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

Pagès, Jean‐Marie; Pages, Catherine; Bernadac, Alain; Princé, P.

doi:10.1016/0161-5890(88)90077-6

Cited by 12 publications

(13 citation statements)

References 31 publications

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“…RESULTS HC1 pH 7.4, 1 50 mM NaCI, 5 mM EDTA, 0.05% methionine) and Triton X-100 (1% final concentration) [20] were added and the samples were pre-incubated for 15 min at room temperature. These conditions have been previously reported to ensure a good solubilization of the native OmpF even at low temperatures [17]. Incubation with the various antibodies were then carried out as previously repqrted [17,18,201.…”

Section: Piilsc2 R I N D Pirlsc4iricc Lrihcllingsmentioning

confidence: 99%

“…These conditions have been previously reported to ensure a good solubilization of the native OmpF even at low temperatures [17]. Incubation with the various antibodies were then carried out as previously repqrted [17,18,201. All the antisera used were decomplemented and preabsorbed with ompF mutant cells in order to remove non-specific antibodies.…”

Section: Piilsc2 R I N D Pirlsc4iricc Lrihcllingsmentioning

confidence: 99%

“…All the antisera used were decomplemented and preabsorbed with ompF mutant cells in order to remove non-specific antibodies. traction protocol which results in complete solubilization of OmpF (see Materials and Methods) [17].…”

Section: Piilsc2 R I N D Pirlsc4iricc Lrihcllingsmentioning

confidence: 99%

“…SDS/PAGE (10.5'h polyacrylamide), protein staining and [18], these various forms should be related to the native confluorography were carried out as previously described [17]. formation of the porin at the cell surface.…”

Section: Sorliutn Tlodcq~l Sulf~rtr~pol~~uc~r~lurnid~~ Gel Electrophmentioning

confidence: 99%

“…A cross-reactivity between these two antisera for the various oligomeric forms of OmpF, extracted by gentle solubilization, has been demonstrated by an immunoprecipitation assay [17]. In a recent study, we described the isolation of several monoclonal antibodies directed against the native form of OmpF [18].…”

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Assembly of the OmpF porin of Escherichia coli B

Pagès

Bolla

1988

European Journal of Biochemistry

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The different conformations of the outer membrane protein OmpF of Escherichia coli B were studied with immunological probes. The antigenic determinants recognized by one monoclonal (MoF3) and two polyclonal antibodies were investigated under various conditions of solubilization which modify the association of OmpF with other membrane components, such as lipopolysaccharide. Several polymeric forms of the protein could be detected after extraction at 37°C or 56°C. The monoclonal antibody, which is specific to an exposed region of native OmpF, recognized various trimeric forms in an immunoprecipitation assay. Under the same conditions, the binding of polyclonal antibodies apparently induced strong conformational rearrangements, since the pattern of trimeric forms detected was greatly modified. The conversion of newly synthesized monomers of OmpF to the various trimer forms was investigated using these antibodies. The trimerization occurred rapidly but the appearance of the native conformation of OmpF was delayed. Some additional step was required to expose the MoF3-specific antigenic site at the surface of the trimeric form. These results are discussed in relation to the structure of OmpF and its association with lipopolysaccharide in the outer membrane.OmpC and OmpF are the major outer membrane proteins of Escherichia coli K12, functioning as passive diffusion pores for small hydrophilic molecules [l -31. These porins are assembled as trimer units at the cell surface [4 -61. The structural genes and the sequence of these proteins have been previously described and the regulation of their syntheses is being intensively studied [l, 21. In E. coli B, only one outer membrane porin, related to OmpF, is present. This protein is identical to the E. coli K12 porin except for three substitutions, at amino acid positions 66, 117 and 262 [7]. The assembly of outer membrane proteins is an intriguing and complex process requiring several steps in various cellular compartments during the export process. During and after synthesis the polypeptide chains are translocated across the cytoplasmic membrane where the cleavage of the signal sequence occurs. Several factors are required during this translocation as demonstrated by genetic and biochemical studies [8 -101. The importance of membrane fluidity, the energy requirement and the kinetics of processing are now well documented for this step.Later steps, which ensure the correct insertion of the proteins into the outer membrane, are less well understood. In the case of the outer membrane protein OmpA, an intermediate form (imp-OmpA) is detected, just after processing, at the external side of the cytoplasmic membrane [ll]. The binding of lipopolysaccharide may induce a conformational change promoting the subsequent integration into, the outer membrane. However, for OmpA neither dimeric nor trimeric assembly is required for the integration of the native protein.The maltoporin LamB [ 121 undergoes several conformational changes in order to achieve its native trimeric form in the Corr...

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Section: Piilsc2 R I N D Pirlsc4iricc Lrihcllingsmentioning

confidence: 99%

Section: Piilsc2 R I N D Pirlsc4iricc Lrihcllingsmentioning

confidence: 99%

Section: Piilsc2 R I N D Pirlsc4iricc Lrihcllingsmentioning

confidence: 99%

Section: Sorliutn Tlodcq~l Sulf~rtr~pol~~uc~r~lurnid~~ Gel Electrophmentioning

confidence: 99%

mentioning

confidence: 99%

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Assembly of the OmpF porin of Escherichia coli B

Pagès

Bolla

1988

European Journal of Biochemistry

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The assembly of the major outer membrane protein OmpF of Escherichia coli depends on lipid synthesis.

1988

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Cerulenin, a drug which specifically blocks lipid synthesis, prevented both the trimerization of OmpF monomers and their assembly into the outer membrane of Escherichia coli B cells. A monoclonal antibody directed against a surface‐exposed epitope of the trimer was used to probe the assembly of OmpF in the presence or absence of the drug. An inhibition level of 80% was reached 16 min after the addition of cerulenin. The accumulated monomeric form could not be assembled even after lipid synthesis was restored. Instead, it was slowly degraded. It was further shown that the inhibition of assembly resulted in a rapid inhibition of OmpF synthesis. These data demonstrate that there is a direct relationship between the synthesis of lipid (most likely lipopolysaccharide) and the correct export of OmpF. This coupling is required to promote the trimerization of the porin monomer and its assembly into the outer membrane.

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