VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)
The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals an...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-09-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1265582/full |
| _version_ | 1797687642249232384 |
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| author | Hiroyuki Takeda Tatsuhiko Ozawa Tatsuhiko Ozawa Hiroki Zenke Yoh Ohnuki Yuri Umeda Wei Zhou Honoka Tomoda Akihiko Takechi Kimiyoshi Narita Takaaki Shimizu Takuya Miyakawa Yuji Ito Tatsuya Sawasaki |
| author_facet | Hiroyuki Takeda Tatsuhiko Ozawa Tatsuhiko Ozawa Hiroki Zenke Yoh Ohnuki Yuri Umeda Wei Zhou Honoka Tomoda Akihiko Takechi Kimiyoshi Narita Takaaki Shimizu Takuya Miyakawa Yuji Ito Tatsuya Sawasaki |
| author_sort | Hiroyuki Takeda |
| collection | DOAJ |
| description | The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning. |
| first_indexed | 2024-03-12T01:21:42Z |
| format | Article |
| id | doaj.art-a00f4a3c1cfb4e7bb5f0d140f48f362c |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-12T01:21:42Z |
| publishDate | 2023-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-a00f4a3c1cfb4e7bb5f0d140f48f362c2023-09-13T05:07:54ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-09-011110.3389/fbioe.2023.12655821265582VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)Hiroyuki Takeda0Tatsuhiko Ozawa1Tatsuhiko Ozawa2Hiroki Zenke3Yoh Ohnuki4Yuri Umeda5Wei Zhou6Honoka Tomoda7Akihiko Takechi8Kimiyoshi Narita9Takaaki Shimizu10Takuya Miyakawa11Yuji Ito12Tatsuya Sawasaki13Proteo-Science Center, Ehime University, Matsuyama, JapanDepartment of Immunology, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, JapanCenter for Advanced Antibody Drug Development, University of Toyama, Toyama, JapanProteo-Science Center, Ehime University, Matsuyama, JapanDepartment of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, JapanProteo-Science Center, Ehime University, Matsuyama, JapanProteo-Science Center, Ehime University, Matsuyama, JapanFisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Iyo, JapanFisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Iyo, JapanFisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Iyo, JapanFisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Iyo, JapanGraduate School of Biostudies, Kyoto University, Kyoto, JapanGraduate School of Science and Engineering, Kagoshima University, Kagoshima, JapanProteo-Science Center, Ehime University, Matsuyama, JapanThe VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1265582/fullVNARJapanese topesharkphage displayyeast displaybiopanningdeep sequencing |
| spellingShingle | Hiroyuki Takeda Tatsuhiko Ozawa Tatsuhiko Ozawa Hiroki Zenke Yoh Ohnuki Yuri Umeda Wei Zhou Honoka Tomoda Akihiko Takechi Kimiyoshi Narita Takaaki Shimizu Takuya Miyakawa Yuji Ito Tatsuya Sawasaki VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica) Frontiers in Bioengineering and Biotechnology VNAR Japanese topeshark phage display yeast display biopanning deep sequencing |
| title | VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica) |
| title_full | VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica) |
| title_fullStr | VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica) |
| title_full_unstemmed | VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica) |
| title_short | VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica) |
| title_sort | vnar development through antigen immunization of japanese topeshark hemitriakis japanica |
| topic | VNAR Japanese topeshark phage display yeast display biopanning deep sequencing |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1265582/full |
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