Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Abstract

SummaryPertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The $1 subunit is responsible for the ADPribosyltransferase activity of the toxin. The B moiety, composed of $2 through $5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits $2 and $3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Ash 105 in $2 and of Lys 105 in $3 resulted in the more drastic reduction of binding to haptogiobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the $1 subunit led to the production of a toxin analog with reduced ADPoribosyltransferasedependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monodonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the $1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.tel/a pertuss/s, the etiologic agent of whooping cough, duces a number of toxins responsible for the local and systemic manifestations of this important childhood disease (1). Among these toxins, pertussis toxin (PTX) 1 plays a major role in the pathogenesis, in that it expresses a wide variety of biological activities, such as histamine sensitization, activation of insulin secretion, promotion of lymphocytoffs, and others (2). In addition, immunization with PTX has been shown to protect mice against subsequent lethal challenge with virulent R pertuss/s (3). It is therefore considered an important constituent in vaccines against pertussis, and is the major component of the new acellnlar vaccines currently being tested or used in several countries (4,5).Paradoxically, this toxin may itself be responsible for the harmful side effects associated with the current vaccines (6). These side effects range in severity from minor local reactions and simple flushing, to permanent neurological damage t Abbre~aons used in this paper: CRAM, cross-reactive mutant; NAD, nicotinamide adenine dinudeotide; FTX, txn'tussis toxin; WT, wild type. and, in some rare cases, death. Before PTX can effectively and safely be used in pertussis vaccines, complete and irreversible detoxification is therefore required.The toxin is composed of five different subunits, named $1 to $5, based on their decreasing molecular weights. The subunits associate in a molar ratio of 1S1:1S2:1S3:2S4:1S5 to form the holotoxin. Functionally, FTX can be...

Bordetella protein toxins

“…The C-terminal half of S2 and of S3 adopt a fold similar to that found in the B subunits of other carbohydrate-binding toxins, and the sialyl group of the oligosaccharide bound to PTX is within hydrogen-bonding distance of the polar and charged groups of Tyr-102, Ser-104, and Arg-125 in both S2 and S3 [44,45]. Site-specific alterations of the residues in that region abolish receptor binding of PTX [45,46]. Interestingly, deletion of Asn-105 in S2 affects haptoglobin binding of the toxin, while keeping its ability to bind to CHO cells intact, whereas the deletion of Lys-105 in S3 has the inverse effect [45], indicating that these residues contribute to receptor-binding specificity.…”

“…Site-specific alterations of the residues in that region abolish receptor binding of PTX [45,46]. Interestingly, deletion of Asn-105 in S2 affects haptoglobin binding of the toxin, while keeping its ability to bind to CHO cells intact, whereas the deletion of Lys-105 in S3 has the inverse effect [45], indicating that these residues contribute to receptor-binding specificity. Other regions, in particular in the N-terminal domain of these subunits, may also be involved in receptor recognition [47].…”

“…However, a few, such as hemagglutination and mitogenicity, are independent of the ADP-transfer reactions, but depend on certain target-cell receptor-binding properties of PTX [45,46]. Some of the biological activities of PTX are hallmarks of whooping cough, which has led to the proposal that pertussis is essentially a toxin-mediated disease, similar to tetanus and diphtheria [12].…”

“…Although both dimers are able to bind to sialocarbohydrates in essentially the same manner, they nevertheless differ in their receptor specificity. For example, Dimer S2/S4 is able to bind to haptoglobin, whereas dimer S3/S4 binds to glycoproteins at the surface of Chinese hamster ovary (CHO) cells [45]. The C-terminal half of S2 and of S3 adopt a fold similar to that found in the B subunits of other carbohydrate-binding toxins, and the sialyl group of the oligosaccharide bound to PTX is within hydrogen-bonding distance of the polar and charged groups of Tyr-102, Ser-104, and Arg-125 in both S2 and S3 [44,45].…”

Investigating Pertussis Toxin and its Impact on Vaccination

“…The crystal structure of PTX bound to a undecasaccharide revealed at least two carbohydrate-binding sites on the S2 and S3 subunits, respectively (Figure 1) [50]. However, the two binding sites display a certain degree of receptor-binding specificities [51]. Surface plasmon resonance studies using carbohydrate ligands showed that binding to sialylated compounds is mediated by the C-terminal binding sites on the S2 and S3 subunits, while binding to nonsialylated N-linked glycans is mediated by their N-terminal sites [52].…”