Anti-CNPase nanobodies light up myelin
CNPase is an enzyme present at high levels in myelinating cells. Novel nanobody tools developed in an international collaboration were used to identify binding epitopes at atomic resolution and visualise myelin. Additionally, the nanobodies were used as intrabodies to bind to CNPase in living cells. These nanotools will be valuable for future research on myelin and its molecules.
By: Petri Kursula
Published:
In a multinational collaboration involving Norway, Germany, Finland, Belgium, and the United States, we developed a set of nanobodies – small proteins derived from the antibodies of alpacas – that bind the myelin enzyme CNPase with ultrahigh affinity. These nanobodies have numerous advantages compared to classical antibodies used in molecular biology, such as their small size, easy recombinant expression, straightforward labelling, as well as expression in live cells as intrabodies. The five nanobodies have different epitopes, as shown by X-ray crystallography (see Figure); yet, they all bind CNPase with nanomolar affinity. One of the nanobodies inhibits the enzymatic activity of CNPase, making it a prospective tool for enzyme inhibition in living systems. The nanobodies can be used for labelling CNPase in both teased nerve fibers and brain slices, and the nanobodies bind and mark endogenous and recombinant CNPase in cell culture. The findings pave way for further experiments, such as the structural characterisation of full-length CNPase using X-ray crystallography or cryo-EM. The nanobodies will be invaluable tools for myelin research in general, given that CNPase is one of the most used myelin markers in various in vitro and in vivo studies.
Original article:
Markússon et al., J Neurochem, in press (external link)
Earlier related work from our group:
Snaidero et al. (2017), Cell Rep (external link)
CNPase
CNPase is an enzyme that is highly expressed in myelinating cells, especially oligodendrocytes. It is present in non-compact myelin and a widely used marker for non-compacted regions of myelin. CNPase is multifunctional – its name derives from its enigmatic enzymatic activity, but additionally the protein is involved in establishing cytoplasmic channels in myelin. The nanobodies developed in our laboratory provide novel tools for myelin research and structural biology.