Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

Xinying Jia,Soohyun Kwon,Ching-I Anderson Wang,Yen-Hua Huang,Lai Y Chan,Chia Chia Tan,K Johan Rosengren,Jason P Mulvenna,Christina I Schroeder,David J Craik

doi:10.1074/jbc.m113.539262

Abstract

Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

Highlights

Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds
The linear peptide [GGG]kalata B1 (kB1)[TGG] was oxidized prior to cyclization with SrtA, which proceeded without the addition of reducing agent to the reaction buffer
Interest has been shown both in the cyclization of linear disulfide-rich peptides and the development of native cyclotides as therapeutics and in their use as scaffolds for the grafting of pharmaceutically relevant epitopes onto the native cyclotide framework [74, 75]

Summary

Background

Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds. We demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, ␣-conotoxin Vc1.1, and sunflower trypsin inhibitor 1 These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Cyclotides are able to accommodate the synthetic introduction of a range of biologically active sequence motifs in the backbone loops between the conserved Cys residues while retaining thermal, chemical, and enzymatic stability This tolerance to sequence substitutions makes them ideal scaffolds for the development of stable peptide-based drugs incorporating epitopes of therapeutic value. This study provides a proof of concept that SrtA-mediated ligation can be utilized to cyclize disulfide-rich peptides ranging from the simple one-disulfidecontaining SFTI-1 to the complex cyclic cystine knot-containing cyclotide structure of kB1 while retaining the native disulfide bond connectivity that is vital for the stability of these proteins.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION