Isolation and Identification of Novel Neutrophil-Activating Cryptides Hidden in Mitochondrial Cytochrome c

Abstract

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on β-hexosaminidase (β-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced β-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

Mitochondrial protein‐derived cryptides: Are endogenous N‐formylated peptides including mitocryptide‐2 components of mitochondrial damage‐associated molecular patterns?

“…MCT‐1 is not N ‐terminally formylated but can efficiently induce not only neutrophilic chemotaxis but also phagocytosis at nanomolar concentrations. Moreover, the existence of many other neutrophil‐activating peptides presumably cleaved from various mitochondrial proteins including acetyl‐CoA dehydrogenase was also proposed . In addition, we recently found that most fragmented peptides derived from mitochondrial localization sequences in nuclear‐encoded mitochondrial proteins exhibit efficient activities in neutrophilic cells .…”

Mitochondria-Derived Damage-Associated Molecular Patterns in Neurodegeneration

“…Cryptides are endogenous, fragmented functional peptides. Pro-inflammatory cryptides from mtDNA and nuclear DNA-encoded mitochondrial proteins were recently described (153–155). As reviewed in Table 1, each of these molecules has been observed to initiate a pro-inflammatory phenotype under various disease and pathological states.…”

Cryptides: latent peptides everywhere

“…Interestingly, both these peptides presented human counterparts (hMTC-1 and hMTC-2) showing identical biological activities (Mukai et al 2009). Similarly, an octadecapeptide named mitocryptide-CYC (MCT-CYC), isolated from porcine heart resulted to be identical to the 68-85 sequence of mitochondrial cytochrome C. It showed features comparable to those of MCT-1 and MCT-2, enhancing the b-hexosaminidase release from neutrophilic-differentiated HL-60 cells (Hokari et al 2012). Bacterial and mitochondrial proteins are the only source of N-formyl peptides in nature being recognized by specific N-formyl-peptide receptors (FPRL-1, 2).…”