Mitocryptide-2: purification, identification, and characterization of a novel cryptide that activates neutrophils.

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.

Cryptide signaling: Amphiphilic peptide-induced exocytotic mechanisms in mast cells.

Amphiphilic peptides with positive charges such as substance P (SP) and mastoparan (MP) are known to induce exocytosis in rat peritoneal mast cells. To elucidate whether and how intracellular Ca(2+) signaling is involved in the peptide-induced exocytosis, here we investigated the relationships between an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and exocytosis caused by SP and MP. SP and MP induced exocytosis coinciding with an initial rapid and transient [Ca(2+)](i) increase, but not with a sustained increase. These stimulations were abolished by pertussis toxin, indicating the involvement of the G(i)-family of G proteins in the peptide signaling. Moreover, the [Ca(2+)](i) increase was shown to accelerate and potentiate exocytosis, suggesting that the transient increase in [Ca(2+)](i) positively modified exocytotic secretion. However, it was indicated that the signal of [Ca(2+)](i) increase was not sufficient for the peptide-induced exocytosis, suggesting the participation of alternative mechanisms other than Ca(2+) signaling in the pathway.

Mammalian copper chaperone Cox17p has an essential role in activation of cytochrome C oxidase and embryonic development.

Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(-/-), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(-/-) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(-/-). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.

Characterization and identification of promoter elements in the mouse COX17 gene.

Cox17p, essential for the assembly of functional cytochrome c oxidase (CCO) in Saccharomyces cerevisiae, has been believed to deliver copper ions to the mitochondrion for insertion into the enzyme. We have recently isolated an approximately 20 kb genomic fragment of the mouse COX17. Reporter assay experiments have shown that most of the promoter activity was restricted to a 0.85 kb fragment flanking the first exon. Further intensive deletion and detailed mutation analysis suggested that the minimal essential region for transactivation was located at bases -155 to -70. This 5'-flanking region did not possess a TATA box, but contained putative Sp1, NRF-1 and NRF-2 binding sites. COX17 basal promoter activity was abrogated by site-directed mutagenesis of Sp1, NRF-1 and NRF-2 binding sites. Electrophoretic mobility shift assays with AtT-20 and NIH3T3 cell nuclear extract revealed that this region binds both a Sp1-like protein and NRF-1 transcription factors. These results indicated that Sp1, NRF-1 and NRF-2 are involved in basal transcription of the COX17 gene, similar to the transcription mechanism of other CCO-related genes.

Isolation and identification of novel neutrophil-activating cryptides hidden in mitochondrial cytochrome C.

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 ß-hexosaminidase (ß-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced ß-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.

Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart.

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing beta-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and G(i2)-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce beta-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.

A mastoparan analog without lytic effects and its stimulatory mechanisms in mast cells.

Mastoparan, a tetradecapeptide isolated from wasp venom, is known to not only induce the secretion of histamine but also cause cell lysis in rat peritoneal mast cells. This lytic effect makes investigations concerning MP-induced signaling mechanisms difficult. Here, we report that a mastoparan derivative peptide, [Lys(10), Leu(13)]mastoparan, also designated "mas 11'', induces exocytosis with greater activity than mastoparan without the undesired lytic effect. The signaling mechanisms triggered by mas 11 were also investigated, and it was clearly demonstrated that mas 11 induced not only the non-lytic release of beta-hexosaminidase but also an increase in the concentration of cytosolic free Ca(2+) in the cells and these effects were mostly prevented by pertussis toxin, suggesting the involvement of G(i)-type G protein in the signaling. Mas 11 is a promising stimulatory molecule with which to investigate the exocytotic mechanisms induced by not only mastoparan but also various amphiphilic peptides in the cells.

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides. Thermolytic digestion demonstrated three disulfide bond pairings of the EGF-like domain in HB-EGF is consistent with that of human-EGF and human-TGF-alpha. HB-EGF(44-86) showed high binding affinity to EGF-receptor, like human-EGF. The truncation of the C-terminal Leu86 residue from HB-EGF(44-86), HB-EGF(45-86) or HB-EGF(46-86) caused a drastic reduction in the binding affinity to the EGF receptor. These results suggest that the EGF-like domain of HB-EGF plays an important role in the binding to the EGF receptor, and its C-terminal Leu86 residue is necessary for binding with the EGF-receptor. In addition, the deletion of the two N-terminal residues (Asp44-Pro45) from HB-EGF(44-86) caused a 10-fold decrease in relative binding affinity to the EGF receptor. This indicates that the two N-terminal residues of the EGF-like domain of HB-EGF are necessary for its optimal binding affinity to the EGF receptor.

A selective requirement for copper-dependent activation of cytochrome c oxidase by Cox17p.

Cox17p is cloned from yeast as a chaperone to deliver copper to the mitochondria of assembly for cytochrome c oxidase (CCO). In mammals, CCO is a key enzyme for cellular respiration and a defect in its function is associated with severe neonatal or infantile lactic acidosis and early death. Recently, we found that Cox17p is not only required for mitochondrial oxidative phosphorylation but also is essential for embryonic growth and development in COX17 gene-deficient mice. To investigate its biochemical features, recombinant human Cox17p was overexpressed and purified without a purification tag. It specifically binds Cu(I) at a molar copper content of 3.3+/-0.04 under reduced conditions and significantly activates the mitochondrial CCO in vitro. Although the Cu-Cox17p complex was maintained between pH values from 5.0 to 7.7, Cu was completely released from Cox17p at pH 8.0. An acute exposure of excess amount of copper ion to mouse cells resulted in a significant reduction of Cox17p mRNA expression, whereas copper starvation maintained the Cox17p transcription level. These results suggest that the stringent selectivity of Cox17p for copper is required for CCO activation, to prevent copper overload, or promote the supply of copper.