Publications by authors named "Koki Tsutsumi"

3 Publications

  • Page 1 of 1

Generation of monoclonal antibodies against mitocryptide-2: toward a new strategy to investigate the biological roles of cryptides.

J Pept Sci 2017 Jul 29;23(7-8):610-617. Epub 2017 Mar 29.

Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.

We recently identified a novel family of neutrophil-activating peptides including mitocryptide-1 and mitocryptide-2 (MCT-2) that are endogenously produced from various mitochondrial proteins. Among them, MCT-2 is an N-formylated pentadecapeptide derived from mitochondrial cytochrome b and is found to promote neutrophilic migration and phagocytosis efficiently. Signaling mechanisms of neutrophil activation by MCT-2 have been investigated at the cellular level, and MCT-2 has been demonstrated to be an endogenous specific ligand for formyl peptide receptor-2 (also referred to as formyl peptide receptor-like 1). It was also found that MCT-2 promoted neutrophilic functions via the activation of G proteins and phosphorylation of ERK1/2 consecutively. However, the physiological production, distribution, and functions of MCT-2 are not yet elucidated. Here, to investigate the roles of MCT-2 in vivo, we generated monoclonal antibodies (mAbs) against human MCT-2 (hMCT-2) that have two different characteristics. One mAb, NhM2A1, not only bound to the region of positions 10-15 of hMCT-2 but also recognized its C-terminal cleavage site that is presumably produced upon enzymatic hydrolysis of cytochrome b, indicating that NhM2A1 specifically interacts with hMCT-2 but not its parent protein. Moreover, we succeeded in acquiring a specific neutralizing mAb, NhM2A5, which blocks the bioactivities of hMCT-2. Specifically, NhM2A5 inhibited hMCT-2-induced β-hexosaminidase release in neutrophilic/granulocytic differentiated HL-60 cells by binding to the region of positions 5-12 of hMCT-2. Functional analysis using obtained mAbs that specifically recognize hMCT-2 but not its parent protein, cytochrome b, and that neutralize bioactivities of hMCT-2 is expected to reveal the physiological roles of MCT-2, which are presently very difficult to investigate. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/psc.3000DOI Listing
July 2017

Accurate quantitation for in vitro refolding of single domain antibody fragments expressed as inclusion bodies by referring the concomitant expression of a soluble form in the periplasms of Escherichia coli.

J Immunol Methods 2017 03 8;442:1-11. Epub 2016 Dec 8.

Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan. Electronic address:

Single domain antibody fragments from two species, a camel VH (PM1) and a shark V (A6), were derived from inclusion bodies of E. coli and refolded in vitro following three refolding recipes for comparing refolding efficiencies: three-step cold dialysis refolding (TCDR), one-step hot dialysis refolding (OHDR), and one-step cold dialysis refolding (OCDR), as these fragments were expressed as 'a soluble form' either in cytoplasm or periplasm, but the amount were much less than those expressed as 'an insoluble form (inclusion body)' in cytoplasm and periplasm. In order to verify the refolding efficiencies from inclusion bodies correctly, proteins purified from periplasmic soluble fractions were used as reference samples. These samples showed far-UV spectra of a typical β-sheet-dominant structure in circular dichroism (CD) spectroscopy and so did the refolded samples as well. As the maximal magnitude of ellipticity in millidegrees (θ) observed at a given wave length was proportional to the concentrations of the respective reference samples, we could draw linear regression lines for the magnitudes vs. sample concentrations. By using these lines, we measured the concentrations for the refolded PM1 and A6 samples purified from solubilized cytoplasmic insoluble fractions. The refolding efficiency of PM1 was almost 50% following TCDR and 40% and 30% following OHDR and OCDR, respectively, whereas the value of A6 was around 30% following TCDR, and out of bound for quantitation following the other two recipes. The ELISA curves, which were derived from the refolded samples, coincided better with those obtained from the reference samples after converting the values from the protein-concentrations at recovery to the ones of refolded proteins using recovery ratios, indicating that such a correction gives better results for the accurate measure of the ELISA curves than those without correction. Our method require constructing a dual expression system, expressed both in periplasm as a soluble form and cytoplasm as an insoluble form; application of the different refolding recipes due to sequence-by-sequence-difference could be precisely monitored using CD spectra with the concomitant soluble samples as a reference.
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http://dx.doi.org/10.1016/j.jim.2016.11.014DOI Listing
March 2017

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

Biopolymers 2016 Nov;106(4):580-7

Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.

Recently, much attention has been paid to "nonclassical" bioactive peptides, which are fragmented peptides simultaneously produced during maturation and degradation of various functional proteins. We identified many fragmented peptides derived from various mitochondrial proteins including mitocryptide-1 and mitocryptide-2 that efficiently activate neutrophils. These endogenous, functionally active, fragmented peptides are referred to as "cryptides." Among them, mitocryptide-2 is an N-formylated cryptide cleaved from mitochondrial cytochrome b that is encoded in mitochondrial DNA (mtDNA). It is known that 13 proteins encoded in mtDNA are translated in mitochondria as N-formylated forms, suggesting the existence of endogenous N-formylated peptides other than mitocryptide-2. Here, we investigated the effects of N-formylated peptides presumably cleaved from mtDNA-encoded proteins other than cytochrome b on the functions of neutrophilic cells to elucidate possible regulation by endogenous N-formylated cryptides. Four N-formylated cryptides derived from cytochrome c oxidase subunit I and NADH dehydrogenase subunits 4, 5, and 6 among 12 peptides from mtDNA-encoded proteins efficiently induced not only migration but also β-hexosaminidase release, which is an indicator of neutrophilic phagocytosis, in HL-60 cells differentiated into neutrophilic cells. These activities were comparable to or higher than those induced by mitocryptide-2. Although endogenous N-formylated peptides that are contained in mitochondrial damage-associated molecular patterns (DAMPs) have yet to be molecularly identified, they have been implicated in innate immunity. Thus, N-formylated cryptides including mitocryptide-2 are first-line candidates for the contents of mitochondrial DAMPs to promote innate immune responses. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 580-587, 2016.
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http://dx.doi.org/10.1002/bip.22788DOI Listing
November 2016
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