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Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide "mitocryptide-2" (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide. Synthetic MCT-2 and its human homolog hMCT-2 induced beta-hexosaminidase release in and chemotaxis of HL-60 cells differentiated into neutrophilic/granulocytic cells. The induction of beta-hexosaminidase release, chemotaxis, and the increase in the intracellular free Ca(2+) concentration by hMCT-2 were completely suppressed by pertussis toxin, indicating the involvement of G(i)- or G(o)-type G proteins in the signaling pathways. Moreover, MCT-2 and hMCT-2 also stimulated beta-hexosaminidase secretion in human neutrophils isolated from peripheral blood in a concentration-dependent manner. Additionally, these peptides partially competed with [(3)H]formyl-Met-Leu-Phe binding to HL-60 cells differentiated into neutrophilic/granulocytic cells, presenting the possibility that the receptor for MCT-2 and hMCT-2 is one of the formyl peptide receptors. These results demonstrate that MCT-2 and its human homolog hMCT-2 are cryptides that activate neutrophils, thus suggesting the presence of regulatory mechanisms involving such mitocryptides in innate immunity.

Mitocryptide-2: Purification, Identification, and Characterization of a Novel Cryptide That Activates Neutrophils