Functional screening system for yeast-secreted peptides acting on G-protein coupled receptors
© Shigemori et al.; licensee Springer. 2015
Received: 24 February 2015
Accepted: 21 April 2015
Published: 13 May 2015
We established a novel functional screening system for peptides acting on G-protein coupled receptors (GPCRs). Peptides are a promising drug scaffold because of their intermediate molecular size between that of therapeutic small molecules and antibodies. They also offer potential advantages of targeting not only membrane proteins but also intracellular protein–protein interactions. Phage display technology has been used for exploring novel peptides acting on GPCRs, but it is unclear whether the identified peptides functionally modulate targets because the technology selects peptides based on binding ability but not functional activity to targets. In a novel screening system that we established, yeast cells were utilized as a peptide producer while mammalian cells stably producing the receptor for glucagon-like peptide 1 (GLP1R) were used as a biosensor for receptor activation. Three kinds of GLP1R agonists secreted by yeasts were successfully detected for their functional activities without any purification and condensation of those peptides. By applying the functional screening system, we were able to identify GLP1R agonist-secreting yeasts based on GLP1R activation from the cell mixture containing a number of background yeasts that produced non-active control peptides. Further applications of this system would include not only activity evaluation of bioactive peptides without chemical synthesis but also discovery of novel peptides activating druggable GPCRs.
KeywordsFunctional screening system G-protein coupled receptors Peptides Yeast Glucagon-like peptide-1
G-protein coupled receptors (GPCRs) are one of critical eukaryotic signal transduction gatekeepers and represent the largest protein family in the human proteome with more than 800 members. They share a common architecture of seven transmembrane helices, and can be classified into five major classes of sequence similarities (Jacoby et al. 2006): rhodopsin receptor family (class A), secretin-like receptor family (class B), glutamate receptor family (class C), frizzled/taste 2 receptor family, and adhesion receptor family. GPCRs recognize a variety of extracellular stimuli, including photons, ions, small molecules, peptides, and proteins; they transmit the resulting extracellular signals across the membrane to elicit intracellular responses. Consequently, GPCRs are involved in physiological and pathophysiological changes in blood pressure, blood sugar, pain, allergies, and so on. Therefore, pharmacological activation or suppression of GPCRs has been effective means to treat various diseases related to GPCR dysregulation.
Peptides are involved in a variety of physiological and pathological processes, and play very important roles in modulating various cell functions such as the absorption of blood glucose into the body through the promotion of insulin secretion in pancreatic β-cells by glucagon-like peptide-1 (GLP1), a peptide hormone that are postprandially secreted in intestinal L-cells and activates one of class B GPCRs, GLP1 receptor (GLP1R) (Baggio and Drucker 2007). Because of their intermediate molecular size ranging from 0.5 to 5 kDa which is between that of small-molecule drugs and therapeutic monoclonal antibodies, peptides potentially have advantages of easy drug design, high safety, accessibility for protein–protein interactions, targeting of intracellular molecules, and low production cost (Craik et al. 2013).
Phage display is the first innovative technology established by Smith (1985) that allows researchers to prepare and screen a large polypeptide library. However, this methodology is not effective at discovering functional peptides such as activators for GPCRs. This is due to the nature of phage display, whose peptides are screened based only on binding ability to targets, resulting frequently in simple binding peptides without any bioactivity (Chen et al. 2007). Furthermore, the identified peptides on the phage are never a functional, soluble form, potentially leading to dissociation in activity between peptides tied up on the phage as a fusion protein and the secretion form of peptides.
The yeast Saccharomyces cerevisiae is suitable as host for production of peptide library; this is due to their abundance in gene manipulation tools, fast growth in a low-cost medium, and protein folding and secretory machinery homologous to that of mammalian cells (Idiris et al. 2010). In this study, we report a novel functional screening method for bioactive peptides acting on GPCRs, which integrated a yeast secretion system and a functional detection system using GPCR-producing mammalian cells. GLP1R produced on mammalian cells was successfully activated by various GLP1R agonistic peptides that were secreted from yeast. We were also able to identify GLP1R agonist-secreting yeasts based on GLP1R activation from the cell mixture containing a number of background yeasts, which produced non-active control peptides, suggesting the effectiveness of our functional screening system to discover novel peptide-based drugs acting on GPCRs.
Materials and methods
Strains and media
Esherichia coli DH5α [F − , ΔlacU169 (φ80lacZΔM15), hsdR17 (r K − , m K + ), recA1, endA1, deoR, thi-1, supE44, gyrA96, relA1, λ −] (Toyobo, Osaka, Japan) was used as a host for DNA manipulation. E. coli transformants were grown in Luria–Bertani (LB) medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride, and 2% (w/v) agarose] containing 100 µg/mL ampicillin or kanamycin depending on the plasmids introduced.
Saccharomyces cerevisiae BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0; EUROSCARF, Frankfurt, Germany) was used to construct the yeasts secreting GLP1R agonists, including GLP1, S2-GLP1 substituted with serine at the position 2 of GLP1, and exendin-4 (Ex4), a naturally occurring peptide found in the saliva of the Gila monster (Furman 2012). Yeast transformants were selected on synthetic dextrose (SDC) solid medium [0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, 1% (w/v) casamino acids, 0.002% (w/v) adenine, 0.002% (w/v) l-tryptophan, and 2% (w/v) agar], and then, the resultant colonies were cultivated in 6-well plate (353046; Thermo Fisher Scientific, Waltham, MA, USA) or 96-well plate (353072; Thermo Fisher Scientific) containing a liquid SDC medium or Dulbecco’s modified Eagle Medium (DMEM) (Nacalai Tesque, Kyoto, Japan) at 30°C.
Chinese hamster ovary (CHO) cells (85050302; European Collection of Cell Cultures, Salisbury, UK) were used as a host cell stably producing human GLP1R and cultivated in Ham-F12 (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Thermo Fisher Scientific) and 400 μg/mL G418 (Nacalai Tesque).
Construction of peptide-secreting yeast
Primers used in this study
Construction of human GLP1R-producing CHO
Human GLP1R gene was PCR-amplified from the human brain cDNA library (BioChain, Newark, CA, USA) using DNA polymerase KOD-Plus-Neo (Toyobo) with primers 3 and 4 (Table 1). The DNA fragment coding human GLP1R was inserted into pIRES (Clontech) digested with EcoRV and BamHI by using In-Fusion (Clontech), resulting in pIRES-hGLP1R. CHO cells were transfected with pIRES-hGLP1R using Xfect (Clontech), and then, selected with G418 for about 2 weeks to construct a stable cell line producing hGLP1R. Single cell cloning of the resistant cells was conducted by limiting dilution, resulting in GLP1R-CHO.
GLP1R activation assay using GLP1R-CHO
GLP1R-CHO was seeded onto a 96-well plate at 5 × 104 cells and cultured at 37°C for 24 h. After the cells were washed with HANKS buffer (Thermo Fisher Scientific), synthetic GLP1R agonists (GLP1 and Ex4; Peptide Institute, Osaka, Japan. S2-GLP1; Bachem, Bubendorf, Switzerland) or culture supernatants of GLP1R agonists-secreting yeast were added and incubated at 37°C for 45 min. Then, the cells were lysed with Assay/Lysis buffer (Thermo Fisher Scientific) and the level of cyclic AMP in the cell lysate was determined by using the cAMP-screen® assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Model screening of Ex4-secreting yeast
Ten yeast cells comprised of Ctrl-yeast and Ex4-yeast in the theoretical ratio of 9:1 were seeded into 16 wells in 96-well plate containing 300 µL of SDC medium and grown for 48 h. Then, the medium was exchanged into 250 µL of DMEM and yeasts were additionally cultivated for 12 h at 30°C. After that, the supernatant was subjected to the GLP1R activation assay mentioned above. Yeasts included in three wells showing or not showing activity were seeded on SDC solid medium to form single colonies. Then, 48 colonies were subjected to colony-direct PCR with primers 5 and 6 (Table 1), and the resultant PCR products were analyzed by agarose gel electrophoresis to identify yeasts with the Ex4 gene.
Construction of stable CHO cells producing human GLP1R
Amino acid sequences of GLP1R agonists used in this study
Amino acid sequence
Medium optimization for GLP1R activation by yeast-secreted peptides and establishment of assay system
We first tested which medium was suitable for yeast growth and evaluation of GLP1R activation in GLP1R-CHO in 6-well plates. Ctrl-yeast and Ex4-yeast were inoculated in a 6-well plate containing SDC medium or DMEM at the initial optical density (OD) which was 600 nm of 0.1 and incubated for 40 h. Then, the growth rates of yeasts and the GLP1R activation in GLP1R-CHO by culture supernatant containing yeast-secreted peptides were evaluated (Figure 2). As a result, SDC gave a higher growth reaching stationary phase at 20 h, showing about 90-fold expansion. On the other hand, yeasts cultivated in DMEM showed much lower growth, reaching only the maximal OD600 of 0.75 at 20 h (Figure 2a). For GLP1R activation potency after the 40-h cultivation, the culture supernatant of Ex4-yeast grown in SDC showed no activity, although exogenously added Ex4 activated GLP1R by 16-fold, compared with the control. In contrast, Ex4-yeast grown in DMEM strongly induced GLP1R activation by 50-fold compared to the Ctrl-yeast (Figure 2b). These results indicate that SDC medium is suitable for yeast proliferation, while DMEM is excellent for GLP1R activation.
Direct functional assay of various yeast-secreted GLP1R peptide agonists
Examination of model screening using our established functional assay system
In this study, we established the novel functional screening system of yeast-secreted peptides acting on GLP1R. The system directly detected the functional activity of yeast secreted-GLP1R agonists based on activation of GLP1R produced on mammalian cells, without any purification and condensation of yeast-produced peptides. In addition, application of the system enabled identification of agonist-secreting yeast in the model screening.
Binding-based peptide screening strategy as represented by phage display has been the most common method used to discover novel peptide-ligands for certain drug targets, including GPCRs (Molek et al. 2011; Vyroubalova et al. 2006). However, with peptides discovered through this methodology, it is uncertain whether they are biologically active or not. In addition, phage-displayed peptides are distinct from functional peptides in their soluble form. These conventional challenges result in additional chemical synthesis of the identified peptides in soluble form to evaluate their true biological activity, which are time-consuming and quite expensive.
In the first optimization step using Ex4-yeasts as a model for our novel functional assay system, we remarkably found that DMEM buffered at neutral pH designed for mammalian cell culture was very suitable for yeast peptide secretion and GLP1R activation in mammalian cells, even though yeast cells could not grow in the medium. Another remarkable point in this culture system is a protection of the target peptides from degradation by yeast-derived proteases. Because heterologous proteins produced in yeasts could be degraded during several steps, including the intracellular secretory pathways and the post-secreted extracellular environment (Idiris et al. 2010), yeast-derived proteases such as Yps1p and Kex2p, which are most active at a mild acidic condition around pH 5.0, are considered accessible to heterologous proteins, especially when yeasts are incubated in SDC medium, which generally has pH 4.5–5.5 (Mizuno et al. 1989; Olsen et al. 1999). In addition, yeasts were a good tool for producing peptides because yeasts swiftly grew in SDC medium; they even in a static 96-well plate setting which would be compatible with high-throughput screening and could easily reach the targeted yeast cell number providing high dynamic range in GLP1R activation assay (Figure 2).
These findings prompted us to conduct the direct functional detection of other GLP1R agonists with weaker activity than Ex4, GLP1, and S2-GLP1 to be secreted by yeasts. It was difficult to detect their GLP1R activation potency at the high level as expected from the difference of the EC50 value of Ex4. Surprisingly, the C-terminus fusion of the FLAG tag with DYKDDDDK in GLP1 and S2-GLP1 much increased the GLP1R activation potency by about tenfold compared to those without the FLAG tag (Figure 4b). In spite of the fact that the precise mechanisms of the FLAG are still uncertain, the beneficial effect of the FLAG fusion was also observed in somatostatin that is endogenous circular peptide agonist acting on class A GPCR SST receptor (data not shown). Accordingly, the FLAG fusion to isolated peptides would be a promising way to increase their activity.
As shown in Figure 5, we successfully identified the wells including Ex4-yeast by evaluating the GLP1R activation potency, and importantly, the wells that did not show activity did not possess any Ex4-yeast, as confirmed by colony-direct PCR detecting Ex4 gene. This successful model screening of yeasts secreting agonists acting on GLP1R encourages us to carry out an actual screening for novel bioactive peptides through our direct functional detection system.
In conclusion, we successfully established a novel system for direct functional assay for yeast-secreted peptides on GLP1R. This system will be applied not only for biological activity assay of sequenced peptides instead of their chemical synthesis but also discovery of novel bioactive peptides.
Chinese hamster ovary
Dulbecco’s modified Eagle medium
G-protein coupled receptor.
TS designed and carried out the experiments, and drafted the manuscript. KK participated in the experimental design and revised the manuscript. MU contributed to the experimental design and revised the manuscript critically. All authors read and approved the final manuscript.
This work was supported by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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