Yuka Hiroshima, Rie Kido, Jun-ichi Kido, Mika Bandou, Kaya Yoshida, Akikazu Murakami and Yasuo Shinohara : Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system, Odontology, Vol.112, No.4, 1103-1112, 2024.
(Summary)
Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.
Yasufumi Nishikawa, Yoritoki Tomotake, Hiromichi Kawano, Koji Naruishi, Jun-ichi Kido, Yuka Hiroshima, Akikazu Murakami, Tetsuo Ichikawa and Hiromichi Yumoto : Effects of Candidalysin Derived from Candida albicans on the Expression of Pro-Inflammatory Mediators in Human Gingival Fibroblasts, International Journal of Molecular Sciences, Vol.24, No.4, 3256, 2023.
Yuta Uemura, Yuka Hiroshima, Ayano Tada, Keiji Murakami, Kaya Yoshida, Yuji Inagaki, Tomomi Kuwahara, Akikazu Murakami, Hideki Fujii and Hiromichi Yumoto : Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways, Biomedicines, Vol.10, No.10, 2643, 2022.
(Summary)
() is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. -OMVs are involved in the pathogenesis of periodontitis. -OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of -OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. -OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. -OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that -OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that -OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.
Narutoshi Tsukahara, Akikazu Murakami, Maiko Motohashi, Hiroshi Nakayama, Yoshiro Kondo, Yuji Ito, Takachika Azuma and Hidehiro Kishimoto : An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens., International Immunology, Vol.34, No.8, 421-434, 2022.
(Summary)
Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.
(Keyword)
Animals / Antigens / Bacteriophages / Camelids, New World / Gene Library / Humans / Immunoglobulin Heavy Chains / Single-Domain Antibodies
Muhammad Reza Pahlevi, Keiji Murakami, Yuka Hiroshima, Akikazu Murakami and Hideki Fujii : pruR and PA0065 Genes Are Responsible for Decreasing Antibiotic Tolerance by Autoinducer Analog-1 (AIA-1) in Pseudomonas aeruginosa, Antibiotics, Vol.11, No.6, 773, 2022.
Akihiro Nishiguchi, Akikazu Murakami, Takachika Azuma and Masayuki Oda : A Trade-off Between Thermostability and Binding Affinity of Anti-(4-hydroxy-3-nitrophenyl)Acetyl Antibodies During the Course of Affinity Maturation., The Protein Journal, Vol.41, No.2, 293-303, 2022.
(Summary)
Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three single-chain Fv fragments (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl Abs, namely 9TG, 9T7, and E11. 9TG has a germline structure that lacks SHM and is an ancestor of 9T7 with 11 mutations. E11, which has 21 mutations, is a mature Ab and has its own ancestor. The thermostabilities and antigen-Ab interactions were analyzed by circular dichroism (CD), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Far-UV CD spectra showed that all scFvs were folded into a structure referred to as immunoglobulin-fold and were unfolded by heating at different melting temperatures. Comparison of thermodynamic parameters obtained from DSC and ITC revealed that the magnitude of stabilization free energy at 37 °C was in the order, 9TG > 9T7 > E11, while that of the free energy of interaction with antigen was 9TG < 9T7 < E11, suggesting that Abs make a trade-off between stability and affinity during affinity maturation.
Akihito Inoue, Takanobu Yasuda, Bo Zhu, Tetsuya Kitaguchi, Akikazu Murakami and Hiroshi Ueda : Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface., Scientific Reports, Vol.11, No.1, 2021.
(Summary)
Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based "mini Q-bodies" by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.
Taro Ikegami, Norimoto Kise, Hidetoshi Kinjyo, Shunsuke Kondo, Mikio Suzuki, Narutoshi Tsukahara, Akikazu Murakami, Asanori Kiyuna, Shinya Agena, Katsunori Tanaka, Narumi Hasegawa, Junko Kawakami, Akira Ganaha, Hiroyuki Maeda and Hitoshi Hirakawa : Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma., Viruses, Vol.13, No.10, 2021.
(Summary)
mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.
Taro Ikegami, Hitoshi Hirakawa, Narutoshi Tsukahara, Akikazu Murakami, Norimoto Kise, Asanori Kiyuna, Takayoshi Kosugi, Shinya Agena, Hidetoshi Kinjyo, Narumi Hasegawa, Masatomo Touyama, Shunsuke Kondo, Hiroyuki Maeda, Mikio Suzuki and Akira Ganaha : Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma., Microorganisms, Vol.9, No.3, 2021.
(Summary)
mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.
Chathuni Jayathilake, Shigefumi Kumachi, Hidenao Arai, Maiko Motohashi, Takuya Terai, Akikazu Murakami and Naoto Nemoto : In vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR., Analytical Biochemistry: Methods in the Biological Sciences, Vol.589, 2019.
(Summary)
Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner.
Graft-versus-host disease (GVHD) constitutes the most frequent complications after the allogeneic hematopoietic stem cell transplantation for a variety of hematological malignancies. In the present study, we explored the prophylactic potential of adipose tissue-derived mesenchymal stem cells (AD-MSCs) in controlling GVHD in murine models with a special focus on bone marrow aplasia related with acute GVHD. The CB6F1 mice were induced GVHD by the injection intravenously of C57BL/6 (B6-Ly-5.1) splenocytes without conditioning irradiation or chemotherapy. AD-MSCs from C3H mice were injected intravenously via tail veins. GVHD was assessed using flowcytometry analysis of peripheral blood cells and histopathologic analysis of target organs. Histopathological analyses revealed that AD-MSCs markedly suppressed the infiltration of lymphocytes into liver as well as the aplasia in bone marrow. This study is the first to clarify the effectiveness of AD-MSCs against bone marrow aplasia in GVHD, supporting a rationale of AD-MSCs for ameliorating bone marrow suppression and infectivity after allo-HSCT in human clinics.
Suzuki Satoko, Oyama Taiji, Yamane Ai, Horiguchi Yasuo, Akao Ken-ichi, Akikazu Murakami, Micsonai András, Kardos József, Nagamori Koushi and Tsumoto Kouhei : Higher Order Structure, Stability, and Similarity Assessment of VHH Antibodies Using CD Spectroscopy, Protein and Antibody Engineering Summit, Nov. 2023.
2.
Raras Ajeng Enggardipta, Minato Akizuki, Kazumitsu Sekine, Kenichi Hamada, Akikazu Murakami and Hiromichi Yumoto : The preliminary study of chitosan nanoparticles as antibacterial agent on Enterococcus faecalis biofilm, 19th Scientific Meeting and Refresher Course in Dentistry (Jakarta, Indonesia), Feb. 2023.
Proceeding of Domestic Conference:
1.
Ito Tomoyuki, Kawada Sakiya, Nakazawa Hikaru, Akikazu Murakami and Umetsu Mitsuo : Machine-learning-assisted affinity maturation of an antibody fragment simultaneously improving the bacterial expression and structural stability, 第75回日本生物工学会, Sep. 2023.