Resonance assignment of13C−15N-labeled snake neurotoxin II fromNaja oxiana

Bocharov, Eduard V.; Lyukmanova, Ekaterina N.; Ermolyuk, Yaroslav S.; Schulga, Alexey; Pluzhnikov, Kirill A.; Долгих, Д. А.; Kirpichnikov, M. P.; Arseniev, Alexander S.

doi:10.1007/bf03166664

Cited by 12 publications

(6 citation statements)

References 10 publications

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“…We have compared the chemical shifts of the solid‐state resonances with the assignment obtained on free NTII from solution NMR [16]. As an example, the 13 C chemical shifts of the threonine in solution are indicated in the solid‐state spectrum, shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid‐state NMR approach

Krabben¹,

Rossum²,

Castellani³

et al. 2004

FEBS Letters

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Solid-state magic-angle spinning nuclear magnetic resonance (NMR) has su⁄cient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptort oxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13 C-, 15 N-labelled neurotoxin II. Despite the large size of the ligand^receptor complex ( s 290 kDa) and the high lipid content of the sample, we were able to detect and identify residues from the ligand. The comparison with solution NMR data of the free toxin indicates that its overall structure is very similar when bound to the receptor, but signi¢-cant changes were observed for one isoleucine.

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Section: Resultsmentioning

confidence: 99%

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid‐state NMR approach

Krabben¹,

Rossum²,

Castellani³

et al. 2004

FEBS Letters

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“…Chimeras were produced by bacterial expression, which was quite successful in the case of NTII and its 15 N-13 C-labeled analogue (36). Introduction of the fifth disulfide into the neurotoxin molecule resulted in a lower yield of the target protein.…”

Section: Discussionmentioning

confidence: 99%

“…First, NTII has low activity against neuronal nAChRs (IC 50 Ͼ 1 M), thus providing reliable reference for detection of even subtle changes in affinity. Second, high level bacterial expression systems for this toxin had previously been developed (32,36) and could be adapted for production of chimeras.…”

Section: Construction Of Short/long-chain ␣-Neurotoxin Chimeras-mentioning

confidence: 99%

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Lyukmanova

Шенкарев

Schulga

et al. 2007

Journal of Biological Chemistry

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Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain ␣-neurotoxins preferentially inhibit muscle-type nAChRs, whereas longchain ␣-neurotoxins block both muscle-type and ␣7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of ␣-and -neurotoxins is essential for their action on the ␣7 and ␣3␤2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfidecontaining loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain ␣-neurotoxins and -neurotoxins. The correct folding and structural stability for both chimeras were shown by 1 H and 1 H-15 N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I block ␣7 nAChRs with similar potency (IC 50 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain ␣-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for ␣7 nAChR (IC 50 126 nM) but did not convey activity at ␣3␤2 nAChRs. Docking of both chimeras to ␣7 and ␣3␤2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of -neurotoxins underlie their selectivity for ␣3␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 belong to the class of ligand-gated ion channels (1-3). All nAChRs are composed of five homologous subunits, which span the plasma membrane to form a transmembrane ion-conducting pore. The muscle-type receptors are composed of four types of subunits, ␣2␤␥␦ in the fetal and ␣2␤⑀␦ in the mature receptor. The best studied nAChR of this type is the receptor from electric organ of ray Torpedo marmorata (4). Neuronal receptors consist of two types of subunits, ␣ (␣2-␣10) and ␤ (␤2-␤4), and can have homooligomeric (for example, ␣7-type nAChR) or heterooligomeric (for example, ␣3␤2-type receptor) composition. Neuronal nAChRs are widely expressed both pre-and postsynaptically, and presynaptic receptors modulate synaptic activity and the release of different neurotransmitters. Neuronal nAChRs are found also in non-neuronal tissues, such as keratinocytes and pulmonary and other tissues (5-8).Because of their role in cellular communication, nAChRs play important roles in the normal functioning of the organism. Dysfunctions of these receptors have been proposed to contribute to a number of disorders of the central and peripheral nervous systems such as schizophrenia, epilepsy, depression, nicotine and alcohol dependence, Alzheimer disease, Parkinson disease, and autis...

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“…coli as described previously [29]. Recombinant analogue of neurotoxin II from Naja oxiana was obtained as in [30]. The purity and homogeneity of the protein preparations were confirmed by HPLC, MALDI-MS, and SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Water-soluble variant of human Lynx1 induces cell cycle arrest and apoptosis in lung cancer cells via modulation of α7 nicotinic acetylcholine receptors

et al. 2019

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Lynx1 is the first three-finger prototoxin found in the mammalian central nervous system. It is a GPI-anchored protein modulating nicotinic acetylcholine receptors (nAChRs) in the brain. Besides the brain, the Lynx1 protein was found in the lung and kidney. Endogenous Lynx1 controls the nicotine-induced up-regulation of the expression of α7 type nAChRs in lung adenocarcinoma A549 cells as well as the cell growth. Here, we analyzed the Lynx1 expression in the set of human epithelial cells. The Lynx1 expression both at the mRNA and protein level was detected in normal oral keratinocytes, and lung, colon, epidermal, and breast cancer cells, but not in embryonic kidney cells. Co-localization of Lynx1 with α7-nAChRs was revealed in a cell membrane for lung adenocarcinoma A549 and colon carcinoma HT-29 cells, but not for breast adenocarcinoma MCF-7 and epidermoid carcinoma A431 cells. The recombinant water-soluble variant of Lynx1 without a GPI-anchor (ws-Lynx1) inhibited the growth of A549 cells causing cell cycle arrest via modulation of α7-nAChRs and activation of different intracellular signaling cascades, including PKC/IP 3 , MAP/ERK, p38, and JNK pathways. A549 cells treatment with ws-Lynx1 resulted in phosphorylation of the proapoptotic tumor suppressor protein p53 and different kinases participated in the regulation of gene transcription, cell growth, adhesion, and differentiation. Externalization of phosphatidylserine, an early apoptosis marker, observed by flow cytometry, confirmed the induction of apoptosis in A549 cells upon the ws-Lynx1 treatment. Our data revealed the ability of ws-Lynx1 to regulate homeostasis of epithelial cancer cells.

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Resonance assignment of13C−15N-labeled snake neurotoxin II fromNaja oxiana

Cited by 12 publications

References 10 publications

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid‐state NMR approach

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid‐state NMR approach

Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Water-soluble variant of human Lynx1 induces cell cycle arrest and apoptosis in lung cancer cells via modulation of α7 nicotinic acetylcholine receptors

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