Novel cathelicidins in horse leukocytes(1).
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.
Scocchi
M
M
Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano, I-34012, Trieste, Italy.
Bontempo
D
D
Boscolo
S
S
Tomasinsig
L
L
Giulotto
E
E
Zanetti
M
M
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics* / Q000378:metabolism*; D015139:Blotting, Southern; D015153:Blotting, Western; D054804:Cathelicidins; D003594:Cytoplasmic Granules / Q000378:metabolism; D006736:Horses / Q000502:physiology*; D007962:Leukocytes / Q000502:physiology*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism*; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D020086:Short Interspersed Nucleotide Elements / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D054804:Cathelicidins; D011498:Protein Precursors; C121625:cathelicidin 2 protein, mammal; C121626:cathelicidin 3 protein, Equus caballus; D013755:Tetradecanoylphorbol Acetate