The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.
A solid phase radioimmunoassay was set up for direct measurement of the binding capacity of human IgG to three lectins recognizing different carbohydrates of the Fc domain, i.e. peanut agglutinin (PNA), Concanavalin A (Con A) and pokeweed mitogen (PWM) which mainly bind to beta-galactose, alpha-mannose and dimers of N-acetyl-beta-glucosamine respectively. The mean specific binding of the 96 normal IgG tested to PNA and to PWM was statistically higher (P less than 0.001) than that to Con A, whereas no significant differences were observed between the mean specific bindings to PNA and to PWM. A statistically significant linear negative correlation could be established only between the relative bindings (expressed in percentage of the total binding to the three lectins) to PNA and to PWM (r = -0.65, P less than 0.001). The mean specific binding of IgG purified from 34 patients suffering from rheumatoid arthritis (RA) to PNA and to Con A was statistically higher (P less than 0.001) than that reached with PWM, whereas no significant differences were noted between their mean binding capacities to PNA and to Con A. When compared to normal IgG, only four out of 34 RA IgG exhibited a significantly higher binding capacity to PNA, whereas all but one RA IgG possessed a significantly higher binding capacity to Con A. Accordingly, the mean specific binding of RA IgG to Con A was significantly higher than that of normal IgG (P less than 0.001). Besides (and contrary to normal IgG), a statistically significant negative linear correlation was noted between the relative bindings of RA IgG to PNA and to Con A (r = -0.89, P less than 0.001). All the five RA IgG tested exhibited an abnormal circular dichroism. Our data suggest that, by altered steric conformation and glycosylation, mannosyl-residues of RA IgG become prominent or terminal or both, and are therefore able to react more effectively with Con A than normal IgG do.
