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A Charbit

Publications and source records attributed to A Charbit.

At least 19 recordsLinked to original sources
PubMed1992

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145-167, 173-187, 197-226, 237-300, 311-329, and 367-387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.

ATP-Binding Cassette Transporters↗
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

Localization and characterization of three different beta-adrenergic receptors expressed in Escherichia coli.

After fusion with the N-proximal portion of the outer membrane protein LamB, three beta-adrenergic receptors, the human beta 1- and beta 2- and turkey beta 1-adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti-peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti-peptide antibodies. The functional expression in E. coli of three beta-adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G-protein-coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.

Animals↗
PubMed1990

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation.

Salmonella typhimurium and S. typhi were transformed with high efficiency by electroporation. Transformation efficiencies of up to 10(10) transformants per microgram of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a ga1E mutation slightly improved transformation efficiency in S. typhimurium (less than tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.

Antigens, Bacterial↗
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↗
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

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↗
PubMed1988

Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria.

A wide variety of peptides in terms of length and sequence can be expressed at the surface of the bacterium Escherichia coli by genetic insertion into a 'permissive' site of the outer membrane protein LamB, used as a carrier. The resulting hybrid proteins essentially keep their biological activities with inserts of up to about 60 amino acid residues, and of a large range of predicted structures or hydrophobicities. This reflects a remarkable flexibility in the organization of the protein, but also in the export machinery. The method used to select such a permissive site is quite general and its potential to generate applications, including a versatile type of live bacterial vaccine, are discussed.

Amino Acid Sequence↗
PubMed1988

Maltose transport and starch binding in phage-resistant point mutants of maltoporin. Functional and topological implications.

The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated. Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions. Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport. It revealed three classes of mutants. Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18). Starch binding was assayed by two different methods that gave compatible results. In class A mutants, binding was normal, while no binding was observed in the class C mutant. Binding was impaired to various extents in category B mutants. There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore. These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct. Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250. Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein. The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.

Bacterial Outer Membrane Proteins↗
PubMed1988

Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane.

Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987). The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers. The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport. This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions. The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins. The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant). Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro. These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter. Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.

Amino Acid Sequence↗
PubMed1987

Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria.

Having developed a genetic procedure to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier, we apply this procedure to express two distinct portions of the preS2 region of hepatitis B virus: region A, residues 132-145, and region B, residues 153-171. The resulting hybrid proteins (LamB-preS2 A and LamB-preS2 B) were normally expressed, stable, and still kept most biologic functions of LamB. The corresponding bacterial strains were used directly as immunogens in rabbits and mice. Both viral sequences were found to be immunogenic in the two animal species. With LamB-preS2 A, antibodies induced were able to react with the viral particles and the immobilized peptide. With LamB-preS2 B, the antibodies raised were not able to recognize the immobilized peptide. However, the results suggest that the B epitope, inserted in LamB, was at least as efficient as the corresponding synthetic peptide in raising antiviral antibodies. Thus, epitope presentation with LamB may present advantages for immunization. We also have shown that peptide A is an essential part of the polymerized human serum albumin receptor. These results, which validate further the LamB vector system for epitope presentation, provide information on the two hepatitis B regions expressed.

Animals↗