Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface.

Charbit, Alain; Boulain, Jean‐Claude; Ryter, A; Hofnung, Maurice

doi:10.1002/j.1460-2075.1986.tb04602.x

Cited by 181 publications

(100 citation statements)

References 25 publications

(11 reference statements)

Supporting

Mentioning

100

Contrasting

Order By: Relevance

“…Insertion of the 16-amino-acid epitope from Semliki Forest virus only strongly affected protein synthesis and folding at four of the 14 sites tested, thus adding it to the list of insertions (Charbit et al, 1986;Agterberg et al, 1990;LeClerc et al, 1991;Struyvé et al, 1993;Newton et al, 1996;Janssen and Tommassen, 1996) whose antigenic properties are fairly independent of the neighbouring amino acids and which only disturb natural protein folding a little.…”

Section: Discussionmentioning

confidence: 99%

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Merker

Tommassen

Kušećek

et al. 1997

Molecular Microbiology

View full text Add to dashboard Cite

SummaryA two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surfaceexposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (ٌ) from Semliki Forest virus were inserted into Bgl II restriction sites created by site-directed mutagenesis. The ٌ epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while ٌ epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

show abstract

Section: Discussionmentioning

confidence: 99%

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Merker

Tommassen

Kušećek

et al. 1997

Molecular Microbiology

View full text Add to dashboard Cite

show abstract

“…Insertions of 15 and 13 amino acids, respectively, in cell-surface-exposed parts of these proteins did not influence the expression and the assembly of these proteins into the outer membrane [40,41].…”

Section: Discussionmentioning

confidence: 99%

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

1987

View full text Add to dashboard Cite

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J . Biochem. 152,. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12 -74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of p-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of P-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.The outer membrane of Enterobacteriaceae functions as a barrier for harmful compounds. The membrane contains a number of proteins, that form transmembrane channels, through which small hydrophilic solutes can pass in a diffusion-like process [I]. Under standard laboratory conditions, Escherichia coli K-12 produces two general pore-forming proteins, OmpC protein and OmpF protein. When cells are grown under phosphate limitation, the synthesis of another pore protein, PhoE protein, is induced [2]. This protein forms pores which are more efficient for phosphate-containing compounds and for negatively charged compounds in general in contrast to the cation-selective OmpC and OmpF pores [3, 41. The primary structures of the OmpC, OmpF and PhoE proteins show an extensive homology of approximately 60% [5].In our laboratory we are studying the structure/function relationship of the porins. In addition, we are aiming to use PhoE protein as a carrier for the transport of foreign antigenic determinants to the cell surface. Korteland et al. [6] have isolated five independent TC45-resistant mutants that produce an altered PhoE protein. In all five mutants the arginine residue in position 158 was substituted by a histidine residue. Thus, arginine-158 is involved in the receptor function of the PhoE protein for phage TC45 and the region around this residue is probably cell-surface-exposed. OmpC protein has an insertion of 14 amino acids in this region when compared to the PhoE protein [5]. Protease-accessibility experiments demonstrated the presence of this insert at the cell surface [7].Correspond...

show abstract

“…To circumvent the problems encountered with reporter enzymes, we started a new and complementary approach that consists of using as a reporter a small peptide corresponding to a foreign antigenic determinant (i.e., a reporter epitope) which we insert genetically into permissive sites of LamB (4,8; reviewed in reference 22). Such sites tolerate insertions without loss of most biological properties of the protein and therefore without major changes in its cellular location and structure.…”

mentioning

confidence: 99%

“…Such sites tolerate insertions without loss of most biological properties of the protein and therefore without major changes in its cellular location and structure. Permissive sites can be identified by a genetic method (8), and the hybrid proteins generated are then probed with a monoclonal antibody (MAb) against the reporter epitope. This approach led us to show that upon insertion after residues 153 and 374 of the mature LamB protein, the C3 epitope was expressed at the bacterial cell surface (8), indicating that these two regions of LamB face the outside of the cell as proposed by our folding model (9,10).…”

mentioning

confidence: 99%

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

et al. 1991

View full text Add to dashboard Cite

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 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.Genetic fusions between proteins have proved to be useful tools in the study of a large number of biological problems. P-Galactosidase, alkaline phosphatase, and 1-lactamase have been used as reporter enzymes to study the location or the folding of proteins and for various biotechnological applications (for recent discussions, see references 7, 27, and 28). For topological studies, such fusions present at least two kinds of limitations. First, they usually delete the COOH-terminal end of the vehicle protein; this end can be critical for the normal biogenesis of the protein. Second, properties of the passenger enzyme (size, sequence, etc.) may alter the location or folding of the vehicle protein (see, for example, references 31 and 42). These two problems occur in the case of the outer membrane protein LamB (3, 4), which is why the topological information on this protein comes from other approaches (10, 21).LamB is representative of a class of integral membrane proteins, the porins (35), which are peculiar for at least two reasons. They are mostly hydrophilic, with no apparent hydrophobic transmembrane segment, and the structured regions are essentially composed of beta-strand segments. LamB plays a role in the penetration of maltose and maltodextrins through the outer membrane and is the cell surface receptor for a number of bacteriophages, including phage lambda; the active form is a trimer (reference 10 and references therein). In the absence of precise crystallo-* Corresponding author. graphic data, most of our knowledge of LamB organization relies on genetic, immunological, and biochemical data. On these bases, we have proposed a two-dimensional folding model that we would like to test and refine (9, 10).To circumvent the problems encountered with reporter enzymes, we started a new and complementary approach that consists of using as a reporter a small peptide corresponding to a foreign antigenic determinant (i.e., a reporter epitope) which we insert genetically into permissive sites of LamB (4,8; reviewed in reference 22). Such sites tolerate insertions without loss of most biological properties of the protein and therefore without major changes in its cellular location and structure. Permissive sites can be identified by a genetic method (8), and the hybrid proteins generated are then probed with a monoclonal antibody (MAb) against the reporter epitope. This approach led us to show that upon insertion after residues 153...

show abstract

Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface.

Cited by 181 publications

References 25 publications

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

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

Contact Info

Product

Resources

About