Kazuko Shimazaki

Improved generation of catalytic antibodies by MRL/MPJ-lpr/lpr autoimmune mice.

To compare the abilities of different strains of mice to elicit catalytic antibodies (Abs), we determined the occurrence of esterolytic Abs in BALB/c (normal strain) and MRL/MPJ-lpr/lpr (MRL/lpr, autoimmune) mice after immunization with the transition state analog (TSA) 1. Hybridoma supernatants elicited against TSA 1 were screened by ELISA for binding to the BSA-conjugated TSA 1 (=3b), and then screened for binding to the BSA-linked short TSA 2 (=4). We obtained eight times more positives from MRL/lpr mice than from BALB/c mice by these screening steps. The monoclonal antibodies (mAbs) obtained here were examined for binding and catalytic activity. Fifteen of 25 mAbs from MRL/lpr had esterolytic activity, compared with only two of 21 mAbs from BALB/c. These results demonstrated that the occurrence of catalytic Abs was much higher in MRL/lpr mice than in BALB/c mice, which is in good agreement with the previous report by Tawfik et al. [Tawfik, D.S., Chap, R., Green, B.S., Sela, M., Eshhar, Z., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 2145-2149] using a different kind of TSA. Thus, these studies strongly suggest that using the appropriate strain can be a key factor in the efficient production of catalytic Abs. Furthermore, these mAbs were characterized to elucidate the mechanism of strain difference, and determine whether MRL/lpr mice can be used with other TSAs for the efficient production of catalytic Abs.

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.

High-resolution crystal structure of the Fab-fragments of a family of mouse catalytic antibodies with esterase activity

The crystal structures of four related Fab fragments of a family of catalytic antibodies displaying differential levels of esterase activity have been solved in the presence and in the absence of the transition-state analogue (TSA) that was used to elicit the immune response. The electron density maps show that the TSA conformation is essentially identical, with limited changes on hapten binding. Interactions with the TSA explain the specificity for the D rather than the L-isomer of the substrate. Differences in the residues in the hapten-binding pocket, which increase hydrophobicity, appear to correlate with an increase in the affinity of the antibodies for their substrate. Analysis of the structures at the active site reveals a network of conserved hydrogen bond contacts between the TSA and the antibodies, and points to a critical role of two conserved residues, HisL91 and LysH95, in catalysis. However, these two key residues are set into very different contexts in their respective structures, with an apparent direct correlation between the catalytic power of the antibodies and the complexity of their interactions with the rest of the protein. This suggests that the catalytic efficiency may be controlled by contacts arising from a second sphere of residues at the periphery of the active site.

Comparison of phosphonate transition state analogs for inducing catalytic antibodies and evaluation of key structural factors by an ab initio study

The relation between the structure of haptens and the esterolytic activities of antibodies was investigated. We synthesized two phenylalanine analogs, the negatively charged phosphonate derivative 1 and the neutral phosphonamidate derivative 2. Seventeen out of 41 monoclonal antibodies generated against the hapten 1 hydrolyzed the relevant phenylalanine ester R-12. On the contrary, none of 27 monoclonal antibodies generated against the hapten 2 had catalytic activity. An ab initio study of the structural and electronic properties of the modeled haptens showed that the value of the negative electrostatic potential around the phosphonyl oxygen was an important factor affecting the induction of esterolytic antibodies.

Behavioral responses of malePeriplaneta americana L. to female sex pheromone components, periplanone-A and periplanone-B.

Bioassays were performed on malePeriplaneta americana L. using synthetic (−)-periplanone-B (P-B) and Hauptmanns (−)-periplanone-A (P-A), and their mixtures at various ratios to estimate the roles of both periplanones for the sexual communication of the species. Both P-A and P-B elicited qualitatively the same responses, such as antennal movement, rapid locomotion, wing raising, and homosexual behavior of male cockroaches, but the threshold of the pheromone activities for P-B was two orders of magnitude lower than that of P-A. Neither synergistic nor inhibitory but only a simple integrated effect on the responses was observed when mixtures of P-A and P-B were applied.

Behavioral and electroantennogram responses of periplanone analogs

The biological activities of synthetic periplanone analogs, including four candidates of periplanone-A (P-A), were evaluated by behavioral and electroantennogram (EAG) assays. Among 16 periplanone analogs, six compounds evoked pheromonal activity from the male American cockroaches. The threshold dosages of these biological active analogs were 10–105 times lower than that of the known periplanone mimic, germacrene-D. The conformation required for eliciting the pheromonal activity is discussed in terms of the structure-activity relationship of these analogs. Hauptmanns P-A elicited the strongest pheromonal activity among four candidates of P-A in our bioassay, suggesting that Hauptmanns P-A is a natural P-A produced from female cockroaches.

Olfactory responses to the sex pheromone component and its behavioural inhibitor in the male cigarette beetle, Lasioderma serricorne

Abstract Electroantennogram and single sensillum recordings were performed on the male cigarette beetle, Lasioderma serricorne , antennae. Strong antennal responses were elicited by not only natural sex pheromone component (4S,5S,7S)-serricornin (SSS-serricornin) but also non-natural behavioural inhibitor, (4S,5S,7R)-serricornin (SSR-serricornin). In the case of mixture application of the SSS- and SSR-serricornin, integrated electroantennogram responses were observed. Single sensillum recording from male flagella sensilla showed that the majority of basiconic type sensilla responded to the behavioural inhibitor as well as to the sex pheromone. However, a few of them responded only to the inhibitor. The behavioural inhibitory action of SSR-serricornin toward the SSS-serricornin results from central integration of the information from separate receptor cells rather than blockage at the peripheral receptors.

Synthesis of a simple analog of periplanone-B

The synthesis of a simple analog of periplanone-B with high biological activity is described mainly on the examination of the conformational property of the key intermediate 10-t-butoxy-6-methyldeca-2,6-dienone by molecular mechanics calculation and dynamic NMR spectroscopy

X-ray crystallographic and NOE studies on the conformation of periplanones and their analogues

The structure of periplanone-A was established by X-ray crystallographic analysis. In comparison of the X-ray and 1H NMR data with those of periplanone-B, it was shown that periphanone-A and -B adopt essentially the same conformation. The epoxy epimer of periplanone-A has l04-times lower biological activity than periplanone-A, and NMR analysis indicated that the molecule exists in a mixture of different conformers. The conformation of the regioisomer, in which the carbonyl and epoxy groups are transposed, was analysed by X-ray crystallography and 1H NMR spectroscopy. It is highly biologically active, and the conformation of the germacranoid skeleton was shown to be almost identical with that of periplanone-A. Simplified analogues and germacrene-D had relatively lower activity. NOE Experiments on these compounds suggested the conformational resemblance of their germacranoid skeletons with those of periplanone-A and -B.

Combined molecular mechanics (MM2) and molecular orbital (AM1) study of periplanone-B and analogues. Evaluation of biological activity from electronic properties and geometries

Combined molecular mechanics (MM) and semiempirical molecular-orbital (MO) calculations have been applied to the investigation on the conformational and electronic properties of periplanone-B (1), a major component of the sex pheromone of American cockroach, and structurally related analogues 2, 3, 4 and 5. In the first step, the geometries of conformers of 1–5 were obtained by MM2 combined with a new algorithm for exhaustive generation of ring conformations, CONFLEX2, and an additional set of MM2 force-field parameters. The global minimum (1A) of the natural pheromone populates predominantly and is superimposable on the X-ray structure. Minor conformers 1B and 1C correspond to the rotamers at the isopropyl group of 1A. The global minimum of the analogue 2 was identified to have the structure 2A, which agrees well with the X-ray data. The structural comparison of the stable conformers of the analogues 2–5 with 1A revealed similar ring conformations in the most stable conformers of 2, 3 and 4, and the third one of 5. These results suggest that the ring structure characteristic of the conformer 1A and the conformer populations must be a significant factor in the biological activity of these analogues. In the next step, the electronic properties were calculated by the semiempirical MO (AM1) method. A significant correlation is found between the biological activity and one of the unoccupied frontier orbitals which is localized around C1–C10–C9 including the carbonyl and spiro-epoxy groups. A newly defined effective frontier parameter: EF(N)(s), which regards the orbital electron density, orbital energy, and ring conformation is found to correlate well with the biological activity of these analogues.