IDENTIFICATION OF CRAMP, A CATHELIN-RELATED ANTIMICROBIAL PEPTIDE EXPRESSED IN THE EMBRYONIC AND ADULT-MOUSE

Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species, Prior work has sug gested that members of this gene family can participate in host defens e through their antimicrobial effects and activate mesenchymal cells d uring wound repair, To permit further study of these proteins a revers e transcriptase-polymerase chain reaction approach was used to identif y potential mouse homologs, A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other c athelicidins and a unique COOH-terminal peptide, This gene, named Cram p for cathelin-related antimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins h ave been mapped in pig and man. Northern blot analysis detected a 1-ki lobase transcript that was expressed in adult bone marrow and during e mbryogenesis as early as E12, the earliest stage of blood development, Reverse transcriptase-polymerase chain reaction also detected CRAMP e xpression in adult testis, spleen, stomach, and intestine but not in b rain, liver, heart, or skeletal muscle. To evaluate further the expres sion and function of CRAMP a peptide corresponding to the predicted CO OH-terminal region was synthesized, CD spectral analysis showed that C RAMP will form an amphipathic alpha-helix similar to other antimicrobi al peptides, Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of b acterial strains (minimum inhibitory concentrations 0.5-8.0 mu M) and by permeabilizing the inner membrane of Escherichia coli directly at 1 mu M. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils, Thus, CRAMP represents the first antibiot ic peptide found in cells of myeloid lineage in the mouse, These data suggest that inflammatory cells in the mouse can use a nonoxidative me chanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair.