A New Antigenic Epitope Appears in the Catalytic Subunit of Viscumin during Intracellular Transport

A. G. Tonevitsky^1*, I. I. Agapov¹, M. M. Moisenovich¹, N. V. Maluchenko¹, V. S. Pashkov², T. A. Balashova², and M. P. Kirpichnikov¹

¹School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 196-0522; E-mail: tonevits@genetika.ru

²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia; fax: (095) 335-2733; E-mail: aars@siobc.ras.ru

^* To whom correspondence should be addressed.

Received November 10, 2002; Revision received December 17, 2002

---

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 ¹H-NMR spectroscopy.

---

KEY WORDS: viscumin, ricin, monoclonal antibodies, epitopic analysis, ¹H-NMR spectroscopy

---