A new protein glosaxin consisting of non-catalytic domains of type piii metalloproteinase from the venom of pit viper Gloydius saxatilis inhibits nicotinic acetylcholine receptor

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Abstract

Previously, we found that the venom of the pit viper Gloydius saxatilis inhibited the muscle-type nicotinic acetylcholine receptor (nAChR). Using liquid chromatography, a protein glosaxin was isolated from the venom that inhibited the binding of the α-bungarotoxin to the nAChR of muscle type from Torpedo californica. The amino acid sequence of the isolated protein was analyzed by high resolution mass spectrometry. Subsequent bioinformatic analysis showed that it is homologous to the amino acid sequences of disintegrin-like proteins, consisting of non-catalytic domains of type PIII metalloproteinases from the venom of pit vipers of genus Gloydius. A study of the biological activity of the isolated protein showed that it inhibits the binding of α-bungarotoxin to Torpedo californica nAChR with IC50 = 51 μM. The protein also inhibited acetylcholine-induced functional responses of the human neuronal nAChR of α3β2 subtype. This is the first evidence of the ability of proteins consisting of non-catalytic domains of snake venom type PIII metalloproteinase to inhibit the nAChR.

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About the authors

A. V. Osipov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. V. Kryukova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

L. O. Ojomoko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

I. V. Shelukhina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

R. H. Ziganshin

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

V. G. Starkov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

T. V. Andreeva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

V. I. Tsetlin

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

Yu. N. Utkin

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Author for correspondence.
Email: utkin@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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Supplementary files

Supplementary Files
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2. Fig. 1. Separation of G. saxatilis venom by gel filtration on a Superdex 75 column (10 × 300 mm) equilibrated with 0.1 M ammonium acetate (pH 6.2) at a flow rate of 1 ml/min. Horizontal lines indicate the collected fractions.

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3. Fig. 2. Separation of fraction I (see Fig. 1) by anion exchange chromatography on a Mono Q column (4.6 × 100 mm) using a concentration gradient of sodium chloride 0.05–0.55 M over 100 min in 20 mM ethanolamine-HCl buffer (pH 9.5) at a flow rate of 0.5 ml/min. Horizontal lines show the collected fractions.

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4. Fig. 3. Fragment of the high-resolution mass spectrum of the protein from fraction 5 (Fig. 2). The signals corresponding to the ions [M + 13H]+13, z = 13 are shown.

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5. Fig. 4. Amino acid sequence of the halysetin protein (UniProtKB: VM3H_GLOHA) and the corresponding peptides found in the hydrolysate of the protein we isolated (shown as blue lines).

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6. Fig. 5. Comparison of amino acid sequences of non-catalytic domains of metalloproteinases from some species of copperhead snakes (genus Gloydius). VM3H_GLOHA is halysetin from the venom of Agkistrodon (Gloydius) halys, A0A0C4ZNF1_GLOIT is a fragment of PIII metalloproteinase from Gloydius intermedius, VM2SA_GLOSA is a fragment of PII metalloproteinase from Gloydius saxatilis, VM2_GLOHA is the disintegrin saxatilin from Gloydius saxatilis. The lines above the sequences indicate the peptides of halysetin detected by mass spectrometry in the hydrolysate of our protein. The residues of the active sites are underlined, identical residues are highlighted in gray.

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7. Fig. 6. Inhibition of binding of radioactive α-Bgt to T. californica nAChR (squares) and the α7-subtype (circle) by glosaxin. For T. californica nAChR, IC50 = 50.9 ± 1.83 μM. M is the molar concentration of the protein.

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8. Fig. 7. Inhibition of acetylcholine-induced currents in α3β2-type nAChR by glossaxin. The response of X. laevis oocytes to 50 μM acetylcholine without addition of protein (100%) and after 1 min of incubation with different protein concentrations (n ​​= 3) was recorded. * p < 0.05 (Student's t-test).

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