A new antigenic epitope appears in the catalytic subunit of viscumin during intracellular transport.
The plant toxin viscumin (60 kD) consists of B- ("binding") and A- ("active") subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody-toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody-toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of (1)H-NMR spectroscopy.
School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia. tonevits@genetika.ru
A G
AG
Tonevitsky
I I
II
Agapov
M M
MM
Moisenovich
N V
NV
Maluchenko
V S
VS
Pashkov
T A
TA
Balashova
M P
MP
Kirpichnikov
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D020134:Catalytic Domain; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D037121:Plant Lectins / Q000737:chemistry / Q000276:immunology; D028321:Plant Preparations / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D010940:Plant Proteins; D011487:Protein Conformation; D054790:Ribosome Inactivating Proteins, Type 2; D012276:Ricin / Q000494:pharmacology; D014118:Toxins, Biological / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum; D012276:Ricin