Antigenic analyses of Sugi basic protein by monoclonal antibodies: II. Detection of immunoreactive fragments in enzyme-cleaved Cry j I.
The 4 anti-Cry j I mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry j I molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry j I N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser- Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry j I or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry j I molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.
Kawashima
T
T
Hayashibara Biochemical Laboratories, Okayama, Japan.
Taniai
M
M
Usui
M
M
Ando
S
S
Kurimoto
M
M
Matuhasi
T
T
D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D010450:Endopeptidases / Q000494:pharmacology; D000939:Epitopes; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology; D011058:Pollen / Q000737:chemistry / Q000276:immunology; D011489:Protein Denaturation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014197:Trees
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C065290:Cry j I protein, Cryptomeria japonica; D000939:Epitopes; D010446:Peptide Fragments; D010940:Plant Proteins; D010450:Endopeptidases