Nanobodies against the myelin enzyme <scp>CNPase</scp> as tools for structural and functional studies
2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, and its loss in mice and humans causes neurodegeneration. Additionally, CNPase is frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognising the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibres and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional studies on myelin formation, dynamics, and disease, including high-resolution imaging of nerve tissue.
Authors
- Arne Raasakka (ORCID: https://orcid.org/0000-0002-9443-9959)
- Shama Sograte‐Idrissi (ORCID: https://orcid.org/0000-0003-4271-7897)
- Petri Kursula (ORCID: https://orcid.org/0000-0001-8529-3751)
- Madeline H. Cooper (ORCID: https://orcid.org/0000-0001-5698-5150)
- Gopinath Muruganandam (ORCID: https://orcid.org/0000-0002-6642-3909)
- Marcel Schröder (ORCID: https://orcid.org/0000-0002-6310-4830)
- Aleksi Sutinen (ORCID: https://orcid.org/0000-0001-6704-1702)
- J. Bradley Zuchero (ORCID: https://orcid.org/0000-0002-4706-2538)
- Hauke B. Werner (ORCID: https://orcid.org/0000-0002-7710-5738)
- Maria A. Eichel‐Vogel (ORCID: https://orcid.org/0000-0002-9925-7249)
- Nicholas Ambiel
- Henrike Körner
- Ommolbanin Asadpour
- Manasi Iyer (ORCID: https://orcid.org/0000-0002-6376-3633)
- Sigurbjörn Markússon (ORCID: https://orcid.org/0000-0003-4364-2997)
- Maya K. Weigel (ORCID: https://orcid.org/0000-0003-0557-5172)
- Felipe Opazo (ORCID: https://orcid.org/0000-0002-4968-9713)
- Risha Chowdhury
- Amir Mohammad Rahimi
- Dmitri Lodygin
Institutions
- Vrije Universiteit Brussel (BE)
- Vlaams Instituut voor Biotechnologie (BE)
- Universitätsmedizin Göttingen (DE)
- Immatics Biotechnologies (Germany) (DE)
- VIB-VUB Center for Structural Biology (BE)
- Max Planck Institute for Multidisciplinary Sciences (DE)
- University of Bergen (NO)
- University of Göttingen (DE)
- Stanford University (US)
- University of Oulu (FI)
Publication Details
- Journal
- Journal of Neurochemistry
- Published
- 2024-12-10
- DOI
- https://doi.org/10.1111/jnc.16274
- Citations
- 1
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 0.40
Funders
- Deutsche Forschungsgemeinschaft
- Jane ja Aatos Erkon Säätiö
- Norges Forskningsråd
- National Institutes of Health