A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Altered protoxin activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia interpunctella.

Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

A laboratory-selected colony of Heliothis virescens displaying a 20- to 70-fold level of resistance to Bacillus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains of H. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2- to 4-fold reduction in binding affinity was accompanied by a 4- to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryIIIA, nor did these proteins compete with the CryIA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli.

Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.

Reaction of antithrombin III with thrombin bound to the vascular endothelium. Analysis in a recirculating perfused rabbit heart preparation.

A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin III (ATIII) with thrombin bound to the surface of the microvascular endothelium. Addition of ATIII to the system after thrombin is equilibrated with its binding sites results in inhibition of the enzyme as measured by disappearance of thrombin enzymatic activity from the circulation or by appearance of 125I-thrombin-ATIII complexes. The rate of inhibition of thrombin as reflected by either method is independent of the bound state of thrombin. Comparable results are obtained with ATIII modified at a single tryptophan residue. This modification does not alter the reaction rate of ATIII with thrombin but abolishes the capacity of heparin or heparan sulfate to enhance the reaction rate. From the kinetics and structural studies and the fit of the kinetics to a theoretical model relating binding equilibrium to thrombin inhibition, it is concluded that glycosaminoglycans are not involved in the reversible, high affinity, high capacity binding of thrombin to the vascular endothelium.