Detection of a Peptide Biomarker by Engineered Yeast Receptors

Abstract

Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.

Keywords: G-protein coupled receptors; diagnostics; directed evolution; receptor engineering; substrate walking.

Figure 1

Substrate walking directed evolution strategy to evolve a receptor to detect a peptide biomarker. Mutant receptors and experimental conditions used in the directed evolution of Ste2 to detect a cystatin C peptide are detailed above. Though evolved to detect peptide Cys5, receptors Cys5R2, Cys5R7, and Cys5R9 can detect the cystatin C peptide Cys7 (ALDFAVGEYNK).

Figure 2

Response of native and mutant receptors to chimeric ligands. Response of mutant receptors to chimeric intermediates throughout the evolutionary pathway is shown here. Responsiveness for Cys7, the diagnostic peptide, is gradually gained through the course of evolution. For peptides Cys1–Cys3, each data point is the average for n = 2 technical replicates. For all other peptides, each data point is the average of n = 3 technical replicates. The experiment was replicated one additional time in our laboratory with comparable results.

Figure 3

Evolved receptors respond in a dose-dependent manner to target ligands. (A) The native Ste2p receptor does not respond to chimeric ligand Cys1. Evolved receptors Cys1H4 and Cys1H5 respond in a dose dependent manner to ligand Cys1. Each data point is the average shown for n = 2 technical replicates. (B) The native Ste2p receptor does not respond to chimeric ligand Cys2, but evolved receptors Cys2K2 and Cys2K3 respond in a dose dependent manner to ligand Cys2. (C) The native Ste2p receptor does not respond to chimeric ligand Cys4. Evolved receptors Cys2K3 and Cys4L3 respond in a dose dependent manner to ligand Cys4. (D) Evolved receptors Cys5R2, Cys5R7, and Cys5R9, and native receptor Ste2p respond to amidated biomarker Cys7. The evolved receptors display significantly higher responsiveness and sensitivity when detecting amidated biomarker Cys7 as compared to the native receptor. (A–D) Unless otherwise noted, each data point is the average shown for n = 3 technical replicates and error bars represent standard error of measurement. For all receptor–ligand pairs, EC50 and Hill slope values are reported in Supplementary Table 1. (E) Snake plot showing mutations acquired during directed evolution for receptor Cys5R7, which displayed the highest sensitivity toward the cystatin C peptide Cys7.

Figure 4

Engineered receptors are sensitive and specific in human urine. Ste2p and the Cys7 responsive receptors were exposed to pooled human urine digested with trypsin and spiked with Cys7. The ability of receptors Cys4L3, Cys5R2, Cys5R7, and Cys5R9 to detect the Cys7 fragment in human urine is comparable to the receptor performance in media. Trypsinized urine does not spontaneously activate the receptor. Each data point is the average shown for n = 3 samples and error bars represent standard error. **p < 0.001. *p < 0.05 as determined by two-way ANOVA. This experiment was not reproduced beyond the technical replicates.

Figure 5

Prediction of receptor responses to chimeric ligands. The results of receptor response modeling for all peptides in the pathway are shown. Increasing predicted normalized response indicates decreasing sensitivity.