Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli

Abstract

Antigen 43 has been identified as a unique protein complex in the outer membrane of Escherichia coli. The complex contains two different polypeptides, a (Mrs 60,000) and ,( (Mrs 53,000), in equal stoichiometry (P. Owen, P. Caffrey, and L.-G. Josefsson, J. Bacteriol. 169:3770-3777, 1987). The a subunit was released in a water-soluble form upon heating of outer membranes to 60°C and was purified to apparent homogeneity by gel filtration and ion-exchange chromatography. The purified protein was acidic (pl 4.6) and had a polarity of 49.2%. The N-terminal sequence showed homology with the N termini of certain enterobacterial fimbrial subunits. In addition, antigen 43 underwent a reversible phase variation similar to that of type 1 fimbriae. By use of subunit-specific antisera, it was shown that the purified a subunit was capable of reassociating with the 0i polypeptide. However, electron microscopic examination indicated that antigen 43 does not form a recognizable surface structure. The available evidence supports the view that antigen 43 is a complex consisting of a peripheral membrane protein (at) anchored to a subunit (Ii) that is integral to the outer membrane.Extensive immunochemical studies have established a reference profile for Escherichia coli ML308-225 envelope components resolved by crossed immunoelectrophoresis (CIE; 37-40, 43). Antigen 43 is often prominent in the array of precipitin arcs generated when outer membranes are analyzed by this technique. In a previous paper (41), we described the initial characterization of this antigen as a unique protein complex in the outer membrane. The antigen complex contains two chemically and immunologically different polypeptides, ot and 1, with Mrs of 60,000 and 53,000, respectively. These occur in equal stoichiometry. The polypeptide is heat modifiable and shows an apparent Mr of 37,000 when solubilized at temperatures lower than 70°C before analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Analysis by CIE performed in conjunction with zymogram staining and radiolabeling failed to detect the presence of enzyme activity, fatty acyl groups, or other cofactors for the complex (41).Antigen 43 provokes interest for a number of reasons. The complex differs from the well-characterized proteins of the outer membrane of E. coli in that it consists of two different subunits (31, 41). In addition, the antigen is surface exposed, and its yield varies in a manner which appears to be independent of external growth conditions. In this communication, we report on the purification and properties of the a subunit of this unique antigen complex.(Preliminary accounts of this work have been presented

Proteolytic Processing Is Not Essential for Multiple Functions of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)

“…This explains why the heat extraction, which is performed at 60°C, results in the release of a mostly unfolded mature protein. In previous studies, the mature forms of AIDA-I or Ag43 were purified from heat extracts by gel filtration rather than the IMAC step that we used (7,29). Presumably, the former procedure can separate correctly folded from denatured protein whereas the latter does not.…”

“…Like TibA and AIDA-I, Ag43 is glycosylated and mediates adhesion to cultured epithelial cells (49), has a similar 19-amino-acid repeat, and promotes autoaggregation (44). Furthermore, like AIDA-I, Ag43 is cleaved by an unknown mechanism after its outer membrane translocation and is not known to mediate invasion (7).…”

Type V Protein Secretion Pathway: the Autotransporter Story

“…As such, antigen 43 was one of the first autotransporters identified, however, it was not until much later that its mode of biogenesis was uncovered (204). In the intervening period, much time was invested in the biochemical characterization of this protein; Owen and coworkers (54,370) demonstrated that antigen 43 existed as a bipartite complex of two proteins (termed ␣ 43 and ␤ 43 ) which could be resolved under denaturing conditions. These subunit proteins were found to exist in a stoichiometric fashion at a ratio of 1:1, and the interaction between the two proteins was noncovalent.…”

Virulence Functions of Autotransporter Proteins

“…The biological function of Ag43 is controversial. In addition to its phase variability, Ag43 displays several properties common to adhesins, as follows: (i) Ag43 possesses two RGD motifs of the type implicated in binding to human integrins, (ii) like many fimbrial subunits the passenger domain (␣ 43 ) can be released from E. coli outer membranes by brief heating to 60°C, (iii) Ag43 displays sequence homology with several known adhesins, (iv) Ag43 mediates bacterial aggregation, and (v) Ag43 confers a low level of adhesion to certain mammalian cells (18,71,74,115). The levels of adhesion to mammalian cells may not be biologically significant (119), but Ag43-mediated autoaggregation apparently contributes to biofilm formation and may enhance colonization of the mammalian intestine.…”