Human Three-Finger Protein Lypd6 Is a Negative Modulator of the Cholinergic System in the Brain

Abstract

Lypd6 is a GPI-tethered protein from the Ly-6/uPAR family expressed in the brain. Lypd6 enhances the Wnt/β-catenin signaling, although its action on nicotinic acetylcholine receptors (nAChRs) have been also proposed. To investigate a cholinergic activity of Lypd6, we studied a recombinant water-soluble variant of the human protein (ws-Lypd6) containing isolated “three-finger” LU-domain. Experiments at different nAChR subtypes expressed in Xenopus oocytes revealed the negative allosteric modulatory activity of ws-Lypd6. Ws-Lypd6 inhibited ACh-evoked currents at α3β4- and α7-nAChRs with IC50 of ∼35 and 10 μM, respectively, and the maximal amplitude of inhibition of 30–50%. EC50 of ACh at α3β4-nAChRs (∼30 μM) was not changed in the presence of 35 μM ws-Lypd6, while the maximal amplitude of ACh-evoked current was reduced by ∼20%. Ws-Lypd6 did not elicit currents through nAChRs in the absence of ACh. Application of 1 μM ws-Lypd6 significantly inhibited (up to ∼28%) choline-evoked current at α7-nAChRs in rat hippocampal slices. Similar to snake neurotoxin α-bungarotoxin, ws-Lypd6 suppressed the long-term potentiation (LTP) in mouse hippocampal slices. Colocalization of endogenous GPI-tethered Lypd6 with α3β4- and α7-nAChRs was detected in primary cortical and hippocampal neurons. Ws-Lypd6 interaction with the extracellular domain of α7-nAChR was modeled using the ensemble protein-protein docking protocol. The interaction of all three Lypd6 loops (“fingers”) with the entrance to the orthosteric ligand-binding site and the loop C of the primary receptor subunit was predicted. The results obtained allow us to consider Lypd6 as the endogenous negative modulator involved in the regulation of the cholinergic system in the brain.

New Three-Finger Protein from Starfish Asteria rubens Shares Structure and Pharmacology with Human Brain Neuromodulator Lynx2

“…Previously described values of IC 50 for human TFPs at different nAChRs expressed in oocytes lay in micromolar ranges. For example: IC 50 of Lypd6 at α3β4-nAChR is ~40 µM [ 15 ], IC 50 of Lynx1 at α7-nAChR is ~50 µM [ 46 ], and IC 50 of SLURP-1 at α7-nAChR is ~10 µM [ 47 ]. Thus, we used here the single concentration of Lystar5 (50 µM).…”

“…Moreover, homologous TFPs of A. rubens and A. planci demonstrate significant sequence similarity with the LYPD6, LYPD6B, and Lynx2 (LYPD1) proteins from different species ( Figure 2 and Figure 3 ). LYPD6 was found in many organisms from fish to human [ 11 , 13 , 22 ]; it is expressed in lung, kidneys, heart, liver, prostate, and brain [ 13 , 52 ], and is involved in the regulation of the cholinergic and Wnt signaling [ 11 , 15 , 22 ]. The function of LYPD6B is not well-studied, although it has high degree of the sequence similarity with LYPD6, so we can propose a similar function.…”

“…It is known that the molecular targets of LYPD6 and Lynx2 include different subtypes of nAChR [ 8 , 9 , 15 ]. Consistent with this, the starfish homologue of Lynx2 - Lystar5 - targets high selective (HS) α4β2-nAChRs ( Figure 8 ).…”

“…Most of the mammalian TFPs studied to date (Lynx1, Lynx2 (LYPD1), Lypd6, Lypd6b, SLURP-1, SLURP-2) belong to the nervous system or to the epithelium [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ] and target different receptors, for example, nicotinic acetylcholine receptors (nAChRs). In contrast to snake toxins, which are typically high-affinity receptor inhibitors, mammalian TFPs do not exhibit complete inhibition of the receptors, but modulate them over the micromolar concentration range [ 7 , 8 , 12 , 13 , 14 , 15 , 16 , 17 ]. Other TFPs regulate the complement system in the mammal blood (CD59 [ 3 ]), are a part of plasminogen activation system (uPAR is urokinase receptor [ 18 ]), or even modulate viral infection (Ly6E [ 19 , 20 , 21 ]).…”

Chrna5 and lynx prototoxins identify acetylcholine super-responder subplate neurons

“…The change in peak and area of the nicotinic responses caused by solube Ly6g6e was significantly different between Chrna5+ and Syt6+ neurons (change in peak : t (14) = 3.43, P = 0.004; Change in area: t (14) = 2.53, P = 0.024, Unpaired t test; Fig 4I - J ). Of note, soluble and endogenous GPI-anchored prototoxins are known to have opposite effects on nicotinic receptors and the exact direction of endogenous modulation of nicotinic receptors by different lynx proteins is still debated (Arvaniti et al, 2016; Kulbatskii et al, 2021; Miwa, 2021). The key outcome of this experiment is the difference in the Ly6g6e modulation of Chrna5+ and Syt6+ neurons, not the direction.…”