Synthesis and antibody recognition of mucin 1 (MUC1)-alpha-conotoxin chimera.
We synthesized and characterized new chimera peptides by inserting an epitope of the mucin 1 glycoprotein (MUC1) as a 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated 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 Thus, the tetramer sequence of alpha-conotoxin, Arg9-His-Tyr-Ser12, has been replaced by PDTR, comprising the minimal epitope for MUC1 specific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 (DTR). Synthesis of the chimera peptide was carried out by Fmoc strategy on (4-(2',4'-dimethoxyphenyl-aminomethyl)phenoxy) (Rink) resin and either 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation of the first Cys3-Cys13 or Cys2-Cys7 disulphide bridge, respectively. For the second disulphide bridge, three different oxidation procedures (iodine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa)3) in TFA) were utilized. The HPLC purified peptides were characterized by electrospray mass spectrometry (ES-MS) and amino acid analysis. The CD spectra of the bicyclic MUC1-alpha-[Tyr1]-conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding studies, the RIA data showed that both the linear and the bicyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera were recognized by MAb HMFG1 specific for PDTR sequence, while no binding was observed between MAb HMFG2 and various forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or native MUC1 as target antigens, recognizes the PDTR motif more efficiently in the linear than in the bicyclic compound, but no reactivity was found with the monocyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera, underlining the importance of certain conformers stabilized by double cyclization.
Drakopoulou
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E
Département d'Ingénierie et d'Etudes des Proteines, CEA, Saclay, Gif-sur-Yvette, France.
Uray
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K
Mezö
G
G
Price
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MR
Vita
C
C
Hudecz
F
F
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D020916:Conotoxins / Q000138:chemical synthesis* / Q000276:immunology*; D000939:Epitopes; D018396:Mucin-1 / Q000737:chemistry / Q000276:immunology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000276:immunology; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D020916:Conotoxins; D000939:Epitopes; D018396:Mucin-1; D010456:Peptides, Cyclic; D011994:Recombinant Proteins