ABSTRACT
The GafD lectin of the G (F17) fimbriae of diarrhea-associatedEscherichia coli was overexpressed and purified from the periplasm of E. coli by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named ΔGafD. Expression of gafD from the clonedgaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of ΔGafD. The peptide was also detected in the periplasm of the wild-type E. coli strain from which the gafgene cluster originally was cloned. We expressed gafDfragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of ΔGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays usinggafD indicated that ΔGafD was processed from GafD and is not a primary translation product. The ΔGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [14C]ΔGafD to GlcNAc-agarose. ΔGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when gafD is expressed alone or from the gaf gene cluster.
Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.