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Xu, Z.-G.

Publications and source records attributed to Xu, Z.-G..

16 recordsLinked to original sources

AtlasMap: enabling low-cost, map-style exploration of million-cell single-cell atlases

Interactive visualization is critical for interpreting single-cell atlases, yet existing web-based tools struggle to handle the growing scale of multimillion-cell datasets, often constrained by browser memory limits and rendering latency. Here, we present AtlasMap, a scalable visualization framework that overcomes these bottlenecks through a multi-resolution, tile-based architecture. Unlike conventional systems that transfer cell-level data to the client, AtlasMap employs an offline preprocessing module to generate quadtree-based spatial aggregations stored in compressed Zarr v3 formats. A high-performance Go backend dynamically renders these summaries as PNG tiles, allowing a lightweight frontend to support fluid pan-and-zoom exploration similar to digital geographic maps. Systematic benchmarking against leading tools (including cellxgene and UCSC Cell Browser) demonstrates that AtlasMap decouples visualization performance from dataset size. While point-based approaches incurred prohibitive client-side memory costs or failed entirely at the 11-million-cell scale, AtlasMap maintained sub-second startup latency and a negligible browser footprint (<5 MB). By shifting computational pressure from the browser to an optimized server-side pipeline, AtlasMap enables accessible, high-fidelity exploration of ultra-scale single-cell datasets on standard hardware.

bioinformatics↗

Ancestral Origin of the CXCL17-GPR25 System Traced to the Lobe-Finned Fish Latimeria chalumnae

The roles of C-X-C motif chemokine ligand 17 (CXCL17) and its receptor, G protein-coupled receptor 25 (GPR25), in immune regulation are gaining recognition, but their evolutionary origins and phylogenetic distributions have yet to be determined. In this study, we identified a CXCL17 gene in the lobe-finned fish Latimeria chalumnae (coelacanth), a close living relative of tetrapods. This gene produces two alternatively spliced secretory proteins, designated Lc-CXCL17a and Lc-CXCL17b, which display limited sequence similarity to known CXCL17s from mammals or ray-finned fishes. Recombinant forms of Lc-CXCL17a and Lc-CXCL17b were generated via bacterial overexpression, purification, and enzymatic processing. Functional assays, including NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment, ligand-receptor binding, and chemotaxis assays, demonstrated that both proteins tightly bound to and potently activated coelacanth GPR25 in a manner dependent on their C-terminal residues, because removal of these residues markedly diminished activities. These findings provide the first evidence that a functional CXCL17-GPR25 system exists in lobe-finned fishes, supporting the hypothesis that this ligand-receptor pair originated in ancient fishes and was transmitted to vertebrate lineages through lobe-finned fish ancestors. This work broadens understanding of chemokine evolution and highlights the coelacanth as a valuable model for tracing conserved immune signaling pathways.

biochemistry↗

Identification and functional characterization of two CXCL17 paralogs from zebrafish

C-X-C motif chemokine ligand 17 (CXCL17) and its receptor G protein-coupled receptor 25 (GPR25) have been identified as a significant pair in regulating immunity, but CXCL17 orthologs have not yet been identified from non-mammalian vertebrates. This study aimed to identify and characterize non-mammalian CXCL17 orthologs based on key features of mammalian CXCL17s, such as a C-terminal Xaa-Pro-Yaa motif, a signal peptide, and six cysteine residues. Two possible CXCL17 paralogs were identified from zebrafish (Danio rerio): Dr-CXCL17 (encoded by zgc:158701) and Dr-CXCL17-like (encoded by si:dkey-112a7.5). Both are previously uncharacterized proteins with unknown functions because they lack overall sequence similarity to known mammalian CXCL17s. For functional characterization, recombinant Dr-CXCL17 and Dr-CXCL17-like were prepared via overexpression in Escherichia coli and subsequent in vitro refolding and their activity was tested using NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assays, NanoBiT-based ligand{square}receptor binding assays, and chemotaxis assays. The results showed that both Dr-CXCL17 and Dr-CXCL17-like tightly bound to and efficiently activated zebrafish GPR25 (Dr-GPR25) and induced chemotactic movement in transfected human embryonic kidney (HEK) 293T cells expressing the receptor. Deletion of three C-terminal residues in both paralogs almost eliminated their binding, activation, and chemotactic effects, which suggests that this fragment is crucial for their function. Homologs of Dr-CXCL17 or Dr-CXCL17-like were retrieved from several other ray-finned fish species, indicating that two CXCL17 paralogs are present in certain fish species and function as endogenous agonists for the fish GPR25 receptor. The identification of fish CXCL17 orthologs suggests that the CXCL17{square}GPR25 pair likely originated in ancient fishes and was conserved across vertebrate lineages. This work represents the first identification of CXCL17 orthologs in non-mammalian vertebrates, paving the way for future functional studies of this ligand{square}receptor pair.

biochemistry↗

Human CXCL17 Activates and Binds to Fish GPR25 Orthologs

C-X-C motif chemokine ligand 17 (CXCL17) functions as a chemoattractant, though its receptor has been controversial. Recent independent studies, including our own, identified CXCL17 as an agonist for the orphan G protein-coupled receptor 25 (GPR25). While GPR25 orthologs are found across fishes to mammals, CXCL17 orthologs appear to be mammalian-specific, leaving the endogenous ligand for non-mammalian GPR25 orthologs unknown. This study unexpectedly found that human CXCL17 exhibits high activity towards GPR25 orthologs from the zebrafish (Danio rerio) and coelacanth (Latimeria chalumnae). Recombinant human CXCL17 efficiently activated both fish GPR25 orthologs in a NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay, and induced chemotactic movement in transfected human embryonic kidney (HEK) 293T cells expressing fish GPR25. A human CXCL17 mutant lacking three C-terminal residues showed no such effect. A NanoBiT-based binding assay revealed that a SmBiT-tagged human CXCL17 C-terminal fragment specifically bound to secretory large NanoLuc fragment (sLgBiT)-fused fish GPR25 orthologs. Fish GPR25 orthologs had significantly higher cell surface expression in transfected HEK293T cells compared to human GPR25, improving {beta}-arrestin recruitment assay data quality. Despite approximately 400 million years of divergence between humans and fishes, the high activity of human CXCL17 on fish GPR25 orthologs suggests that the CXCL17-GPR25 pair may be conserved across all vertebrates, even though non-mammalian CXCL17 orthologs remain unidentified.

biochemistry↗

CXCL17 activates three MAS-related G protein-coupled receptors independently of its conserved C-terminal fragment

C-X-C motif chemokine ligand 17 (CXCL17) is a chemoattractant whose receptor remains controversial. While recent studies identified CXCL17 as an agonist of G protein-coupled receptor 25 (GPR25), it was also reported to activate MAS-related GPR family member X2 (MRGPRX2), a member of MAS-related G protein-coupled receptors (MRGPRs), though this finding has not yet been reproduced by other laboratories. In this study, we confirmed that micromolar concentrations of human CXCL17 activate human MRGPRX2 in transfected human embryonic kidney (HEK) 293T cells using a NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay. We further demonstrated that human CXCL17 also activates MRGPRX1 and MAS1 among 10 human MRGPRs in the same assay. CXCL17 could also induce chemotactic movement of transfected HEK293T cells expressing MRGPRX2, MRGPRX1, or MAS1. However, removal of C-terminal residues from CXCL17 did not affect its activation of these three MRGPRs, even though this region is essential for GPR25 activation. These results suggest that CXCL17 activates MRGPRX2, MRGPRX1, and MAS1 through a mechanism distinct from GPR25 activation. Further investigation is needed to determine whether these MRGPRs mediate the in vivo functions of CXCL17.

biochemistry↗

Identification of the orphan GPR25 as a receptor of the chemokine CXCL17

C-X-C motif chemokine ligand 17 (CXCL17) is a small secretory protein primarily expressed in mucosal tissues and likely functions as a chemoattractant, but its receptor is still controversial. In the present study, we identified the rarely studied orphan G protein-coupled receptor 25 (GPR25) as a receptor of CXCL17 via prediction by the newly developed AlphaFold 3 algorithm and validation by the NanoBiT-based {beta}-arrestin recruitment assay. In the {beta}-arrestin recruitment assay, recombinant human CXCL17 could activate human GPR25 in transfected human embryonic kidney (HEK) 293T cells with an EC50 value around 100 nM, but it had no activation effect on 17 other tested G protein-coupled receptors (GPCRs). Deletion of three conserved C-terminal residues of human CXCL17 almost abolished its activity, and alanine replacement of W95 or R178 of human GPR25, two conserved residues in the predicted orthosteric ligand binding pocket, almost abolished its response towards CXCL17. These results are consistent with the AlphaFold 3-predicted binding model in which the highly conserved C-terminal fragment of CXCL17 inserts into the orthosteric ligand binding pocket of the receptor GPR25. According to the expression pattern of CXCL17 and GPR25 shown at the Human Protein Atlas, CXCL17 might be an endogenous agonist of GPR25 in human and other mammals, but this hypothesis needs to be tested in future studies via more assays. The present deorphanization paves the way for further functional characterization of the orphan receptor GPR25 and the orphan ligand CXCL17.

biochemistry↗

Is myeloid-derived growth factor a ligand of the sphingosine-1-phosphate receptor 2?

Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF. In the NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay and the cAMP-response element (CRE)-controlled NanoLuc reporter assay, S1P could efficiently activate human S1PR2 overexpressed in human embryonic kidney (HEK) 293T cells; however, recombinant human MYDGF, overexpressed either from Escherichia coli or HEK293 cells, had no detectable effect. Thus, the results demonstrated that human MYDGF is not a ligand of human S1PR2. Considering the high conservation of MYDGF and S1PR2 in evolution, MYDGF is also probably not a ligand of S1PR2 in other vertebrates.

biochemistry↗

Force-sensing LPHN2 is indispensable for normal hearing and mediates MET in cochleae through Gs and CNGA3 coupling

The conversion of force sensation into electrical signals via mechanoelectrical transduction (MET) is considered the key step in auditory perception. Here, we found that G protein-coupled receptor (GPCR) LPHN2/ADGRL2 was expressed at the tips of stereocilia in cochlear hair cells and was associated with MET channel components. Hair cell-specific LPHN2 deficiency caused hearing loss and impaired MET responses. A specific inhibitor of LPHN2, developed by in silico screening and pharmacological characterization, reversibly blocked the MET response. Mechanistically, administration of force to LPHN2 activated TMC1 through direct interaction and caused conformational changes in TMC1 in vitro. Furthermore, the sensing of force by LPHN2 stimulated Ca2+ responses and neurotransmitter release in hair cells. Finally, hearing loss in LPHN2-deficient mice was reversed by the re-expression of LPHN2-GAIN in cochlear hair cells. The important roles of LPHN2 in auditory perception and a TMC-coupled force sensor indicated that GPCRs could be candidate auditory receptors.

physiology↗

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↗

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↗

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↗

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↗

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↗

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↗

Development of a novel 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 labeling and cyclic peptide synthesis. To characterize known peptide ligases or identify new ones, we propose a novel bioluminescent activity assay via the genetic fusion of a recognition motif of a peptide ligase to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase to the N-terminus of the low-affinity SmBiT complementation tag. When the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase, its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the novel bioluminescent activity assay using bacterial sortase A and plant 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 novel 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.

biochemistry↗

Integration of gut microbiome, host biomarkers, and urine metabolome data reveals networks of interactions associated with distinct clinical phenotypes

BackgroundComprehensive analyses of multi-omics data may provide insights into interactions between different biological layers in relation to distinct clinical features. Here we integrated data on the gut microbiota, blood parameters and urine metabolites of treatment-naive individuals presenting a wide range of metabolic disease phenotypes to delineate clinically meaningful associations.\n\nResultsTrans-omics correlation networks revealed that candidate gut microbial biomarkers and urine metabolite features covaried with distinct clinical phenotypes. Integration of the gut microbiome, the urine metabolome and the phenome revealed that variations in one of these three systems correlated with changes in the other two. Of specific note in relation to clinical parameters of liver function, we identified Eubacterium eligens, Faecalibacterium prausnitzii and Ruminococcus lactaris to be associated with a healthy liver function, whereas Clostridium bolteae, Tyzzerella nexills, Ruminococcus gnavus, Blautia hansenii, and Atopobium parvulum were associated with blood biomarkers for liver diseases. Variations in these microbiota features paralleled changes in specific urine metabolites. Network modeling yielded two core clusters including one large gut microbe-urine metabolite close-knit cluster and one triangular cluster composed of a gut microbe-blood-urine network, demonstrating close inter-system crosstalk especially between the gut microbiome and the urine metabolome.\n\nConclusionsDistinct clinical phenotypes are manifested in both the gut microbiome and the urine metabolome, and inter-domain connectivity takes the form of high-dimensional networks. Such networks may further our understanding of complex biological systems, and may provide a basis for identifying biomarkers for diseases.

microbiology↗