Discovery of a Beetroot Protease Inhibitor to Identify and Classify Plant-Derived Cystine Knot Peptides

Abstract

Plant peptide protease inhibitors are important molecules in seed storage metabolism and to fight insect pests. Commonly they contain multiple disulfide bonds and are exceptionally stable molecules. In this study, a novel peptide protease inhibitor from beetroot ( Beta vulgaris ) termed bevuTI-I was isolated, and its primary structure was determined via mass spectrometry-based amino acid sequencing. By sequence homology analysis a few peptides with high similarity to bevuTI-I, also known as the Mirabilis jalapa trypsin inhibitor subfamily of knottin-type protease inhibitors, were discovered. Hence, we assessed bevuTI-I for inhibitory activity toward trypsin (IC 50 = 471 nM) and human prolyl oligopeptidase (IC 50 = 11 μM), which is an emerging drug target for neurodegenerative and inflammatory disorders. Interestingly, using a customized bioinformatics approach, bevuTI-I was found to be the missing link to annotate 243 novel sequences of M. jalapa trypsin inhibitor-like peptides. According to their phylogenetic distribution they appear to be common in several plant families. Therefore, the presented approach and our results may help to discover and classify other plant-derived cystine knot peptides, a class of plant molecules that play important functions in plant physiology and are currently being explored as lead molecules and scaffolds in drug development.

Cyclotides Isolated From Violet Plants of Cameroon Are Inhibitors of Human Prolyl Oligopeptidase

“…A Michaelis-Menten plot was generated and K m of 42.4 µM and V max of 0.00015 μmol/min were obtained ( Figure 3A ), which are in agreement with published data. The performed protease inhibition assay is based on the system described by Toide et al (1995) and updated protocols were described previously ( Hellinger et al, 2015a ; Retzl et al, 2020 ). Using a 96-well plate assay, enzyme activity was determined in the presence of different concentrations of plant extracts.…”

“…To date, it has not been studied if cyclotide inhibitors of POP take advantage of the cyclic backbone similar as cyclic trypsin inhibitors, or if the ligation loop itself is the inhibition motif for protease. However, a cyclic backbone appeared not mandatory for inhibition of POP by the knottin peptide bevuTI-I, which showed overall very similar inhibition activity compared to cyclotide inhibitors ( Retzl et al, 2020 ). Since cyclotides comprise up to three prolines it will be necessary to consider all of them for the identification of the putative binding site to POP.…”

“…Bioactivity-guided fractionation of plant extracts from A. cauliflora were performed with the aim to isolate bioactive plant peptides. The technique has been applied successfully in the past to identify and isolate bioactive cyclotides from plants ( Tarrago et al, 2007 ; Hellinger et al, 2015a ; Retzl et al, 2020 ). Several purification steps yielded two A. cauliflora peptides, denoted alca 1 and alca 2, which obtained an IC 50 between 4 and 8 µM for POP.…”

“…The POP inhibition assays were carried out similarly as recently described ( Hellinger et al, 2015a ; Retzl et al, 2020 ) using a bacterial expressed POP enzyme. Human POP cDNA (accession NM:002726.4) was inserted into a pGEX-6P1 expression vector using the EcoRI and XhoI sites (Genescript, Leiden, Netherlands) and expressed in Escherichia coli (BL21 D3 strain).…”

“…balcalein) inhibit POP activity ( Tarrago et al, 2007 ; Tarragó et al, 2008 ; Cahlíková et al, 2015 ). Importantly, plant peptides have been shown to inhibit POP such as the trypsin inhibitor peptide bevuTI-I isolated from Beta vulgaris L. ( Retzl et al, 2020 ), or cyclotides. The cyclotides psysol 2 ( Psychotria solitudinum Standl.)…”