Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>

The <i>Sphenophorus levis</i> (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old la...

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Autores principales:	Priscila Yumi Tanaka Shibao, Milene Ferro, Fernando Fonseca Pereira de Paula, Bruno Salata Lima, Flávio Henrique-Silva
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	pseudoenzyme cysteine protease coleoptera protease inhibitor insect digestion Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/c9da468f32fc45858344913abfd4c4fc
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spelling	oai:doaj.org-article:c9da468f32fc45858344913abfd4c4fc2021-11-11T16:56:37ZIdentification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>10.3390/ijms2221114761422-00671661-6596https://doaj.org/article/c9da468f32fc45858344913abfd4c4fc2021-10-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11476https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The <i>Sphenophorus levis</i> (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC (V<sub>max</sub> = 1017.60 ± 135.55, K<sub>m</sub> = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (K<sub>i</sub> = 38.52 ± 1.20 μM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.Priscila Yumi Tanaka ShibaoMilene FerroFernando Fonseca Pereira de PaulaBruno Salata LimaFlávio Henrique-SilvaMDPI AGarticlepseudoenzymecysteine proteasecoleopteraprotease inhibitorcysteine proteaseinsect digestionBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11476, p 11476 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	pseudoenzyme cysteine protease coleoptera protease inhibitor cysteine protease insect digestion Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	pseudoenzyme cysteine protease coleoptera protease inhibitor cysteine protease insect digestion Biology (General) QH301-705.5 Chemistry QD1-999 Priscila Yumi Tanaka Shibao Milene Ferro Fernando Fonseca Pereira de Paula Bruno Salata Lima Flávio Henrique-Silva Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>
description	The <i>Sphenophorus levis</i> (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC (V<sub>max</sub> = 1017.60 ± 135.55, K<sub>m</sub> = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (K<sub>i</sub> = 38.52 ± 1.20 μM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.
format	article
author	Priscila Yumi Tanaka Shibao Milene Ferro Fernando Fonseca Pereira de Paula Bruno Salata Lima Flávio Henrique-Silva
author_facet	Priscila Yumi Tanaka Shibao Milene Ferro Fernando Fonseca Pereira de Paula Bruno Salata Lima Flávio Henrique-Silva
author_sort	Priscila Yumi Tanaka Shibao
title	Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>
title_short	Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>
title_full	Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>
title_fullStr	Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>
title_full_unstemmed	Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>
title_sort	identification and functional analysis of a pseudo-cysteine protease from the midgut transcriptome of <i>sphenophorus levis</i>
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/c9da468f32fc45858344913abfd4c4fc
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Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of <i>Sphenophorus levis</i>

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