Preparation of MUC-1 Oligomers Using an Improved Convergent Solid-phase Peptide Synthesis

Abstract

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.

V3 Induces in Human Normal Cell Populations an Accelerated Macrophage-Mediated Proliferation- Apoptosis Phenomenon of Effector T Cells When They Respond to Their Cognate Antigen

“…The HIV-1 gp120 peptides V3 (LAI strain, a.a. 304 -318) RKSIRIQRGPGRAFY, C4 (CD4 receptor, binding site) SFINMWQEVGKAMYAPPISG, tetanus toxin peptide (a.a. 830 -844) QYIKANSKFIGITEL and the control mucin core antigen MUC-1 PAHGVTSAPDTRPAPGSTAP were synthesized using F-moc/tBu chemistry (13). These were converted to lipopeptides according to the manufacturer's instructions by covalent binding of serine-S-[2,3-bis(palmitoyloxy)-(2rs)-propyl]-n-palmitoyl-(r)-cysteine, (Boehringer Mannheim Biochemica, Germany) to the above peptides.…”

Dys-Regulation of Effector CD4+ T Cell Function by the V3 Domain of the HIV-1 gp120 during Antigen Presentation

“…284, No. 4,2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS sure. The phenomenon was more pronounced when CD4ϩ T cells were primary-activated with antigen ( Fig.…”

Molecular Evolution of Specific Human Antibody against MUC1 Mucin Results in Improved Recognition of the Antigen on Tumor Cells

“…These peptides were all obtained from Thermo Electron (Ulm, Germany). A biotinylated peptide made up of three TR of MUC1 (biotin-ala 3 -MUC1 3 ; biotin-ala 3 -[GVTSAPDTRPAP GSTAPPAH] 3 ) was kindly provided by Elias Krambovitis (Institute of Molecular Biology and Biotechnology, Heraklion, Greece) [28] .…”