Monoclonal antibodies against the Tn-specific isolectin B4 from Vicia villosa seeds: characterization of the epitope of the blocking antibody VV34.
Vicia villosa isolectin B4 (VVLB4) recognizes the Tn antigen (GalNAc-O-Ser/Thr) exposed in certain human carcinomas. We have produced anti-VVLB4 monoclonal antibodies (MAbs), and their lectin recognition selectivity was assessed by ELISA and Western blot against the purified Gal/GalNAc-specific lectins from Vicia villosa, Salvia sclarea, Helix pomatia, Arachis hypogaea, Glycine max, and Dolichos biflorus. The antibodies were also tested for their ability to block the binding of VVLB4 to the Tn antigen expressed on immobilized asialo ovine submaxillary mucin. Two MAbs, VV34 and VV2, specifically recognized VVLB4 and impaired the binding of the lectin to the Tn antigen by 98% and 21%, respectively. On the other hand, MAbs VV1 and VV22 cross-reacted with other purified lectins. The four antibodies recognized native and periodate-oxidized nonreduced as well as reduced VVLB4 after SDS-PAGE and Western blot, suggesting that they were recognizing continuous polypeptide epitopes. The VV34 antibody recognized two tryptic peptides (7-29 and 96-106) from VVLB4, which are contiguous in the three-dimensional structure of the lectin. The minimum structural determinant of the epitope was mapped to the polypeptide stretch (18)LILQED(23) using a set of overlapping synthetic peptides. This region of the molecule encompasses the C-terminal part of the loop joining strands beta1 and beta2 and the N-terminal part of beta2, and is located about 20-25 A away from the center of the Tn-combining site.
Medeiros
Andrea
A
Dept. de Bioquímica, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay.
Berois
Nora
N
Balter
Henia
H
Robles
Ana
A
Perez-Payá
Enrique
E
Gimenez
Ana
A
Calvete
Juan J
JJ
Osinaga
Eduardo
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D037102:Lectins / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D012639:Seeds / Q000737:chemistry*; D031306:Vicia / Q000737:chemistry* / Q000196:embryology
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes; D037102:Lectins; C040286:Tn antigen