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

Autor(es)

Shibao, Priscila Yumi Tanaka ^[1] ; Ferro, Milene ^[1] ; de Paula, Fernando Fonseca Pereira ^[1] ; Lima, Bruno Salata ^[1] ; Henrique-Silva, Flavio ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Dept Genet & Evolut, Mol Biol Lab, Rodovia Washington Luis, Km 235, BR-13565905 Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 22, n. 21 NOV 2021.
• Citações Web of Science: 0

Resumo

The Sphenophorus levis (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-max = 1017.60 \& PLUSMN; 135.55, K-m = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (K-i = 38.52 \& PLUSMN; 1.20 mu M), 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. (AU)

• Processo FAPESP: 17/16118-1 - Produção de de canacistatina em vacúolos de cana-de-açúcar transgênica
• Beneficiário: Priscila Yumi Tanaka Shibao
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/11641-7 - Silenciamento gênico aplicado ao desenvolvimento de plantas de cana-de-açúcar transgênicas resistentes ao inseto Sphenophorus levis, o bicudo da cana-de-açúcar
• Beneficiário: Fernando Fonseca Pereira de Paula
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado