title: Synthesis and immunological studies of alpha-conotoxin chimera containing an immunodominant epitope from the 268-284 region of HSV gD protein.
abstract: We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrophilic and adopts a beta-turn. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, stabilized by two disulfide bridges at positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, 8Arg-His-Tyr-Ser12 has been replaced by Asp-Pro-Val-Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3-13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys2-Cys7 three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 M HCl or Tl(tfa)3 in trifluoroacetic acid (TFE)] were compared. The high-performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha-[Tyr1]-conotoxin chimeric peptide and native alpha-conotoxin GI showed similar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type antibody responses showed that the bicyclic HSV-alpha-[Tyr1]-conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV-alpha-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha-[Tyr1]-conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.
authors.affiliation: Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, Budapest, Hungary.
authors.forename: G
authors.initials: G
authors.lastname: Mezo
authors.forename: E
authors.initials: E
authors.lastname: Drakopoulou
authors.forename: V
authors.initials: V
authors.lastname: Paál
authors.forename: E
authors.initials: E
authors.lastname: Rajnavölgyi
authors.forename: C
authors.initials: C
authors.lastname: Vita
authors.forename: F
authors.initials: F
authors.lastname: Hudecz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002942:Circular Dichroism; D020916:Conotoxins / Q000138:chemical synthesis* / Q000235:genetics / Q000276:immunology*; D016056:Immunodominant Epitopes / Q000235:genetics*; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D020916:Conotoxins; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; C029960:conotoxin GI