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

Immunocastration targeting gonadotropin releasing hormone (GnRH) can be obt ained in male piglets using native GnRH conjugates. However. due to insuffi cient efficacy of these conjugates. improved GnRH antigens, like peptides e xisting 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 p osition 6 of the native GnRH sequence (G6k-TD) being highly effective. To e valuate the contribution of each individual amino acid of the GnRH decapept ide to the efficacy of the G6k-TD peptide, each amino acid was replaced con secutively by alanine (Ala-scan). The G6k-TD peptides were conjugated to ov albumin. used for immunisation and tested for their ability to elicit GnRH antibodies and to immunocastrate male piglets. The results show that four o ut of nine amino acids (pGlu-1. Ser-4, Arg-8 and Gly-10) can be replaced by alanine without negatively affecting immunocastration efficacy. Replacemen t of amino acids in other positions (Tyr-5, Leu-7 and Pro-9) gave partial d ecrease of efficacy, respectively, five, six and six out of seven piglets w ere 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 w ithout affecting immunocastration efficacy. (C) 2001 Elsevier Science Ltd. All rights reserved.