Pertussis toxin has eukaryotic-like carbohydrate recognition domains.

Abstract

Bordetela peinussis is bound to glycocoijugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite >80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.During the clinical course of whooping cough, Bordetella pertussis specifically attaches to the cilia of the respiratory epithelium (1), establishes an intracellular state within alveolar macrophages (2), and produces systemic disease by elaborating several toxins (3). At least two adhesins, filamentous hemagglutinin and pertussis toxin (PT), are known to mediate bacterial attachment to human cells (4, 5). Both adhesins are required for attachment to cilia, but each can act independently in the adherence of the organism to macrophages. These adhesins have several unusual features of interest. They are large, nonfimbrial molecules that contain multiple binding domains (1, 5) and, when shed from the cell surface, retain the ability to mediate adherence even for other bacterial species (6).PT is a major virulence determinant of B. pertussis, which induces metabolic changes in the host (7), alters immune responses (8), and is the only toxin known to bind whole bacteria to eukaryotic cells (4). The toxin is a hexameric protein with an A-B architecture (9). The A protomer is composed of a single S1 subunit (Mr, 26,026) containing the catalytic site for toxic ADP-ribosylation of cellular signaltransducing guanine nucleotide regulatory proteins. The B oligomer possesses the cellular recognition domains; it is a complex pentamer containing subunits S2 (Me,21,925), S3 (Mr,21,873), S4 (Mr,12,059), and S5 (Mr, 11,013) in a respective molar ratio of 1:1:2:1 (9). By interacting with glycoproteins and glycolipids on many types of eukaryotic cells (4,(10)(11)(12)(13)(14)(15)(16), the B oligomer serves several distinct and independent functions (4, 15): mitogenicity for T lymphocytes, adherence of the bacteria to eukaryotic cells, and delivery of the toxic S1 subunit to its target. Several findings suggest that the B oligomer contains at least two distinct binding specificit...

Localisation of a receptor‐recognition domain on the S3 subunit of pertussis toxin by peptide mapping

“…Both N-acetylneuramin-lactose (1 -80 pM) and the cerebroside lactosylceramide (1 -30 pM), which lacks the Neu and galactosyl(P1-3)N-acetylgalactosamine moieties of the ganglioside, were found not to perturb (E)S3c fluorescence when mixed with the peptide (data not shown). The findings with lactosylceramide are similar to those of Saukkonen et al (1992), who found by TLC that this glycolipid was only recognised by the S2 subunit and not the S3 subunit of PT.…”

“…Previously, this technique has primarily been applied to the identification of peptides which represent subsites of anti-protein serum epitopes (Getzoff et al, 1987). The localised glycoprotein-binding region of S3 is closely analogous to the recently defined region I1 of a carbohydraterecognition domain proposed for the S3 subunit (Saukkonen et al, 1992). The possibility of multiple regions of S3 contributing to receptor binding cannot be excluded on the basis of our peptide-mapping results.…”

“…PT can function as one of the B. pertussis adhesins, which facilitate the first stage of infection by enabling attachment of the bacteria to the cilia of the respiratory tract (Relman et al, 1990;Saukkonen et al, 1992). In addition, several of the systemic disease effects which may result from infection, such as lymphocytosis, enhanced vascular permeability and stimulation of insulin secretion, can be reproduced by PT in experimental models (Ui, 1988).…”

“…The interaction of the B-oligomer with eukaryotic membrane glycoproteins and glycolipids is primarily mediated by subunits S2 and S3 (Capiau et d., 1986;Schmidt et al, 1991), which are both closely associated in the B-oligomer with subunit S4 (Tamura et al, 1982). S2 and S3 differ in their range of receptorbinding specificities, but also possess some receptors in common (Witvliet et al, 1989;Saukkonen et al, 1992). Attachment of the holotoxin to a target cell is considered to be followed by penetration of the target cell membrane by the catalytic S1 subunit (Ui, 1988).…”

“…Optimal binding of PT to glycoprotein depends upon the presence of N-acetylneuraminic acid and lactosamine constituents in the oligosaccharide (Armstrong et al, 1988;Witliviet et al, 1989). Furthermore, the existence of carbohydrate recognising domains in both the S2 and S3 subunits, has recently been c o n f i e d using site-directed mutants of PT (Saukkonen et al, 1992). Since certain carbohydrate-recognising domains of other proteins, such as fibronectin (Pierschbacher and Ruoslahti, 1984), have been mimicked by linear-peptide analogues, we have chosen to assess whether a peptide from the amino acid sequence of the S3 subunit of PT could also bind either glycoprotein or glycolipid.…”