How Protein Recognizes Ladder-like Polycyclic Ethers

Abstract

Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein.Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 Å , respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10°C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.Ciguatoxins are a family of ladder-like polycyclic ethers that causes ciguatera seafood poisoning in tropical and subtropical regions; more than 50,000 people suffer annually from this type of food poisoning (1-7). CTX3C, which contains 13 ether rings, is probably the best known the member of polycyclic ether family. Although CTX3C is known to bind to voltage-sensitive sodium channels, like other polycyclic ether toxins (8 -10), the precise pathogenic mechanism remains to be elucidated, and methods for the treatment of ciguatera poisoning have not yet been developed (5).Oguri et al. (11) established a direct sandwich enzyme-linked immunosorbent assay using two distinct monoclonal antibodies (10C9 and 3D11) to detect CTX3C at the parts per billion level with no cross-reactivity against other related marine toxins. 10C9 IgG is prepared by immunization of mice with a carrier protein (keyhole limpet hemocyanin (KLH) 3 ) conjugated to the ABCDE-ring of CTX3C. 10C9 IgG binds not only to CTX3C-ABCDE but also to full-length CTX3C (Fig. 1), with dissociation constants of 0.8 and 2.8 nM, respectively. Because the skeletons of polycyclic ethers are so rigid and because the compounds are so large (12-15) (they extend beyond the binding surface of the paratope of the antibody (200 -400 Å 2 ) (16 -21)), how the antibody recognizes the polycyclic ethers within the limited areas of the antigen-bindin...

Update on Methodologies Available for Ciguatoxin Determination: Perspectives to Confront the Onset of Ciguatera Fish Poisoning in Europe

“…In a CIEA study, three carefully selected rFabs displayed K D values for free ABCD in the range of 2.4 × 10 −5 M to 5.0 × 10 −5 M. Moreover, rFab 1C49, exhibiting the highest affinity for ABCD, was also found to react to CTX-3C, though to a lesser extent. These last experiments and other studies [211,214] confirmed the importance of using more than four-ring fragments as synthetic haptens in designing valuable hapten-carrier conjugates for the production of high affinity and specific MAbs to CTX congeners.…”

“…Up to now, in spite of many attempts, there is no fully validated immunoassay screening that is commercially available for ciguateric fish, but promising results have been obtained by Hirama’s group in Japan [207–214]. As a general rule, in the course of producing Abs specific to Ags in laboratory animals, several critical steps must be carefully evaluated and planned such as: (i) the preparation and analysis of the Ags; (ii) the selection of the animal species in order to raise either only PAbs (e.g., sheep or rabbit) or PAbs plus MAbs (e.g., Balb/c mice); (iii) the selection of the adjuvant (e.g., Freund’s adjuvant); (iv) the development of an immunization schedule ( i.e., determine the dose, volume, route, number and site of injection, and intervals between injections); and (v) the monitoring of the Ab response (e.g., ELISA format assays).…”

“…Following this first demonstration of the relevance of using synthetic ring fragments of CTX-1B instead of the natural congeners, Hirama’s group [207–214] pursued their synthetic work to prepare suitable immunogenic conjugates of carboxylic derivatives of various CTX ring fragments via the bulk activated ester method. Analysis of the conjugates was performed by MALDI-TOFMS and the epitope density of the conjugates was always found to be compatible with good PAb response and production of MAbs in Balb/c mice.…”

Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

“…This result suggests that mAb 10C9 recognizes the entire pentacyclic skeleton (ABCDE ring) of CTX3C, and that the surface area required for molecular recognition of 10C9 is consistent with the prediction of 400 Å 2 . Probable recognition of mAb 10C9 and the pentacyclic ring moiety (ABCDE ring) of CTX3C was confirmed by crystal structure analysis of 10C9 complexed with ABCDE ring 15 [65] .…”

Total synthesis and related studies of large, strained, and bioactive natural products

“…6). 71,72) Antibody 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCDE fragment binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragment by means of rotational motion in the variable region, and furthermore its recognition requires molecular rearrangements over the entire antibody structure.…”