In vitro digestion of Cry1Ab proteins and analysis of the impact on their immunoreactivity.
A pepsin resistance test performed at pH 1.2 and with high pepsin to protein ratio is one of the steps of the weight-of-evidence approach used for assessment of allergenicity of new proteins. However, the use of other in vitro digestibility tests, performed in more physiologically relevant conditions and in combination with immunological assays so as to increase the value of the information gained from the studies of stability of a novel protein to digestion for the overall allergenicity assessment, has been proposed. This study then aimed to investigate the stability to digestion of Cry1Ab protoxin and toxin, insecticidal proteins expressed in genetically modified crops, using simulated gastric fluid (SGF) at different pH values and pepsin-to-substrate ratios, in the presence or absence of physiological surfactant phosphatidylcholine (PC). Electrophoresis and immunoblot patterns and residual immunoreactivity of digesta were analyzed. Although Cry1Ab protoxin is extensively degraded at pH 1.2 with high pepsin-to-protein ratio, it is only slightly degraded at pH 2.0 and conserved its immunoreactivity. Furthermore, Cry1Ab proteins were demonstrated to be stable in a more physiologically relevant in vitro digestibility test (pH 2.5, pepsin-to-substrate ratio 1:20 (w/w) with PC). Factors such as pH, SGF composition, and pepsin-to-substrate ratio then greatly influence the digestion of Cry1Ab proteins, confirming that new and more physiologically relevant in vitro digestibility tests should be also considered to study the relationship between the resistance of a protein to digestion and its allergenicity.
Guimaraes
Valeria
V
INRA, UR496, Laboratoire d'Immuno-Allergie Alimentaire, F-78352 Jouy-en-Josas, France.
Drumare
Marie-Françoise
MF
Lereclus
Didier
D
Gohar
Michel
M
Lamourette
Patricia
P
Nevers
Marie-Claire
MC
Vaisanen-Tunkelrott
Marie-Lisa
ML
Bernard
Hervé
H
Guillon
Blanche
B
Créminon
Christophe
C
Wal
Jean-Michel
JM
Adel-Patient
Karine
K
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013379:Substrate Specificity
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis