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• Gladkikh, I
• Monastyrnaya, M
• Leychenko, E
• Zelepuga, E
• Chausova, V
• Isaeva, M
• Anastyuk, S
• Andreev, Y
• Peigneur, S
• Tytgat, J
• Kozlovkaya, E
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• Gladkikh, I
• Monastyrnaya, M
• Leychenko, E
• Zelepuga, E
• Chausova, V
• Isaeva, M
• Anastyuk, S
• Andreev, Y
• Peigneur, S
• Tytgat, J
• Kozlovkaya, E
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• Gladkikh, I
• Monastyrnaya, M
• Leychenko, E
• Zelepuga, E
• Chausova, V
• Isaeva, M
• Anastyuk, S
• Andreev, Y
• Peigneur, S
• Tytgat, J
• Kozlovkaya, E

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Mar. Drugs 2012, 10(7), 1545-1565; doi:10.3390/md10071545

Article

Atypical Reactive Center Kunitz-Type Inhibitor from the Sea Anemone Heteractis crispa

Irina Gladkikh ^1,* , Margarita Monastyrnaya ¹ , Elena Leychenko ¹ , Elena Zelepuga ¹ , Victoria Chausova ¹ , Marina Isaeva ¹ , Stanislav Anastyuk ¹ , Yaroslav Andreev ² , Steve Peigneur ³ , Jan Tytgat ³  and Emma Kozlovkaya ¹

¹ G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation ² Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10, Miklukho-Maklaya Str., Moscow 117997, Russian Federation ³ Laboratory of Toxicology, University of Leuven (K.U. Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven B-3000, Belgium

* Author to whom correspondence should be addressed.

Received: 18 April 2012; in revised form: 4 July 2012 / Accepted: 11 July 2012 / Published: 19 July 2012

(This article belongs to the Special Issue Sea Anemone Toxins)

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Abstract: The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the K_i of InhVJ to trypsin and α-chymotrypsin (7.38 × 10⁻⁸ M and 9.93 × 10⁻⁷ M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.

Keywords: sea anemone; Kunitz-type protease inhibitor; structure; function; channels

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