Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12

Abstract

Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100°C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydratespecific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not aHl of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.The structure and interactions of the outer membrane porin proteins OmpF and OmpC from Escherichia coli are very similar. They have similar molecular weights and amino acid sequences (22), the native proteins are very high in beta structure (25, 32), both are tightly associated with the peptidoglycan (9, 19, 32), and they are very tightly associated with lipopolysaccharide (LPS) (10,12,36,40). Porins are known to form water-filled channels (2, 33) and are unusually stable to a wide variety of perturbants such as sodium dodecyl sulfate (SDS) detergent (24, 25, 41), pH extremes from 2 to 12 (8), and temperatures up to 70°C (32). Porins are also resistant to many proteases, perhaps because of their tight association with LPS (12); the proteins are thought to be functional only in the presence of LPS (12,20,33). Nakae et al. (24) have suggested that only the lipid component of LPS is essential for porin function. In contrast, Parr and co-workers (30) have reported that porin is functional in the absence of LPS. Thus, the importance of bound LPS for porin structure and function has yet to be resolved.The OmpF porin exists as a trimeric complex (24, 29) with three identical subunits with approximate molecular weights of 36,000 (13, 32). Low SDS binding coupled with the tight, compact shape of the trimer results in more rapid migration on polyacrylamide gels than the aggregate molecular weight would suggest. Investigator...

Evolution of the ferric enterobactin receptor in gram-negative bacteria

“…For external cell surface residues, associations with the core and G-antigen sugars of LPS may exert further requirements for hydrophilicity. Polypeptides that traverse the OM bilayer, on the other hand, associate with phospholipid and LPS fatty acids (54,62) and must therefore include residues of sufficient hydrophobicity to stabilize this interaction. Enteric bacterial porins, for example, contain transmembrane ,B-strands that possess a hydrophobic face that interacts with the OM lipids on the porin exterior and a hydrophilic face that lines the surface of the water-filled channel in the porin interior (11,35,68).…”

“…SDS-PAGE was performed and analyzed by ammoniacal silver staining as described previously (35,51). Cell envelopes and OM fractions from wild isolates of Salmonella, Citrobacter, Serratia, and Enterobacter resolved poorly in SDS-PAGE, probably because of the association of OM proteins with LPS (54,62) and capsule. The incubation of OM or cell envelopes with lysozyme (1 mg/ml) for 30 min at 0°C and the addition 0.03 M EDTA and 0.2 M NaCl to the SDS-PAGE sample buffer improved electrophoretic separation.…”

Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis

“…Hib, Haernophilus influenzae type b; mAb, monoclonal antibody; LPS, lipopolysaccharide; CD, circular dichroism; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline Porins of Gram-negative bacteria bind tightly to lipopolysaccharide (LPS) [7,8]. Purified OmpF of E. coli was shown to have up to nine molecules of LPS associated with each trimer [8,9]. While most of this LPS was loosely bound and easily dissociated, it was estimated that one LPS molecule remained tightly bound to each OmpF trimer [9].…”

Assembly of the OmpF porin of Escherichia coli B

“…These results indicated that several trimeric forms exist after cell solubilization. Moreover, immunological differences between these native forms have been demonstrated using antibodies directed against lipopolysaccharide [27] and against OmpF [32]. In addition, it has been demonstrated that several monoclonal antibodies prepared against the unfolded monomer of OmpC and OmpF cannot recognize the OmpF trimer, indicating the masking of these epitopes in the native form [33].…”