Development of a VHH that inhibits the binding of neuronal pentraxin 2 to a postsynaptic glutamate receptor, AMPAR

Takanori Yokoo; Makoto Nakakido; Keiko Matsuda; Jose M M Caaveiro; Jorge Fernandez-Perez; Michisuke Yuzaki; Kouhei Tsumoto

doi:10.1016/j.jbc.2025.110975

Development of a VHH that inhibits the binding of neuronal pentraxin 2 to a postsynaptic glutamate receptor, AMPAR

J Biol Chem. 2026 Jan;302(1):110975. doi: 10.1016/j.jbc.2025.110975. Epub 2025 Nov 25.

Authors

Takanori Yokoo¹, Makoto Nakakido², Keiko Matsuda³, Jose M M Caaveiro⁴, Jorge Fernandez-Perez⁵, Michisuke Yuzaki³, Kouhei Tsumoto⁶

Affiliations

¹ Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
² Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. Electronic address: nakakido@g.ecc.u-tokyo.ac.jp.
³ Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.
⁴ Department of Protein Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.
⁵ Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
⁶ Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; The institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan. Electronic address: tsumoto@bioeng.t.u-tokyo.ac.jp.

Abstract

Neurons connect to each other via synapses to form neural circuits. Recent research has shown that neuropsychiatric disorders and neurological disorders, such as autism spectrum disorders and Alzheimer's disease, are synaptic diseases caused by abnormalities of synapses. Synaptic organizers are molecules responsible for synapse formation. Neuronal pentraxin 2 (NP2) is a synaptic organizer and a secreted protein that is expressed mainly in the hippocampus and cerebellum, and it contributes to synaptic plasticity. NP2 forms clusters with its family proteins, NP1 and neuronal pentraxin receptor, and binds to postsynaptic amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type receptors. In recent years, research has revealed the disease relevance of NP2. For example, it can be a biomarker of Alzheimer's disease, and its overexpression in the peripheral nervous system has been reported to cause chronic itch. However, the mechanism of NP2 function has not been well described at the molecular level. In this study, we developed a variable domain of a heavy-chain antibody (VHH) against NP2 to elucidate its molecular mechanism of action and to regulate its function of NP2. The obtained VHH N1 showed high specificity and affinity to NP2, and its binding mechanism was elucidated by X-ray crystallography. Furthermore, VHH N1 inhibited the binding of NP2 to amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type receptors, and this inhibitory activity was confirmed in cells. These results provide useful insights into the molecular mechanism of NP2 function and highlight the potential application of VHH N1 as a detection agent for NP2 or as a therapeutic agent for chronic itch.

Keywords: AMPAR; Alzheimer's disease; ESPs; X-ray crystallography; chronic itch; mass photometry; nanobody; neuronal pentraxin; synaptic organizer.

MeSH terms

Animals
C-Reactive Protein* / antagonists & inhibitors
C-Reactive Protein* / chemistry
C-Reactive Protein* / genetics
C-Reactive Protein* / metabolism
HEK293 Cells
Humans
Nerve Tissue Proteins* / antagonists & inhibitors
Nerve Tissue Proteins* / chemistry
Nerve Tissue Proteins* / genetics
Nerve Tissue Proteins* / metabolism
Protein Binding
Receptors, AMPA* / chemistry
Receptors, AMPA* / metabolism
Synapses / metabolism

Substances

Nerve Tissue Proteins
C-Reactive Protein
Receptors, AMPA