and their products function in maltose transport. This shows that the malB products from the three species are interchangeable, at least in the combinations tested. The general genetic organization of the malB region is conserved. Potential binding sites and distances between them are highly conserved in the regulatory intervals. An unexpected conserved region was detected, which we call the U box, and which could be another target for a regulatory protein. This hypothesis is supported by the presence of the U box in the regulatory region of the pulA-malX operon in Klebsiella pneumoniae. The intergenic region between malE and malF from S. typhimurium and E. aerogenes, contains inverted repeats similar to the palindromic units (PU or REP) found at the same location in E. coli. The predicted amino acid sequence of the encoded proteins showed 90% or more identity in every pairwise comparison of species.

Amino Acid Sequence↗
PubMed1989

Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents.

The effects of 3 types of ionizing radiation, gamma-rays, neutrons and accelerated alpha-particles, were examined using the SOS Chromotest, a bacterial colorimetric assay for genotoxic agents based on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses being in the order of 2.5-5 Gy. We discuss the possible use of these observations to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals.

DNA Repair↗
PubMed1989

Antibody response to a foreign epitope expressed at the surface of recombinant bacteria: importance of the route of immunization.

A genetic procedure has been previously established to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier. A portion of the pre-S2 region of hepatitis B virus, residues 132-145, has been inserted at amino acid position 153 of the LamB protein, in a cell surface exposed loop. In the present study, we have analysed the antibody responses induced by these recombinant bacteria (live, heat-killed or sonicated) depending upon the route of immunization. The intravenous (i.v.) or intraperitoneal (i.p.) administration of the live recombinant bacteria to mice induced the synthesis of antibodies against both the inserted peptide and the native LamB protein. The antibodies raised recognized HBsAg particles. These mice also had high titres of antibodies against E. coli antigens (as determined using a crude bacterial sonicate). In contrast, mice immunized subcutaneously (s.c.) did not develop antibodies against the pre-S2 peptide nor against the HBsAg particles. Their anti-LamB responses were low compared with the response of mice immunized by the parenteral route. Interestingly, s.c. or i.v. immunizations induced comparable levels of anti-E. coli antibodies. Thus, the antibody response to the inserted peptide generally parallels the response to the LamB protein (and not to the bulk of E. coli antigens). However, this treatment corresponding to a 'pre-processing' of the recombinant bacteria was not sufficient to obtain an anti-peptide response following s.c. immunization.

Animals↗
PubMed1989

Beta-galactosidase overexpression in SV40-transformed Chinese hamster fibroblasts exposed to mutagens as a result of amplification of transfected bacterial lacZ DNA sequences.

Genetic constructions in which the bacterial lacZ gene, encoding the enzyme beta-galactosidase, is fused to a viral (SV40) origin of replication have been introduced in an SV40-transformed hamster cell line (C1102). We have studied in detail 3 clones in which beta-galactosidase-specific activity increases after treatment with genotoxic agents. We show that this increase is dependent on the activity of the viral T protein and correlates with an amplification of lac sequences. This system provides a basis for the study of the induction of gene amplification by genotoxic agents in mammalian cells.

Animals↗
PubMed1989

DNA adduct formation by 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), an extremely potent mutagen.

The effects on DNA, in bacteria, of 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), a very potent genotoxic product from the 2-nitronaphthofuran series, were investigated with two different approaches: (i) measurement of the binding of the radiolabelled mutagen to DNA and (ii) detection by the '32P-postlabelling' method of DNA adducts following treatment with unlabelled mutagens. The covalent binding of R7000 to DNA in Escherichia coli was demonstrated by both methods, and in the latter case it was found to involve the formation of nine different adducts. Formation of adducts by R7000 was shown to require metabolic activation of the compound.

Chromatography, Thin Layer↗