{"insert":{"user_id":"R000014847","type":"published_papers"},"similar_merge":{"see_also":[{"@id":"https://www.ncbi.nlm.nih.gov/pubmed/40763016","label":"url"},{"@id":"https://www.scopus.com/pages/publications/105013074954","label":"url"},{"@id":"https://web.db.tokushima-u.ac.jp/cgi-bin/edb_browse?EID=454146","label":"url"}],"paper_title":{"en":"Exploring glycoform-dependent dynamic modulations in human immunoglobulin G via computational and experimental approaches","ja":"Exploring glycoform-dependent dynamic modulations in human immunoglobulin G via computational and experimental approaches"},"authors":{"en":[{"name":"Yanaka Saeko"},{"name":"Sakae Yoshitake"},{"name":"Miyanoiri Yohei"},{"name":"Yamaguchi Takumi"},{"name":"Isono Yukiko"},{"name":"Kondo Sachiko"},{"name":"Iwasaki Miyuki"},{"name":"Onitsuka Masayoshi"},{"name":"Yagi Hirokazu"},{"name":"Kato Koichi"}],"ja":[{"name":"Yanaka Saeko"},{"name":"Sakae Yoshitake"},{"name":"Miyanoiri Yohei"},{"name":"Yamaguchi Takumi"},{"name":"Isono Yukiko"},{"name":"Kondo Sachiko"},{"name":"Iwasaki Miyuki"},{"name":"鬼塚 正義"},{"name":"Yagi Hirokazu"},{"name":"Kato Koichi"}]},"description":{"en":"We investigate the impact of glycoform alterations on the dynamic structure of the human immunoglobulin G1 (IgG1) Fc region using integrated computational and experimental approaches. Four distinct IgG1-Fc glycoforms, varying in core fucosylation and nonreducing terminal galactosylation, were generated through a combination of cell engineering and in vitro enzymatic reactions. Stable-isotope-assisted NMR spectroscopy, incorporating both glycan and protein signals, revealed that galactosylation induces chemical shift perturbations extending from the glycan-protein interface to the CH2-CH3 domain boundary. Molecular dynamics simulations demonstrated that the absence of galactose enhances the mobility of both the glycan and the CH2 domain, broadening the conformational landscape of the Fc quaternary structure. This increased flexibility likely contributes to a greater entropic penalty upon binding to effector molecules, which constrain the Fc in an asymmetric conformation. Conversely, the effects of fucosylation are more localized, primarily influencing the dynamics of residues involved in Fcγ receptor IIIa binding. These findings provide atomic-level insights into the distinct yet synergistic mechanisms by which galactosylation and fucosylation modulate IgG1-Fc dynamics and effector functions, offering crucial information for the optimization of therapeutic antibodies.","ja":"We investigate the impact of glycoform alterations on the dynamic structure of the human immunoglobulin G1 (IgG1) Fc region using integrated computational and experimental approaches. Four distinct IgG1-Fc glycoforms, varying in core fucosylation and nonreducing terminal galactosylation, were generated through a combination of cell engineering and in vitro enzymatic reactions. Stable-isotope-assisted NMR spectroscopy, incorporating both glycan and protein signals, revealed that galactosylation induces chemical shift perturbations extending from the glycan-protein interface to the CH2-CH3 domain boundary. Molecular dynamics simulations demonstrated that the absence of galactose enhances the mobility of both the glycan and the CH2 domain, broadening the conformational landscape of the Fc quaternary structure. This increased flexibility likely contributes to a greater entropic penalty upon binding to effector molecules, which constrain the Fc in an asymmetric conformation. Conversely, the effects of fucosylation are more localized, primarily influencing the dynamics of residues involved in Fcγ receptor IIIa binding. These findings provide atomic-level insights into the distinct yet synergistic mechanisms by which galactosylation and fucosylation modulate IgG1-Fc dynamics and effector functions, offering crucial information for the optimization of therapeutic antibodies."},"publication_date":"2025-08-05","publication_name":{"en":"Proceedings of the National Academy of Sciences of the United States of America","ja":"Proceedings of the National Academy of Sciences of the United States of America"},"volume":"122","number":"32","starting_page":"e2505473122","ending_page":"e2505473122","languages":["eng"],"referee":true,"identifiers":{"doi":["10.1073/pnas.2505473122"],"issn":["0027-8424"]},"published_paper_type":"scientific_journal"},"priority":"input_data"}
{"insert":{"user_id":"R000014847","type":"published_papers"},"similar_merge":{"see_also":[{"@id":"https://tokushima-u.repo.nii.ac.jp/records/2010248","label":"url"},{"@id":"https://www.ncbi.nlm.nih.gov/pubmed/34739851","label":"url"},{"@id":"https://web.db.tokushima-u.ac.jp/cgi-bin/edb_browse?EID=390159","label":"url"}],"paper_title":{"en":"Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells.","ja":"Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells."},"authors":{"en":[{"name":"Senga Yukako"},{"name":"Doi Motomichi"},{"name":"Onitsuka Masayoshi"},{"name":"Honda Shinya"}],"ja":[{"name":"Senga Yukako"},{"name":"Doi Motomichi"},{"name":"鬼塚 正義"},{"name":"Honda Shinya"}]},"description":{"en":"Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.","ja":"Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality."},"publication_date":"2022-01-20","publication_name":{"en":"Cell Chemical Biology","ja":"Cell Chemical Biology"},"volume":"29","number":"1","starting_page":"120","ending_page":"132","languages":["eng"],"referee":true,"identifiers":{"doi":["10.1016/j.chembiol.2021.08.010"],"issn":["2451-9448"]},"published_paper_type":"scientific_journal"},"priority":"input_data"}
{"insert":{"user_id":"R000014847","type":"published_papers"},"similar_merge":{"see_also":[{"@id":"https://www.ncbi.nlm.nih.gov/pubmed/34978013","label":"url"},{"@id":"https://cir.nii.ac.jp/crid/1360576118730018560/","label":"url"},{"@id":"https://www.scopus.com/pages/publications/85122130696","label":"url"},{"@id":"https://web.db.tokushima-u.ac.jp/cgi-bin/edb_browse?EID=390175","label":"url"}],"paper_title":{"en":"Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1","ja":"Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1"},"authors":{"en":[{"name":"Yanaka Saeko"},{"name":"Yagi Hirokazu"},{"name":"Yogo Rina"},{"name":"Onitsuka Masayoshi"},{"name":"Kato Koichi"}],"ja":[{"name":"Yanaka Saeko"},{"name":"Yagi Hirokazu"},{"name":"Yogo Rina"},{"name":"鬼塚 正義"},{"name":"Kato Koichi"}]},"description":{"en":"Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled L-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.","ja":"Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled L-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1."},"publication_date":"2022-01-03","publication_name":{"en":"Journal of Biomolecular NMR","ja":"Journal of Biomolecular NMR"},"volume":"76","number":"1-2","starting_page":"17","ending_page":"22","languages":["eng"],"referee":true,"identifiers":{"doi":["10.1007/s10858-021-00387-5"],"issn":["1573-5001"]},"published_paper_type":"scientific_journal"},"priority":"input_data"}
{"insert":{"user_id":"R000014847","type":"published_papers","id":"31219007"},"force":{"see_also":[{"@id":"https://www.ncbi.nlm.nih.gov/pubmed/29970568","label":"url"},{"@id":"https://www.scopus.com/pages/publications/85049804969","label":"url"},{"@id":"https://web.db.tokushima-u.ac.jp/cgi-bin/edb_browse?EID=348369","label":"url"}],"paper_title":{"en":"Construction of Anti-HER2 Recombinants as Targeting Modules for a Drug-delivery System Against HER2-positive Cells","ja":"Construction of Anti-HER2 Recombinants as Targeting Modules for a Drug-delivery System Against HER2-positive Cells"},"authors":{"en":[{"name":"Tang Qing"},{"name":"Onitsuka Masayoshi"},{"name":"Tabata Atsushi"},{"name":"Tomoyasu Toshifumi"},{"name":"Nagamune Hideaki"}],"ja":[{"name":"唐 卿"},{"name":"鬼塚 正義"},{"name":"田端 厚之"},{"name":"友安 俊文"},{"name":"長宗 秀明"}]},"description":{"en":"Recombinant antibodies have been investigated and used in applications such as targeting modules of drug-delivery systems (DDS) against cancers. This study aimed to prepare recombinant antibodies against HER2, containing sortase A (SrtA) recognition sequence, that are applicable as targeting modules in DDS after linkage with the drug-carrier containing oligoglycine-acceptor peptide by SrtA transpeptidation. The recombinant trastuzumab fragment antibodies (scFvs and Fab) with the SrtA-recognition motif (LPXTG) at their C-terminal were constructed and expressed in Escherichia coli and Chinese hamster ovary (CHO) cells, respectively. The reactivity of the purified recombinant antibodies towards HER2-expressing cells was also evaluated via immunofluorescent staining. Fab demonstrated higher yield and purity and better reactivity towards HER2-expressing cells (HCT-15 and HeLa) when compared to scFvs. The CHO expression system possesses superior yield and purity when compared to the E. coli expression system with respect to the preparation of recombinant antibodies applicable in targeting modules for DDS (DDS-TM). Moreover, a Fab variant prepared in this study demonstrated the potential to be a DDS-TM against HER2-expressing cancer cells.","ja":"Recombinant antibodies have been investigated and used in applications such as targeting modules of drug-delivery systems (DDS) against cancers. This study aimed to prepare recombinant antibodies against HER2, containing sortase A (SrtA) recognition sequence, that are applicable as targeting modules in DDS after linkage with the drug-carrier containing oligoglycine-acceptor peptide by SrtA transpeptidation. The recombinant trastuzumab fragment antibodies (scFvs and Fab) with the SrtA-recognition motif (LPXTG) at their C-terminal were constructed and expressed in Escherichia coli and Chinese hamster ovary (CHO) cells, respectively. The reactivity of the purified recombinant antibodies towards HER2-expressing cells was also evaluated via immunofluorescent staining. Fab demonstrated higher yield and purity and better reactivity towards HER2-expressing cells (HCT-15 and HeLa) when compared to scFvs. The CHO expression system possesses superior yield and purity when compared to the E. coli expression system with respect to the preparation of recombinant antibodies applicable in targeting modules for DDS (DDS-TM). Moreover, a Fab variant prepared in this study demonstrated the potential to be a DDS-TM against HER2-expressing cancer cells."},"publication_date":"2018-07","publication_name":{"en":"Anticancer Research","ja":"Anticancer Research"},"volume":"38","number":"7","starting_page":"4319","ending_page":"4325","languages":["eng"],"referee":true,"identifiers":{"doi":["10.21873/anticanres.12731"],"issn":["1791-7530"]},"published_paper_type":"scientific_journal"},"priority":"input_data"}