Zeng-Guang Xu

A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase

Insulin-like peptide 3 (INSL3) is a reproduction-related peptide hormone belonging to the insulin/relaxin superfamily, which mediates testicular descent in the male fetus, suppresses male germ cell apoptosis and promotes oocyte maturation in adults by activating the relaxin family peptide receptor 2 (RXFP2). To establish an ultrasensitive receptor-binding assay for INSL3-RXFP2 interaction studies, in the present work we labeled a recombinant INSL3 peptide with a newly developed nanoluciferase (NanoLuc) reporter through a convenient chemical conjugation approach, including the introduction of an active disulfide bond to INSL3 by chemical modification and engineering of a 6× His-Cys-NanoLuc carrying a unique exposed cysteine at the N-terminus. The bioluminescent NanoLuc-conjugated INSL3 retained high binding affinity with the target receptor RXFP2 (Kd = 2.0 ± 0.1 nM, n = 3) and was able to sensitively monitor the receptor-binding of a variety of ligands, representing a novel ultrasensitive tracer for non-radioactive receptor-binding assays. Our present chemical conjugation approach could readily be adapted for conjugation of NanoLuc with other proteins, even other macrobiomolecules, for various highly sensitive bioluminescent assays.

The highly conserved negatively charged Glu141 and Asp145 of the G-protein-coupled receptor RXFP3 interact with the highly conserved positively charged arginine residues of relaxin-3

Relaxin-3 is a newly identified insulin/relaxin superfamily peptide that plays a putative role in the regulation of food intake and stress response by activating its cognate G-protein-coupled receptor RXFP3. Relaxin-3 has three highly conserved arginine residues, B12Arg, B16Arg and B26Arg. We speculated that these positively charged arginines may interact with certain negatively charged residues of RXFP3. To test this hypothesis, we first replaced the negatively charged residues in the extracellular domain of RXFP3 with arginine, respectively. Receptor activation assays showed that arginine replacement of Glu141 or Asp145, especially Glu141, significantly decreased the sensitivity of RXFP3 to wild-type relaxin-3. In contrast, arginine replacement of other negatively charged extracellular residues had little effect. Thus, we deduced that Glu141 and Asp145, locating at the extracellular end of the second transmembrane domain, played a critical role in the interaction of RXFP3 with relaxin-3. To identify the ligand residues interacting with the negatively charged EXXXD motif of RXFP3, we replaced the three conserved arginines of relaxin-3 with negatively charged glutamate or aspartate, respectively. The mutant relaxin-3s retained the native structure, but their binding and activation potencies towards wild-type RXFP3 were decreased significantly. The compensatory effects of the mutant relaxin-3s towards mutant RXFP3s suggested two probable interaction pairs during ligand–receptor interaction: Glu141 of RXFP3 interacted with B26Arg of relaxin-3, meanwhile Asp145 of RXFP3 interacted with both B12Arg and B16Arg of relaxin-3. Based on these results, we proposed a relaxin-3/RXFP3 interaction model that shed new light on the interaction mechanism of the relaxin family peptides with their receptors.

The electrostatic interactions of relaxin‐3 with receptor RXFP4 and the influence of its B‐chain C‐terminal conformation

Relaxin‐3 (also known as insulin‐like peptide 7) is an insulin/relaxin‐superfamily peptide hormone that can bind and activate three relaxin‐family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin‐3 and its cognate receptor RXFP3 by using a charge‐exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin‐3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100–104) motif that is essential for RXFP4 activation by relaxin‐3. Mutagenesis of the conserved positively charged Arg residues of relaxin‐3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B26Arg of relaxin‐3, and Asp104 of RXFP4 versus both B12Arg and B16Arg of relaxin‐3. For interaction with the essential EXXXD motifs of both RXFP3 and RXFP4, a folding‐back conformation of the relaxin‐3 B‐chain C‐terminus seems to be critical, because it brings B26Arg sufficiently close to B12Arg and B16Arg. To test this hypothesis, we replaced the conserved B23Gly‐B24Gly dipeptide of relaxin‐3 with an Ala‐Ser dipeptide that occupied the corresponding position of insulin‐like peptide 5 and resulted in an extended helical conformation. The mutant relaxin‐3 showed a significant decrease in receptor‐activation potency towards both RXFP3 and RXFP4, suggesting that a folding‐back conformation of the B‐chain C‐terminus was important for relaxin‐3 to efficiently interact with the EXXXD motifs of both receptors.

A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin

Hepcidin is a liver‐secreted small disulfide‐rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin (Fpn). To study hepcidin–Fpn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin‐induced Fpn internalization by fusing a small nanoluciferase (NanoLuc, 171 amino acids) at the Fpn C‐terminus. Once the NanoLuc‐tagged Fpn was internalized, the measured luminescence was significantly decreased when assayed with the intact transiently transfected cells and an inducible expression system. Through the coexpression of a NanoLuc‐tagged Fpn and an enhanced green fluorescent protein (EGFP)‐tagged Fpn by the use of an inducible bidirectional promoter, we could measure the hepcidin‐induced Fpn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged NanoLuc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin–Fpn interaction qualitatively and quantitatively. Through coexpression of a NanoLuc‐tagged wild‐type Fpn and an EGFP‐tagged hepcidin‐insensitive mutant [C326S]Fpn, we demonstrated that the mutant Fpn had no effect on hepcidin‐induced internalization of wild‐type Fpn, suggesting that wild‐type Fpn and mutant Fpn are functionally independent.

Identification of hydrophobic interactions between relaxin-3 and its receptor RXFP3: implication for a conformational change in the B-chain C-terminus during receptor binding

Relaxin-3 is an insulin/relaxin superfamily neuropeptide implicated in the regulation of food intake and stress response via activation of the G protein-coupled receptor RXFP3. Their electrostatic interactions have been recently identified, and involves three positively charged B-chain residues (B12Arg, B16Arg, and B26Arg) of relaxin-3 and two negatively charged residues (Glu141 and Asp145) in a highly conserved ExxxD motif at the extracellular end of the second transmembrane domain of RXFP3. To investigate their hydrophobic interactions, in the present work we deleted the highly conserved B-chain C-terminal B27Trp residue of relaxin-3, and mutated four highly conserved aromatic residues (Phe137, Trp138, Phe146, and Trp148) around the ExxxD motif of RXFP3. The resultant [∆B27W]relaxin-3 exhibited approximately tenfold lower binding potency and ~1000-fold lower activation potency towards wild-type RXFP3, confirming its importance for relaxin-3 function. Although the RXFP3 mutants could be normally trafficked to cell membrane, they had quite different activities. [F137A]RXFP3 could normally distinguish wild-type relaxin-3 and [∆B27W]relaxin-3 in binding and activation assays, whereas [W138A]RXFP3 lost most of this capability, suggesting that the Trp138 residue of RXFP3 forms hydrophobic interactions with the B27Trp residue of relaxin-3. The hydrophobic Trp138 residue and the formerly identified negatively charged Glu141 and Asp145 residues in the highly conserved WxxExxxD motif may thus form a functional surface that is important for interaction with relaxin-3. We hypothesize that the relaxin-3 B-chain C-terminus changes from the original folding-back conformation to an extended conformation during binding with RXFP3, to allow its B27Trp and B26Arg residues to interact with the Trp138 and Glu141 residues of RXFP3, respectively.

Identification of important residues of insulin-like peptide 5 and its receptor RXFP4 for ligand–receptor interactions

Insulin-like peptide 5 (INSL5) is an insulin/relaxin superfamily peptide involved in the regulation of glucose homeostasis by activating its receptor RXFP4, which can also be activated by relaxin-3 in vitro. To determine the interaction mechanism of INSL5 with its receptor RXFP4, we studied their electrostatic interactions using a charge-exchange mutagenesis approach. First, we identified three negatively charged extracellular residues (Glu100, Asp104 and Glu182) in human RXFP4 that were important for receptor activation by wild-type INSL5. Second, we demonstrated that two positively charged B-chain Arg residues (B13Arg and B23Arg) in human INSL5 were involved in receptor binding and activation. Third, we proposed probable electrostatic interactions between INSL5 and RXFP4: the B-chain central B13Arg of INSL5 interacts with both Asp104 and Glu182 of RXFP4, meanwhile the B-chain C-terminal B23Arg of INSL5 interacts with both Glu100 and Asp104 of RXFP4. The present electrostatic interactions between INSL5 and RXFP4 were similar to our previously identified interactions between relaxin-3 and RXFP4, but had subtle differences that might be caused by the different B-chain C-terminal conformations of relaxin-3 and INSL5 because a dipeptide exchange at the B-chain C-terminus significantly decreased the activity of INSL5 and relaxin-3 to receptor RXFP4.

Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4.

The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1–4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr, and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-chain of relaxin-3 and the A-chain of INSL5, and retains full activation potency at RXFP3 and RXFP4. All resultant R3/I5 mutants retained high activation potency towards RXFP4, but most displayed significantly decreased or even abolished activation potency towards RXFP3, confirming the role of these four INSL5 determinants in distinguishing RXFP4 from RXFP3.

Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model.

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the so far brightest bioluminescence. In recent studies, we developed NanoLuc as an ultrasensitive probe for novel bioluminescent receptor-binding assays of some protein/peptide hormones. In the present study, we proposed a simple method for quick preparation of the NanoLuc-based protein tracers using erythropoietin (Epo) as a model. Epo is a glycosylated cytokine that promotes erythropoiesis by binding and activating the cell membrane receptor EpoR. For quick preparation of a bioluminescent Epo tracer, an Epo-Luc fusion protein carrying a NanoLuc-6 × His-tag at the C-terminus was secretorily overexpressed in transiently transfected human embryonic kidney (HEK) 293 T cells. The Epo-Luc fusion protein retained high-binding affinities with EpoR either overexpressed in HEK293T cells or endogenously expressed in mouse erythroleukemia cells, representing a novel ultrasensitive bioluminescent tracer for non-radioactive receptor-binding assays. Sufficient Epo-Luc tracer for thousands of assays could be quickly obtained within 2 days through simple transient transfection. Thus, our present work provided a simple method for quick preparation of novel NanoLuc-based bioluminescent tracers for Epo and some other protein hormones to facilitate their ligand-receptor interaction studies.

Efficient oxidative folding and site‐specific labeling of human hepcidin to study its interaction with receptor ferroportin

Hepcidin is a small disulfide‐rich peptide hormone that plays a key role in the regulation of iron homeostasis by binding and mediating the degradation of the cell membrane iron efflux transporter, ferroportin. Since it is a small peptide, chemical synthesis is a suitable approach for the preparation of mature human hepcidin. However, oxidative folding of synthetic hepcidin is extremely difficult due to its high cysteine content and high aggregation propensity. To improve its oxidative folding efficiency, we propose a reversible S‐modification approach. Introduction of eight negatively charged sulfonate moieties into synthetic hepcidin significantly decreased its aggregation propensity and, under optimized conditions, dramatically increased the refolding yield. The folded hepcidin displayed a typical disulfide‐constrained β‐sheet structure and could induce internalization of enhanced green fluorescent protein (EGFP) tagged ferroportin in transfected HEK293 cells. In order to study interactions between hepcidin and its receptor ferroportin, we propose a general approach for site‐specific labeling of synthetic hepcidin analogues by incorporation of an l‐propargylglycine during chemical synthesis. Following efficient oxidative refolding, a hepcidin analogue with Met20 replaced by l‐propargylglycine was efficiently mono‐labeled by a red fluorescent dye through click chemistry. The labeled hepcidin was internalized into the transfected cells together with the EGFP‐tagged ferroportin, suggesting direct binding between hepcidin and ferroportin. The labeled hepcidin was also a suitable tool to visualize internalization of overexpressed or even endogenously expressed ferroportin without tags. We anticipate that the present refolding and labeling approaches could also be used for other synthetic peptides.

Interaction mechanism of insulin-like peptide 5 with relaxin family peptide receptor 4

Insulin-like peptide 5 (INSL5) is a gut peptide hormone belonging to the insulin/relaxin superfamily. It is implicated in the regulation of food intake and glucose homeostasis by activating relaxin family peptide receptor 4 (RXFP4). Previous studies have suggested that the B-chain is important for INSL5 activity against RXFP4. However, functionalities of the B-chain residues have not yet been systematically studied. In the present work, we conducted alanine-scanning mutagenesis of the B-chain residues of human INSL5 to obtain an overview of their contributions. Binding and activation assays of these INSL5 mutants with human RXFP4 identified two essential exposed B-chain C-terminal residues (B23Arg and B24Trp) and one important exposed central B-chain residue (B16Ile). These three determinant residues together with the C-terminal carboxylate moiety probably constitute a central receptor-binding patch that forms critical hydrophobic and electrostatic interactions with RXFP4 during INSL5 binding. Some other exposed residues, including B10Glu, B12Ile, B13Arg, B17Tyr, B21Ser, and B22Ser, made minor contributions to INSL5 function. These auxiliary residues are scattered around the edge of the central receptor-binding patch, and thus form a peripheral receptor-binding patch on the surface of INSL5. Our present work provides new insights into the interaction mechanism of INSL5 with its receptor RXFP4.