The effects of phospholipids and fatty acids on the oligomer formation of NAP-22

• Published in: Neuroscience letters Vol. 736; p. 135288
• Main Authors: Odagaki, Sin-Ichi, Maekawa, Shohei, Hayashi, Fumio, Suzaki, Toshinobu, Morigaki, Kenichi
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 25-09-2020
• Subjects: Acidic phospholipid; Bacteria - metabolism; Calmodulin - metabolism; Calmodulin-Binding Proteins - metabolism; Fatty Acids - metabolism; Lysophospholipid; Membrane Microdomains - metabolism; Membrane Proteins - metabolism; NAP-22; Nerve Tissue Proteins - metabolism; Neuron; Phospholipids - metabolism; Protein Binding; PUFA; Repressor Proteins - metabolism; PS; NAP-22; CHL; LPS; cPA; GA; LLPS; AA; PUFA; ID protein; CL; HMEN buffer; PA; PC; Neuron; Acidic phospholipid; PE; PG; EPA; Lysophospholipid; PI; CTL; DHA; LPA; DRM; PIP2; LPC
• ISSN: 0304-3940; 1872-7972
• DOI: 10.1016/j.neulet.2020.135288

•Oligomerization of NAP-22 was observed by SDS-PAGE after cross-linking using glutaraldehyde.•Acidic phospholipids and fatty acids affected the oligomer species.•Polyunsaturated fatty acids (PUFAs) induced much higher oligomer formation of NAP-22.•Tangled rope-like structures and their packing by PUFAs were observed with TEM.•Annular-shaped oligomers of ∼10 nm diameter could be the assembly unit of the rope-like structure.
Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM fraction of the neuronal cell membrane. Previous studies showed tight binding activity of NAP-22 to acidic membrane lipids and the self-interaction of NAP-22, i.e., oligomerization. In this study, the effect of various phospholipids, lysophospholipids and fatty acids on the oligomerization of NAP-22 was studied through SDS-PAGE after chemical cross-linking and electron microscopic observation. High molecular mass oligomers were detected by SDS-PAGE after incubation in solutions containing over 20 mM NaCl at pH 6.5–8.5, even in the absence of lipid addition, and the addition of Ca2+/calmodulin abolished oligomerization. Higher molecular mass oligomer formation after incubation with acidic phospholipids was detected with gradient SDS-PAGE. Much higher mass oligomers were detected in the presence of polyunsaturated fatty acids. Electron microscopic analysis of the samples after SDS treatment showed tangled rope-like structures. Liposome-bound NAP-22 showed small oval or annular structures after cross-linking and SDS treatment. These oligomers were suggested to make the tangled rope-like structures, for annular structures of the same size were observed in the structure. These results suggest the participation of NAP-22 to liquid-liquid phase separation through oligomerization.