Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies
Abstract
:1. Introduction
2. Materials and Methods
2.1. DNA Constructs
2.2. Cell Culture and Transfection
2.3. Immunoblotting Analysis
2.4. Protein Purification
2.5. Enzyme-Linked Immunosorbent Assays (ELISA)
2.6. Liquid Chromatography–Mass Spectrometry (LC-MS)
2.7. Statistical Analysis
3. Results
3.1. Producing a GBi Antibody as a Functional Mimic for Restoring the Linkage between the Laminin and the Sarcolemma
3.2. Glyco-Optimized Procedure Produced the GBi Antibody with a Dramatically Enhanced Matriglycan Modification
3.3. The Glyco-Optimized Procedure-Derived GBi Antibody Displayed Dramatically Improved ELISA Activities of the β-DG-Laminin Bridging ELISA and IIH6C4 ELISA
3.4. Further Characterization of the GBi Antibody with a Large-Scale Production under the Glyco-Optimized Procedure
3.5. It Is the Addition of Sugar Feeds That Increases Matriglycan Modification of GBi Antibody
3.6. Mn2+ Played a Critical Role in Producing Functionally Active GBi Antibody
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Sanes, J.R. The basement membrane/basal lamina of skeletal muscle. J. Biol. Chem. 2003, 278, 12601–12604. [Google Scholar] [CrossRef] [PubMed]
- Candiello, J.; Balasubramani, M.; Schreiber, E.M.; Cole, G.J.; Mayer, U.; Halfter, W.; Lin, H. Biomechanical properties of native basement membranes. FEBS J. 2007, 274, 2897–2908. [Google Scholar] [CrossRef] [PubMed]
- Ibraghimov-Beskrovnaya, O.; Ervasti, J.M.; Leveille, C.J.; Slaughter, C.A.; Sernett, S.W.; Campbell, K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 1992, 355, 696–702. [Google Scholar] [CrossRef] [PubMed]
- Barresi, R.; Campbell, K.P. Dystroglycan: From biosynthesis to pathogenesis of human disease. J. Cell Sci. 2006, 119 Pt 2, 199–207. [Google Scholar] [CrossRef] [PubMed]
- Endo, T. Glycobiology of alpha-dystroglycan and muscular dystrophy. J. Biochem. 2015, 157, 1–12. [Google Scholar] [CrossRef]
- Michele, D.E.; Campbell, K.P. Dystrophin-glycoprotein complex: Post-translational processing and dystroglycan function. J. Biol. Chem. 2003, 278, 15457–15460. [Google Scholar] [CrossRef]
- Ervasti, J.M.; Campbell, K.P. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 1993, 122, 809–823. [Google Scholar] [CrossRef]
- Longman, C.; Brockington, M.; Torelli, S.; Jimenez-Mallebrera, C.; Kennedy, C.; Khalil, N.; Feng, L.; Saran, R.K.; Voit, T.; Merlini, L.; et al. Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Hum. Mol. Genet. 2003, 12, 2853–2861. [Google Scholar] [CrossRef]
- Inamori, K.; Hara, Y.; Willer, T.; EAnderson, M.; Zhu, Z.; Yoshida-Moriguchi, T.; Campbell, K.P. Xylosyl- and glucuronyltransferase functions of LARGE in alpha-dystroglycan modification are conserved in LARGE2. Glycobiology 2013, 23, 295–302. [Google Scholar] [CrossRef]
- Inamori, K.; Yoshida-Moriguchi, T.; Hara, Y.; Anderson, M.E.; Yu, L.; Campbell, K.P. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE. Science 2012, 335, 93–96. [Google Scholar] [CrossRef]
- Ashikov, A.; Buettner, F.F.; Tiemann, B.; Gerardy-Schahn, R.; Bakker, H. LARGE2 generates the same xylose- and glucuronic acid-containing glycan structures as LARGE. Glycobiology 2013, 23, 303–309. [Google Scholar] [CrossRef] [PubMed]
- Han, R.; Kanagawa, M.; Yoshida-Moriguchi, T.; Rader, E.P.; Ng, R.A.; Michele, D.E.; Muirhead, D.E.; Kunz, S.; Moore, S.A.; Iannaccone, S.T.; et al. Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan. Proc. Natl. Acad. Sci. USA 2009, 106, 12573–12579. [Google Scholar] [CrossRef] [PubMed]
- Brinkmann, U.; Kontermann, R.E. The making of bispecific antibodies. mAbs 2017, 9, 182–212. [Google Scholar] [CrossRef] [PubMed]
- Zhong, X.; D’Antona, A.M. Recent Advances in the Molecular Design and Applications of Multispecific Biotherapeutics. Antibodies 2021, 10, 13. [Google Scholar] [CrossRef] [PubMed]
- Gumlaw, N.; Sevigny, L.M.; Zhao, H.; Luo, Z.; Bangari, D.S.; Masterjohn, E.; Chen, Y.; McDonald, B.; Magnay, M.; Travaline, T.; et al. biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for alpha-Dystroglycanopathies. Mol. Ther. 2020, 28, 664–676. [Google Scholar] [CrossRef]
- Klimpel, K.R.; Molloy, S.S.; Thomas, G.; Leppla, S.H. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc. Natl. Acad. Sci. USA 1992, 89, 10277–10281. [Google Scholar] [CrossRef]
- Peyrard, M.; Seroussi, E.; Sandberg-Nordqvist, A.-C.; Xie, Y.-G.; Han, F.-Y.; Fransson, I.; Collins, J.; Dunham, I.; Kost-Alimova, M.; Imreh, S.; et al. The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family. Proc. Natl. Acad. Sci. USA 1999, 96, 598–603. [Google Scholar] [CrossRef]
- Winder, S.J. The complexities of dystroglycan. Trends Biochem. Sci. 2001, 26, 118–124. [Google Scholar] [CrossRef]
- Zhou, J.; Yan, G.G.; Cluckey, D.; Meade, C.; Ruth, M.; Sorm, R.; Tam, A.S.; Lim, S.; Petridis, C.; Lin, L.; et al. Exploring Parametric and Mechanistic Differences between Expi293F(TM) and ExpiCHO-S(TM) Cells for Transient Antibody Production Optimization. Antibodies 2023, 12, 53. [Google Scholar] [CrossRef]
- Zhang, P.; Hu, H. Differential glycosylation of alpha-dystroglycan and proteins other than alpha-dystroglycan by like-glycosyltransferase. Glycobiology 2012, 22, 235–247. [Google Scholar] [CrossRef]
- D’Antona, A.M.; Lee, J.M.; Zhang, M.; Friedman, C.; He, T.; Mosyak, L.; Bennett, E.; Lin, L.; Silverman, M.; Cometa, F.; et al. Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4. Int. J. Mol. Sci. 2024, 25, 1931. [Google Scholar] [CrossRef]
- Kanagawa, M.; Saito, F.; Kunz, S.; Yoshida-Moriguchi, T.; Barresi, R.; Kobayashi, Y.M.; Muschler, J.; Dumanski, J.P.; Michele, D.E.; Oldstone, M.B.; et al. Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell 2004, 117, 953–964. [Google Scholar] [CrossRef] [PubMed]
- Bozzi, M.; Morlacchi, S.; Bigotti, M.G.; Sciandra, F.; Brancaccio, A. Functional diversity of dystroglycan. Matrix Biol. 2009, 28, 179–187. [Google Scholar] [CrossRef] [PubMed]
- Yoshida-Moriguchi, T.; Campbell, K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane. Glycobiology 2015, 25, 702–713. [Google Scholar] [CrossRef] [PubMed]
- Grewal, P.K.; Hewitt, J.E. Glycosylation defects: A new mechanism for muscular dystrophy? Hum. Mol. Genet. 2003, 12, R259–R264. [Google Scholar] [CrossRef] [PubMed]
- Zhong, X.; Ma, W.; Meade, C.L.; Tam, A.S.; Llewellyn, E.; Cornell, R.; Cote, K.; Scarcelli, J.J.; Marshall, J.K.; Tzvetkova, B.; et al. Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins. Biotechnol. Prog. 2019, 35, e2724. [Google Scholar] [CrossRef]
- Zhong, X.; Schwab, A.; Ma, W.; Meade, C.L.; Zhou, J.; D’Antona, A.M.; Somers, W.; Lin, L. Large-Scale Transient Production in ExpiCHO-S with Enhanced N-Galactosylation-Sialylation and PEI-Based Transfection. Methods Mol. Biol. 2022, 2313, 143–150. [Google Scholar]
- Evans, C.; Conney, A.; Trousof, N.; Burns, J. Metabolism of D-galactose to D-glucuronic acid, L-gulonic acid and L-ascorbic acid in normal and barbital-treated rats. Biochim. Biophys. Acta 1960, 41, 9–14. [Google Scholar] [CrossRef]
- Dammen-Brower, K.; Epler, P.; Zhu, S.; Bernstein, Z.J.; Stabach, P.R.; Braddock, D.T.; Spangler, J.B.; Yarema, K.J. Strategies for Glycoengineering Therapeutic Proteins. Front. Chem. 2022, 10, 863118. [Google Scholar] [CrossRef]
- Righino, B.; Bozzi, M.; Pirolli, D.; Sciandra, F.; Bigotti, M.G.; Brancaccio, A.; De Rosa, M.C. Identification and Modeling of a GT-A Fold in the alpha-Dystroglycan Glycosylating Enzyme LARGE1. J. Chem. Inf. Model. 2020, 60, 3145–3156. [Google Scholar] [CrossRef]
- Brockington, M.; Torelli, S.; Prandini, P.; Boito, C.; Dolatshad, N.F.; Longman, C.; Brown, S.C.; Muntoni, F. Localization and functional analysis of the LARGE family of glycosyltransferases: Significance for muscular dystrophy. Hum. Mol. Genet. 2005, 14, 657–665. [Google Scholar] [CrossRef] [PubMed]
- Inamori, K.; Willer, T.; Hara, Y.; Venzke, D.; Anderson, M.E.; Clarke, N.F.; Guicheney, P.; Bönnemann, C.G.; Moore, S.A.; Campbell, K.P. Endogenous glucuronyltransferase activity of LARGE or LARGE2 required for functional modification of alpha-dystroglycan in cells and tissues. J. Biol. Chem. 2014, 289, 28138–28148. [Google Scholar] [CrossRef] [PubMed]
- Lommel, M.; Strahl, S. Protein O-mannosylation: Conserved from bacteria to humans. Glycobiology 2009, 19, 816–828. [Google Scholar] [CrossRef] [PubMed]
- Chiba, A.; Matsumura, K.; Yamada, H.; Inazu, T.; Shimizu, T.; Kusunoki, S.; Kanazawa, I.; Kobata, A.; Endo, T. Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin. J. Biol. Chem. 1997, 272, 2156–2162. [Google Scholar] [CrossRef]
- Sheikh, M.O.; Venzke, D.; Anderson, M.E.; Yoshida-Moriguchi, T.; Glushka, J.N.; Nairn, A.V.; Galizzi, M.; Moremen, K.W.; Campbell, K.P.; Wells, L. HNK-1 sulfotransferase modulates alpha-dystroglycan glycosylation by 3-O-sulfation of glucuronic acid on matriglycan. Glycobiology 2020, 30, 817–829. [Google Scholar] [CrossRef]
- Harrison, D.; Hussain, S.-A.; Combs, A.C.; Ervasti, J.M.; Yurchenco, P.D.; Hohenester, E. Crystal structure and cell surface anchorage sites of laminin alpha1LG4-5. J. Biol. Chem. 2007, 282, 11573–11581. [Google Scholar] [CrossRef]
- Pham, P.L.; Kamen, A.; Durocher, Y. Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol. Biotechnol. 2006, 34, 225–237. [Google Scholar] [CrossRef]
- Geisse, S.; Voedisch, B. Transient expression technologies: Past, present, and future. Methods Mol. Biol. 2012, 899, 203–219. [Google Scholar] [CrossRef]
- Baldi, L.; Hacker, D.L.; Adam, M.; Wurm, F.M. Recombinant protein production by large-scale transient gene expression in mammalian cells: State of the art and future perspectives. Biotechnol. Lett. 2007, 29, 677–684. [Google Scholar] [CrossRef]
- Ghetie, V.; Ward, E.S. Transcytosis and catabolism of antibody. Immunol. Res. 2002, 25, 97–113. [Google Scholar] [CrossRef]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Zhong, X.; Yan, G.G.; Chaturvedi, A.; Li, X.; Gao, Y.; Girgenrath, M.; Corcoran, C.J.; Diblasio-Smith, L.; LaVallie, E.R.; de Rham, T.; et al. Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies. Antibodies 2024, 13, 83. https://doi.org/10.3390/antib13040083
Zhong X, Yan GG, Chaturvedi A, Li X, Gao Y, Girgenrath M, Corcoran CJ, Diblasio-Smith L, LaVallie ER, de Rham T, et al. Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies. Antibodies. 2024; 13(4):83. https://doi.org/10.3390/antib13040083
Chicago/Turabian StyleZhong, Xiaotian, Guoying Grace Yan, Apurva Chaturvedi, Xiuling Li, Yijie Gao, Mahasweta Girgenrath, Chris J. Corcoran, Liz Diblasio-Smith, Edward R. LaVallie, Teresse de Rham, and et al. 2024. "Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies" Antibodies 13, no. 4: 83. https://doi.org/10.3390/antib13040083
APA StyleZhong, X., Yan, G. G., Chaturvedi, A., Li, X., Gao, Y., Girgenrath, M., Corcoran, C. J., Diblasio-Smith, L., LaVallie, E. R., de Rham, T., Zhou, J., Abel, M., Riegel, L., Lim, S. K. H., Bloom, L., Lin, L., & D’Antona, A. M. (2024). Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies. Antibodies, 13(4), 83. https://doi.org/10.3390/antib13040083