PURIFICATION AND STRUCTURAL CHARACTERIZATION OF BOVINE CATHELICIDINS,PRECURSORS OF ANTIMICROBIAL PEPTIDES

Cathelicidins are a novel family of antimicrobial peptide precursors f rom mammalian myeloid cells. They are characterized by a conserved N-t erminal region while the C-terminal antimicrobial domain can vary cons iderably in both primary sequence and length. Four cathelicidins, proB ac5, proBac7, prododecapeptide and proBMAP-28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies . The correlation of ES-MS data from the purified proteins with their cDNA-deduced sequences has revealed several common features of their p rimary sequence, such as the presence of N-terminal 5-oxoproline (pyro glutamate) residues and two disulfide bridges in a 1-2, 3-4 arrangemen t. The N-terminal domains of the cathelicidins present one or two Asp- Pro bonds, which are particularly acid-labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organiz ation around these bonds may vary in different cathelicidins, and Favo ur hydrolysis in some cases. An unexpected feature of the prododecapep tide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine-containing antimicrobial dodecapept ide, indicates that dimerization occurs via disulfide bridge formation at the level of the C-terminal domain and that the dodecapeptide is l ikely released as a dimer from its precursor. Sequence-based secondary structure predictions and CD results indicate for cathelicidins a 30- 50% content of extended conformation and <20% content of alpha-helical conformation, with the alpha-helical segment placed near the N-termin us. Finally, similarity searching and topology-based structure predict ion underline a significant sequential and structural similarity betwe en the conserved N-terminal domain of cathelicidins and cystatin-like domains, placing this family within the cystatin superfamily. When ass ayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor- inhibitory activity (K-i = 0.6-3 mu M).