Biology subjects

Li, H.-Z.

Publications and source records attributed to Li, H.-Z..

6 recordsLinked to original sources

bioRxiv2023

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolysis activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To solve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via an ion-exchange chromatography. After the non-covalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolysis activity, and could efficiently catalyze inter-molecular protein ligation and intra-molecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

biochemistry↗

bioRxiv2023

The ghrelin receptor GHSR has two efficient agonists in an ancient fish species

The gastric peptide ghrelin and its receptor GHSR have important functions in energy metabolism. Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous antagonist for GHSR. Ghrelin, LEAP2, and GHSR are ubiquitously present from fishes to mammals and are highly conserved in evolution. However, our recent study suggested that GHSRs from the Actinopterygii fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost their binding to ghrelin, despite binding normally to LEAP2. Do these fish GHSRs use another peptide as their agonist? To answer this question, in the present study, we tested to two fish motilins that are closely related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the GHSR from the Sarcopterygii fish Latimeria chalumnae (coelacanth) bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in D. rerio and L. crocea, but is an efficient agonist for GHSR in L. chalumnae, which is known as a living fossil and is believed to be one of the closest fish ancestors of tetrapods. The results of present study suggested that in ancient fishes, GHSR had two efficient agonists, ghrelin and motilin; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins. Our present work shed new light on the ligand usage of GHSR in different fish species and in evolution.

biochemistry↗

bioRxiv2023

Nanomolar range of FAM237B can activate receptor GPR83

The orphan G protein-coupled receptor 83 (GPR83) is implicated in the regulation of energy metabolism and certain anxiety-related behaviors. Our recent study confirmed that family with sequence similarity 237 member A (FAM237A), also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83, but did not support the proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N, also known as proSAAS)-derived peptide PEN and the procholecystokinin-derived peptide proCCK56-63 as ligands of this receptor. FAM237B (also known as NPGM) is a paralog of FAM237A that was previously reported as a weak agonist for GPR83 with approximately 100-fold lower activity in an inositol 1-phosphate accumulation assay. In the present study, we prepared mature human FAM237B via an intein-fusion approach and measured its activity towards human GPR83 via a NanoLuc Binary Technology (NanoBiT)-based ligand{square}receptor binding assay and a NanoBiT-based {beta}-arrestin recruitment assay. Mature FAM237B displayed moderately lower activity than its paralog FAM237A in these binding and activation assays, but could cause a significant activation effect at the nanomolar range (1{square}10 nM). Thus, FAM237B appears to be another endogenous agonist for receptor GPR83.

biochemistry↗

bioRxiv2022

FAM237A, rather than peptide PEN and proCCK56-63, is a ligand of the orphan receptor GPR83

G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some behaviors. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A, raising doubts that all of them are ligands of GPR83. To identify its actual ligand(s), in the present study we developed a NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and a NanoBiT-based {beta}-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, which activated this receptor and induced its internalization in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, the results confirmed that FAM237A is an agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of its actual endogenous agonist will pave the way for further functional studies of this brain-specific receptor. The present study also provided an efficient approach for the preparation of mature FAM237A, which would facilitate further functional studies of this difficult-to-make peptide in the future.

biochemistry↗

bioRxiv2022

LEAP2 is a more conserved ligand than ghrelin for fish GHSRs

Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous competitive antagonist and an inverse agonist of the ghrelin receptor GHSR. However, its functions in lower vertebrates are not well understood. Our recent study demonstrated that both LEAP2 and ghrelin are functional towards a fish GHSR from Latimeria chalumnae, an extant coelacanth believed to be one of the closest ancestors of tetrapods. However, amino acid sequence alignment identified that the 6.58 position (Ballesteros-Weinstein numbering system) of most fish GHSRs are not occupied by an aromatic Phe residue, which is absolutely conserved in all known GHSRs from amphibians to mammals, and is responsible for human GHSR binding to its agonist, ghrelin. To test whether these unusual fish receptors are functional, we studied the ligand binding properties of three representative fish GHSRs, two from Danio rerio (zebrafish) and one from Larimichthys crocea (large yellow croaker). After overexpression in human embryonic kidney 293T cells, the three fish GHSRs retained normal binding to all tested LEAP2s, except for a second LEAP2 from L. crocea. However, they displayed almost no binding to all chemically synthesized n-octanoylated ghrelins, despite these ghrelins all retaining normal function towards human and coelacanth GHSRs. Thus, it seems that LEAP2 is a more conserved ligand than ghrelin towards fish GHSRs. Our results not only provided new insights into the interaction mechanism of GHSRs with LEAP2s and ghrelins, but also shed new light on the functions of LEAP2 and ghrelin in different fish species.

biochemistry↗

bioRxiv2022

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 cellular homeostasis by activating the growth hormone secretagogue receptor type 1a (GHSR1a), and thus has therapeutic potential to treat certain diseases. Native ghrelin carries an essential O-fatty acyl moiety at the side-chain of its third Ser residue; however, this posttranslational modification is susceptible to hydrolysis by certain esterases in circulation, representing a major route of in vivo inactivation of ghrelin. In the present study, we developed a novel approach to prepare various esterase-resistant ghrelin analogs via photo-induced thiol-ene click chemistry. A recombinant unacylated human ghrelin mutant carrying a unique Cys residue at the third position was reacted with commercially available end alkenes, thus various alkyl moieties were introduced to the side-chain of its unique Cys residue via a thioether bond. Among eleven S-alkylated ghrelin analogs, analog 11, generated by reacting with 2-methyl-1-octene, not only acquired much higher stability in human serum and fetal bovine serum, but also acquired moderately higher activity compared with native human ghrelin. Thus, the present study not only provided an efficient approach to prepare various esterase-resistant ghrelin analogs, but also produced a novel highly stable and highly active ghrelin analog with therapeutic potential.

biochemistry↗