Novel cathelicidins in horse leukocytes

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Abstract
Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra‐ and inter‐species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription‐PCR‐based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH‐1, eCATH‐2 and eCATH‐3. Putative antimicrobial domains of 26, 27 and 40 residues with no significant sequence homology to other peptides were inferred at the C‐terminus of the sequences. Southern analysis of genomic DNA using a probe based on the cathelicidin‐conserved propiece revealed a polymorphic DNA region with several hybridization‐positive fragments and suggested the presence of additional genes. A null eCATH‐1 allele was also demonstrated with a frequency of 0.71 in the horse population analyzed and low amounts of eCATH‐1‐specific mRNA were found in myeloid cells of gene‐positive animals. A Western analysis using antibodies to synthetic eCATH peptides revealed the presence of eCATH‐2 and eCATH‐3 propeptides, but not of eCATH‐1‐related polypeptides, in horse neutrophil granules and in the secretions of phorbol myristate acetate‐stimulated neutrophils. These results thus suggest that eCATH‐2 and eCATH‐3 are functional genes, whereas eCATH‐1 is unable to encode a polypeptide.