Synthesis and antibody recognition of mucin 1 (MUC1)-alpha-conotoxin chimera

We synthesized and characterized new chimera peptides by inserting an epito pe of the mucin 1 glycoprotein (MUC1) as a 'guest' sequence in the 'host' s tructure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolat ed from the venom of Conus geographus. The Pro-Asp-Thr-Arg (PDTR) sequence of MUC1 selected for these studies is highly hydrophilic and adopts a beta- turn conformation. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, which is stabilized by two disulphide bridges in positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, Arg(9)-His-Tyr-S er(12). has been replaced by PDTR comprising the minimal epitope for MUC1 s pecific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 [DTR]. Synthesi s of the chimera peptide was carried out by Fmoc strategy on (4-(2',4'-dime thoxyphenyl-aminomethyl)phenoxy) (Rink) resin and either 5,5'-dithio-bis-(2 -nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation o f the first Cys(3)-Cys(13) or Cys(2)-Cys(7) disulphide bridge, respectively . For the second disulphide bridge, three different oxidation procedures (i odine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa) (3)) in TFA) were utilized. The HPLC purified peptides were characterized b y electrospray mass spectrometry (ES-MS) and amino acid analysis. The CD sp ectra of the bicyclic MUCl-alpha-[Tyr(1)]-conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding stu dies, the RIA data showed that both the linear and the bicyclic forms of MU C1-alpha-[Tyr(1)]-conotoxin chimera were recognized by MAb HMFG1 specific f or PDTR sequence, while no binding was observed between MAb HMFG2 and vario us forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or n ative MUC1 as target antigens, recognizes the PDTR motif more efficiently I n the linear than in the bicyclic compound, but no reactivity was found wit h the monocyclic forms of MUC1-alpha-[Tyr(1)]-conotoxin chimera, underlinin g the importance of certain conformers stabilized by double cyclization. Co pyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.