The role of the individual amino acids of a GnRH-tandem-dimer peptide used as an antigen for immunocastration of male piglets determined with systematic alanine replacements

Abstract

Immunocastration targeting gonadotropin releasing hormone (GnRH) can be obtained in male piglets using native GnRH conjugates. However, due to insufficient efficacy of these conjugates, improved GnRH antigens, like peptides existing of repeats of the GnRH amino acid sequence, have been designed. We previously reported about a dimerised GnRH-tandem peptide with a d-Lys at position 6 of the native GnRH sequence (G6k-TD) being highly effective. To evaluate the contribution of each individual amino acid of the GnRH decapeptide to the efficacy of the G6k-TD peptide, each amino acid was replaced consecutively by alanine (Ala-scan). The G6k-TD peptides were conjugated to ovalbumin, used for immunisation and tested for their ability to elicit GnRH antibodies and to immunocastrate male piglets. The results show that four out of nine amino acids (pGlu-1, Ser-4, Arg-8 and Gly-10) can be replaced by alanine without negatively affecting immunocastration efficacy. Replacement of amino acids in other positions (Tyr-5, Leu-7 and Pro-9) gave partial decrease of efficacy, respectively, five, six and six out of seven piglets were immunocastrated. Replacements at two other positions (His-2 and Trp-3) completely negated immunocastration activity. Thus, seven out of nine amino acid positions in the basic unit of G6k-TD can be substituted by alanine without affecting immunocastration efficacy.