Preparation of MUC-1 oligomers using an improved convergent solid-phase peptide synthesis.
The sequentially repeating nature of the core mucin polypeptide chain MUC-1 on the surface of malignant cells makes it an excellent target for cancer immunotherapy. We describe a reliable and efficient method of synthesizing oligomers, up to five tandem repeats and oligomer heterotope derivatives with a 15-amino acid epitope from tetanus toxin using an improved convergent solid-phase peptide synthesis. The different oligomers were easily distinguishable by reverse-phase high pressure liquid chromatography, but they were poorly fixed and migrated with the same migration rate, irrespective of size, in electrophoretic studies. In contrast, the oligomer heterotopes exhibited size-dependent electrophoretic behavior but in high pressure liquid chromatography chromatograms the different heterotopes were eluted simultaneously in two peaks representing the L- and D-enantiomers of the derivatives. The oligomer heterotopes were recognized as antigens in Western blotting with a murine monoclonal antibody against the epitope APDTR. In enzyme immunoassay studies with the same antibody an increasing reactivity was observed against the larger oligomers and confirmed by inhibition assays as the MUC-1 pentamer was the most efficient inhibitor. These results support the suggestion that the pentamer attains a structure closer to the native conformation and is more immunogenic. In conclusion, large composite peptides can be reliably synthesized with the convergent solid-phase peptide strategy offering an attractive option to vaccine designing and development.
Krambovitis
E
E
Department of Applied Biochemistry and Immunology, Institute of Molecular Biology and Biotechnology, P. O. Box 1527, Heraklion GR 71110, Crete, Greece. Krambo@nefeli.imbb.forth.gr
Hatzidakis
G
G
Barlos
K
K
D000595:Amino Acid Sequence; D001704:Biopolymers; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry*; D013237:Stereoisomerism; D013744:Tetanus Toxin / Q000737:chemistry
D001704:Biopolymers; D000939:Epitopes; D018396:Mucin-1; D013744:Tetanus Toxin