Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase.

Karen S Harris,Thomas Durek,David J Craik,Norelle L Daly,Nicole L Van Der Weerden,Quentin Kaas,Ivana Saska,Edward K Gilding,Aaron G Poth,Marilyn A Anderson,Brendon F Conlan

doi:10.1038/ncomms10199

Abstract

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

Highlights

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive
In the two other classes of plant-derived cyclic peptides, strong Asx sequence conservation at the C-terminal P1 site implicates as possible cyclases the asparaginyl endopeptidases (AEPs), a group of cysteine proteases known as vacuolar processing enzymes or Legumains, and this hypothesis is supported by studies in transgenic plants[8,9,21,22]
Three expressed AEP isoforms were identified in an O. affinis complementary DNA library (OaAEP1-3) and a fourth sequence, with a single nucleotide change from OaAEP1, was identified from genomic DNA (OaAEP1b) (Fig. 2a; Supplementary Fig. 1)

Summary

Introduction

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Proteases can ligate polypeptides, producing new or alternatively spliced variants This unusual function has been reported for processes such as the maturation of the lectin concanavalin A1, peptide presentation by major histocompatibility complex class I molecules[2], and anchoring of bacterial proteins to the cell wall[3]. Cyclotides are a well-studied class of gene-encoded cyclic peptides that are expressed in plants and exhibit a range of bioactivities including insecticidal, nematicidal and molluscicidal activity against agricultural pests[11,12,13,14] They are characterized by a cyclic cystine knot motif that confers exceptional stability. Its specificity for model peptides mirrors the sequence requirements for cyclization of kB1 in transgenic plants, supporting a native function in the maturation of O. affinis cyclotides[8,21]

Methods

Results

Conclusion