Jean-Marie Pages

A quantitative immunochemical technique for evaluation of the extent of integration of membrane proteins and of protein conformational changes and homologies

Abstract A methodology that allows an estimation of the number of antigenic determinants that remain exposed or accessible on a protein antigen after conformational changes, evolution, or integration in a membrane matrix has been developed. We have observed that a protein antigen, used as an immunogen, will stimulate the production of antibodies directed specifically against the exposed antigenic determinants of its surface, and then the maximum number of antibody molecules that can simultaneously bind to this antigen corresponds to the total covering of its surface. Surface modifications are shown to occur on Escherichia coli alkaline phosphatase upon noncovalent binding of inorganic phosphate and upon Zn2+ removal. The homology between pig intestinal and kidney brush border membrane aminopeptidases has been studied. When probed with anti-intestinal aminopeptidase (free form) the surfaces of both enzymes have been shown to bear six identical antigenic determinants, two of which are located in the area that is masked upon integration into the membranes. A technique to determine precisely the number of determinants rendered inaccessible by integration of aminopeptidases into the membranes is described. Two determinants out of the 12 of the “free intestinal aminopeptidase” surface are masked by integration in the membrane matrix. Thus, the methodology presented should be a valuable tool in all processes of molecular biology that involve protein surface probing.

Improving the stability of a foreign protein in the periplasmic space of Escherichia coli.

An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed [1]. The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space. However, in this location proteolytic degradation occurred at the C-terminal region. Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage. The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections. Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.

Preferential sensitivity of syntheses of exported proteins to translation inhibitors of low polarity in Escherichia coli

SummaryWe have compared the sensitivities to different translation inhibitors of the syntheses of enzymes from various cell compartments. Alkaline and acid phosphatases were chosen as representative of periplasmic enzymes. Aminopeptidase N and β-galactosidase represented peripheral membrane protein and cytoplasmic enzyme respectively. Antibiotics of low polarity such as spectinomycin and tetracycline, when used at low concentrations, caused a preferential inhibition of exported proteins compared to total cell proteins, to aminopeptidase N and to β-galactosidase. Synthesis itself was inhibited as demonstrated by immunochemical assay of the amount of proteins synthesized in the presence or absence of spectinomycin. Temperature-dependance of the penetration of spectinomycin as reported by its effect, indicated that this drug does not enter the cell by simple dissolution in the membrane lipids but rather enters at specific sites of the cell envelope. In contrast to low polarity inhibitors, a polar antibiotic such as kanamycin did not cause any preferential inhibition of the syntheses of exported proteins. By using radioactive tetracycline (0.025, 0.1 and 0.2 μg/ml) we have investigated the distribution of this antibiotic between free and membrane-bound polysomes isolated without the use of lysozyme. Three times as much tetracycline was found in the membrane-bound compared to free polysomes fraction.Therefore we propose that preferential sensitivity of synthesis of exported proteins to translation inhibitors of low polarity reflects a preferential binding of these antibiotics to membrane-bound polysomes fractions in Escherichia coli. We thus suggest that this preferential binding occurs since the site of entry of weakly ionized antibiotics used in this study, correspond to the site of synthesis and transfer of exported proteins.

Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli

Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein. This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.

Immunological analysis of porin polymorphism in Escherichia coli B and K-12.

Two sets of monoclonal antibodies (MoF type I and MoF type II) directed against the OmpF protein were used to analyze the immunological reactivity of the major outer membrane porins of E. coli B and K-12. All these antibodies present a specificity to the native OmpF protein. In addition, among the type II antibodies, MoF 18, 19 and 20 could recognize an epitope present on both monomeric and trimeric forms of the porin as demonstrated by immunoblotting analyses. The use of two different screening methods led to the isolation of two different sets of MoF, one specific for a native conformation accessible only on E. coli B strain and the second directed against epitopes present on OmpF of the two strains, B and K-12. These various responses are discussed in relation to the lipopolysaccharide binding to OmpF and with respect to the screening test used.

Immunological comparison of major outer membrane proteins from different strains of Escherichia coli

Rabbits were immunized with either the native trimer or the denatured monomer of OmpF porin of Escherichia coli. The specificity of the two antisera was checked by two different techniques. In liquid immunorecognition assays, the antisera detected the presence of common antigenic determinants at the surface of the monomer and trimer forms. A high response was observed between the two antisera and the native form of OmpF. Although cross-reactivity between the antiserum directed against the OmpF monomer and a denatured form of PhoE was obtained, this denatured porin was weakly recognized by the antiserum specific to the native protein. In immunoblotting experiments, though antiserum directed against the native porin detected the two forms of OmpF, antiserum specific to the monomer recognized only the denatured protein. These two antisera were used to check the immunological relationships of E. coli strains isolated from various contaminated waters. Proteins from several wild strains were compared to the immunoblotting patterns of E. coli reference strains. Porins from the majority of these wild strains were immunorelated to porins of the ML30 strain. In two strains, porins were related to the OmpC/OmpF of the K12 strain, and one strain contained a porin identical to the OmpF of the B strain. Thus, an immunological approach using polyclonal antibodies directed against a major outer membrane protein appears to be fruitful for an accurate classification of various E. coli strains. Moreover, the ecological habitat of a strain can also be investigated by this analysis.

Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12

Nine monoclonal antibodies (MoF 0-8) directed against the native form (trimeric) of outer membrane protein OmpF of Escherichia coli B were obtained and characterized. All these antibodies bind to OmpF porin in intact E. coli B cells but not OmpF from E. coli K-12 cells which only differ at positions 66, 117 and 262 in the sequence. These antibodies exhibit a specificity to the native form, failing to recognize the denatured form in a liquid immunorecognition assay. Four tested antibodies are able to protect against colicin A, a bacteriotoxin using OmpF as receptor. One monoclonal antibody (MoF 0) is specific to the external topology of native porin in the outer membrane and three antibodies could recognize epitopes present in each conformation of subunits of trimer form. It is concluded that the region around the 66th and more probably around the 262nd amino acids are involved in cell-surface exposed epitopes. Moreover, these results support the assumption that the conformation of protruding regions of OmpF from E. coli B and K-12 are different.

Export and secretion of overproduced OmpA-β-lactamase in Escherichia coli

The export of β‐lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN‐III. The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains. However, when the processing starts, no alteration in the rate of cleavage itself is observed. Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported.

Preparation of active iodinated specific antibodies

Lactoperoxydase-catalyzed iodination of antibodies is very convenient since it is simple to perform and can be carried out in a short-time as previously pointed out by Morrison et al. [ 1 ]. Furthermore the radioiso-, topes of iodine are inexpensive, easy to detect, and iodinated proteins of high specific radioactivities can be obtained. However, it has been shown [1,2] that iodination of antibodies results in the partial inactivation of IgG molecules. Many problems in molecular biology are currently approached by immunochemical techniques. In most instances inactivated antibodies must be discarded since active iodinated antibodies are required to obtain quantitative results. In this regard, we have recently described the use of immunochemical techniques in investigations of protein conformational change [3] and in topological studies on membrane bound enzymes [4]. Trace iodination of immunoglobulins, catalyzed by lactoperoxydase has been described by Marchalonis [5]. However, in our hands, using rabbit immunoglobulins, we obtained very little iodine incorporation in the conditions used by this author. In this report we describe a method that allows the obtention of iodinated active antibodies of high specific radioactivity. This technique involves a lactoperoxydase-catalyzed iodination followed by the reisolation of specific antibodies on immuno-adsorbent columns. By this way all the IgG molecule that have been inactivated in the process of iodination are eliminated. Thus, we have been able to evaluate precisely the extent of inactivation as a function of the number of iodine atoms incorporated. 2. Materials and methods

Membrane potential (Δψ) depolarizing agents inhibit maturation

Precursor forms of exported proteins were first accumulated in the envelope of phenethyl alcohol (PEA)‐treated cells. After removal of PEA, a complete processing could be obtained in a few minutes. In this work, we demonstrate that colicins A and E1, that act on the electrical gradient in the cytoplasmic membrane, prevent the processing of precursor forms previously accumulated. Concentrations of colicins accounting for ∼1 killing unit (50–3000 molecules/cell) were found to be sufficient for inhibition of processing. Therefore our results strongly suggest that in intact cells the electrical gradient across the cytoplasmic membrane is required for maturation of exported proteins.