Unusual orthologs shed new light on the binding mechanism of ghrelin to its receptor GHSR1a.

The gastric peptide ghrelin has important functions in energy metabolism and cellular homeostasis by activating growth hormone secretagogue receptor type 1a (GHSR1a). The N-terminal residues of ghrelin orthologs from all vertebrates are quite conserved; however, in orthologs from Cavia porcellus and Phyllostomus discolor, Ser2 and Leu5 are replaced by a smaller Ala and a positively charged Arg, respectively. In the present study, we first demonstrated that the hydrophobic Leu5 is essential for the function of human ghrelin, because Ala replacement caused an approximately 100-fold decrease in activity. However, replacement of Leu5 by an Arg residue caused much less disruption; further replacement of Ser2 by Ala almost restored full activity, although the [S2A] mutation itself showed slight detriments, implying that the positively charged Arg5 in the [S2A,L5R] mutant might form alternative interactions with certain receptor residues to compensate for the loss of the essential Leu5. To identify the responsible receptor residues, we screened GHSR1a mutants in which all conserved negatively charged residues in the extracellular regions and all aromatic residues in the ligand-binding pocket were mutated separately. According to the decrease in selectivity of the mutant receptors towards [S2A,L5R]ghrelin, we deduced that the positively charged Arg5 of the ghrelin mutant primarily interacts with the essential aromatic Phe286 at the extracellular end of the sixth transmembrane domain of GHSR1a by forming cation-π and π-π interactions. The present study provided new insights into the binding mechanism of ghrelin with its receptor, and thus would facilitate the design of novel ligands for GHSR1a.

LEAP2 has antagonized the ghrelin receptor GHSR1a since its emergence in ancient fish.

Recent studies have demonstrated that liver-expressed antimicrobial peptide 2 (LEAP2) antagonizes the ghrelin receptor GHSR1a in mammals. However, its antagonistic function in lower vertebrates has not yet been tested. LEAP2 orthologs have been identified from a variety of fish species; however, previous studies all focused on their antimicrobial activity. To test whether LEAP2 functions as a GHSR1a antagonist in the lowest vertebrates, we studied the antagonism of a fish LEAP2 from Latimeria chalumnae, an extant coelacanth that is one of the closest living fish relatives of tetrapods. Using binding assays, we demonstrated that the coelacanth LEAP2 and ghrelin bound to the coelacanth GHSR1a with IC50 values in the nanomolar range. Using activation assays, we demonstrated that the coelacanth ghrelin activated the coelacanth GHSR1a with an EC50 value in the nanomolar range, and this activation effect was efficiently antagonized by a nanomolar range of the coelacanth LEAP2. In addition, we also showed that the human LEAP2 and ghrelin were as effective as their coelacanth orthologs towards the coelacanth GHSR1a; however, the coelacanth peptides had moderately lower activity towards the human GHSR1a. Thus, LEAP2 serves as an endogenous antagonist of the ghrelin receptor GHSR1a in coelacanth and the ghrelin-LEAP2-GHSR1a system has evolved slowly since its emergence in ancient fish.

Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin-GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved Asp99 of GHSR1a probably interacts with the essential Arg6 of LEAP2. After five conserved Phe residues in the predicted ligand-binding pocket of human GHSR1a were mutated, three of them were identified as important for GHSR1a binding to LEAP2. According to a structural model of GHSR1a, we deduced that the adjacent Phe279 and Phe312 might interact with the essential Phe4 of LEAP2, while Phe119 might interact with the aromatic Trp5 of LEAP2. The present study provided new insights into the interaction of LEAP2 with its receptor, and would facilitate the design of novel ligands for GHSR1a in future studies.

Development of a general bioluminescent activity assay for peptide ligases.

In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labelling and cyclic peptide synthesis. To characterize various known peptide ligases or identify new ones, we propose a general bioluminescent activity assay via the genetic fusion of a recognition motif of peptide ligase(s) to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase(s) to the N-terminus of the low-affinity SmBiT complementation tag. After the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase(s), its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the bioluminescent activity assay using bacterial sortase A and plant-derived butelase-1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT-SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase-type peptide ligase and four of them likely express prolyl endopeptidase-type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The present bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies.

Development of Esterase-Resistant and Highly Active Ghrelin Analogs via Thiol-Ene Click Chemistry.

The orexigenic peptide ghrelin exerts important functions in energy metabolism and has therapeutic potential to treat certain diseases. Native ghrelin carries an essential O-fatty acyl moiety; however, this post-translational modification is susceptible to hydrolysis by certain esterases in circulation, representing a major route of its in vivo inactivation. In the present study, we developed a novel approach to prepare various esterase-resistant ghrelin analogs via photoinduced thiol-ene click chemistry. A recombinant unacylated human ghrelin mutant was reacted with commercially available terminal alkenes; thus, various alkyl moieties were introduced to the side chain of its unique Cys3 residue via a thioether bond. Among 11 S-alkylated ghrelin analogs, analog 11, generated by reacting with 2-methyl-1-octene, not only acquired much higher stability in serum but also retained full activity compared with native human ghrelin. Thus, the present study provided an efficient approach to prepare highly stable and highly active ghrelin analogs with therapeutic potential.