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

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
(IC50 = 471 nM) and human prolyl oligopeptidase (IC50 = 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.