Hololectin Interdomain Linker Determines Asparaginyl Endopeptidase-Mediated Maturation of Antifungal Hevein-Like Peptides in Oats

Abstract

Heveins and hevein-containing (hev-) lectins play important roles in stress and pathogenic responses in plants but cause health concerns in humans. Hev-hololectins contain multiple modular hev-peptide domains and are abundantly present in cereals and pseudocereals. However, it is unclear why some cereal hev-hololectins are presented as different forms of proteolytically processed proteoforms. Here we show the precursor architectures of hev-hololectins lead to different processing mechanisms to give either hololectins or hevein-like peptides. We used mass spectrometry and datamining to screen hev-peptides from common cereals, and identified from the oat plant Avena sativa nine novel hevein-like peptides, avenatide aV1–aV9. Bioinformatic analysis revealed that asparaginyl endopeptidase (AEP) can be responsible for the maturation of the highly homologous avenatides from five oat hev-hololectin precursors, each containing four tandemly repeating, hev-like avenatide domains connected by AEP-susceptible linkers with 13–16 residues in length. Further analysis of cereal hev-hololectins showed that the linker lengths provide a distinguishing feature between their cleavable and non-cleavable precursors, with the cleavables having considerably longer linkers (>13 amino acids) than the non-cleavables (<6 amino acids). A detailed study of avenatide aV1 revealed that it contains eight cysteine residues which form a structurally compact, metabolic-resistant cystine-knotted framework with a well-defined chitin-binding site. Antimicrobial assays showed that avenatide aV1 is anti-fungal and inhibits the growth of phyto-pathogenic fungi. Together, our findings of cleavable and non-cleavable hololectins found in cereals expand our knowledge to their biosynthesis and provide insights for hololectin-related health concerns in human.

Decoding the Cure-all Effects of Ginseng

“…The identification of ginsentides as bioactive and proteolytic-stable peptides underscore the emergence of constrained peptides as active compounds in herbal medicines. Ginsentides, similar to other cysteine-rich peptides, including roseltides, bleogens, chenotides, and avenatides 14,15,57,58 , are structurally constrained by multiple disulfides to render them resistant to proteolytic degradation, making them potentially orally active 14,15,16,17,29 . Because ginsentides have a cystine-dense core, they have an "inside-out" conformation, causing the side chain of hydrophobic amino acids to be exposed 17,29 .…”

Ginsentide TP1 Protects Hypoxia-Induced Dysfunction and ER Stress-Linked Apoptosis

“…TP1. The cell-penetrating property can also be found in certain highly cystine-braced microproteins, such as the plant-derived 8C-hevein-like peptides (20)(21)(22)(23)(24)26). Thus, hyperstability and cell-penetrating properties of ginsentides are desirable attributes for drug development.…”

“…TP1 and other members of ginsentides (TP2-14) form a new CRP family. They contain eight cysteine residues and a cysteine motif similar to heveins found in Hevea brasiliensis and hevein-like peptides, but they can be distinguished from heveins by distinctly different disulfide connectivity, biosynthesis, and the absence of a chitin-binding domain which is essential for the host-defense functions of heveins and hevein-like peptides (19)(20)(21)(22)(23)(24). Because ginsentide TP1 is a prototype, its biological functions have not been explored.…”

Hyperstable EGF-like bleogen derived from cactus accelerates corneal healing in rats

“…Unlike EGF and its related family, bleogen pB1 has a single domain and a cystine-knotted structure. Similar to other cysteine-rich peptides and due to its highly compact cystine-knot scaffold, bleogen pB1 is more than 100 times more resistant to proteolysis than the mammalian EGF ( Loo et al, 2016 ; Loo et al, 2017 ; Tam et al, 2018 ; Kam et al, 2019b ; Loo et al, 2021a ; Loo et al, 2021b ; Loo et al, 2022 ). Structural stability and proteolytic-resistant properties are highly desirable features for growth factors: they reduce their degradation and improve their cost-effectiveness, formulation feasibility, shelf life, and commercial viability for the development of ophthalmic formulations for corneal injury.…”