Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species

Hernández de la Torre, Martha; Covaleda-Cortés, Giovanni; Montesinos, Laura; Covaleda, Daniela; Ortiz, Juan C.; Piñol, Jaume; Bautista, José M.; Castillo, J. Patricio; Reverter, David; Avilés, Francesc Xavier

doi:10.3390/ijms26031150

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Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species

by

Martha Hernández de la Torre

^1,†

,

Giovanni Covaleda-Cortés

^2,†

,

Laura Montesinos

³

,

Daniela Covaleda

^4,5

,

Juan C. Ortiz

⁵

,

Jaume Piñol

⁴,

José M. Bautista

⁶

,

J. Patricio Castillo

⁷

,

David Reverter

⁴

and

Francesc Xavier Avilés

^4,*

¹

Facultad de Ciencias Forestales, Universidad de Concepción, Concepción 4030000, Chile

²

Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain

³

Institute of Food and Agricultural Technology-CIDSAV-XaRTA, University of Girona, 17004 Girona, Spain

⁴

Institut de Biotecnologia i Biomedicina, and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain

⁵

Institut de Biotecnologia i Biomedicina, and Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain

⁶

Department of Biochemistry and Molecular Biology, Universidad Complutense de Madrid, 28040 Madrid, Spain

⁷

Departamento de Ciencias Nucleares, Escuela Politécnica Nacional, Quito 170143, Ecuador

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Int. J. Mol. Sci. 2025, 26(3), 1150; https://doi.org/10.3390/ijms26031150

Submission received: 9 December 2024 / Revised: 21 January 2025 / Accepted: 21 January 2025 / Published: 28 January 2025

(This article belongs to the Special Issue Advances in Enzyme Inhibitors: Mechanisms, Applications, and Future Perspectives)

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Abstract

Protease inhibitors are biomolecules with growing biotechnological and biomedical relevance, including those derived from plants. This study investigated strong trypsin inhibitors in quinoa, amaranth, and lupine seeds, plant grains traditionally used in Andean South America. Amaranth seeds displayed the highest trypsin inhibitory activity, despite having the lowest content of aqueous soluble and thermostable protein material. This activity, directly identified by enzymatic assay, HPLC, intensity-fading mass spectrometry (IF-MS), and MS/MS, was attributed to a single protein of 7889.1 Da, identified as identical in Amaranthus caudatus and A. hybridus, with a K_i of 1.2 nM for the canonical bovine trypsin. This form of the inhibitor, which is highly homogeneous and scalable, was selected, purified, and structurally–functionally characterized due to the high nutritional quality of amaranth seeds as well as its promising agriculture–biotech–biomed applicability. The protein was crystallized in complex with bovine trypsin, and its 3D crystal structure resolved at 2.85 Å, revealing a substrate-like transition state interaction. This verified its classification within the potato I inhibitor family. It also evidenced that the single disulfide bond of the inhibitor constrains its binding loop, which is a key feature. Cell culture assays showed that the inhibitor did not affect the growth of distinct plant microbial pathogen models, including diverse bacteria, fungi, and parasite models, such as Mycoplasma genitalium and Plasmodium falciparum. These findings disfavour the notion that the inhibitor plays an antimicrobial role, favouring its potential as an agricultural insect deterrent and prompting a redirection of its functional research.

Keywords: plant trypsin inhibitors; quinoa; amaranth; lupine seeds; Amaranthus hybridus and Amaranthus caudatus; HPLC; MS and X-ray analysis; structure–function characterisation; plant defence

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MDPI and ACS Style

Hernández de la Torre, M.; Covaleda-Cortés, G.; Montesinos, L.; Covaleda, D.; Ortiz, J.C.; Piñol, J.; Bautista, J.M.; Castillo, J.P.; Reverter, D.; Avilés, F.X. Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species. Int. J. Mol. Sci. 2025, 26, 1150. https://doi.org/10.3390/ijms26031150

AMA Style

Hernández de la Torre M, Covaleda-Cortés G, Montesinos L, Covaleda D, Ortiz JC, Piñol J, Bautista JM, Castillo JP, Reverter D, Avilés FX. Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species. International Journal of Molecular Sciences. 2025; 26(3):1150. https://doi.org/10.3390/ijms26031150

Chicago/Turabian Style

Hernández de la Torre, Martha, Giovanni Covaleda-Cortés, Laura Montesinos, Daniela Covaleda, Juan C. Ortiz, Jaume Piñol, José M. Bautista, J. Patricio Castillo, David Reverter, and Francesc Xavier Avilés. 2025. "Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species" International Journal of Molecular Sciences 26, no. 3: 1150. https://doi.org/10.3390/ijms26031150

APA Style

Hernández de la Torre, M., Covaleda-Cortés, G., Montesinos, L., Covaleda, D., Ortiz, J. C., Piñol, J., Bautista, J. M., Castillo, J. P., Reverter, D., & Avilés, F. X. (2025). Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species. International Journal of Molecular Sciences, 26(3), 1150. https://doi.org/10.3390/ijms26031150

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Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species

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