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Summary

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

Abstract

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4.

These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis.

Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Introduction

Dietary obtained Vitamin A/all-trans retinol/ROL is an important component playing a role in visual function1,2. The chromophore 11-cis retinal, a metabolite of dietary vitamin A, binds to the G protein-coupled receptor (GPCR) opsin to generate the retinylidene protein, rhodopsin, in the photoreceptors. When light falls on the eye, the configuration of rhodopsin undergoes a fundamental change via the conversion of its 11-cis-retinal component to the all-trans-retinal. This configuration change triggers a phototransduction cascade within the rod photoreceptors, converting light into an electrical signal, which is transmitted to the visual cortex in the brain via the optic nerve3,4,5,6,7,8,9,10. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn causes opsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor OS degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis, which can cause blindness3,10.

All-trans retinol is the fundamental transport form of dietary vitamin A and it is the source from which all functional retinoids and dietary vitamin A metabolites are derived. The liver serves as the primary organ for dietary vitamin A storage. Hepatic retinol is transported via the serum as its complex with retinol-binding protein 4 (RBP4). RBP4, primarily expressed in the liver, forms a holo-complex with the retinol substrate and transthyretin (TTR), which enters the circulation11,12,13,14,15,16,17. The report of a cell surface receptor for RBP4 in the 1970s led to the hypothesis of membrane transport proteins aiding the transport of retinoids in and out of cells. The cell surface receptor for RBP4-bound retinol (RBP4-ROL) was identified as STimulated by Retinoic Acid 6 Retinol (STRA6) in the retinal pigment epithelium (RPE) of the eye. STRA6 binds to the circulatory holo-RBP4 complex and shuttles the RBP4-bound retinol across the RPE to be utilized by photoreceptors18,19. Mutations in STRA6 can lead to a myriad of diseases and phenotypes associated with reduced ocular ROL concentrations. STRA6 mutations during development can lead to anophthalmia, microphthalmia, and other non-ocular symptoms that overlap with phenotypes associated with Matthew-Wood syndrome20,21,22,23,24,25,26,27. STRA6 is expressed in different organs and tissues, such as the RPE in the eye, but not in all tissues26,27. Although the primary site of retinoid storage is the liver, STRA6 is not expressed in the liver.

Alapatt and colleagues discovered that the Retinol Binding Protein 4 Receptor 2 (RBPR2) bound RBP4 with high affinity and was responsible for the uptake of RBP4-bound retinol in the liver, similar to STRA6 in the RPE28. RBPR2 has been reported to share structural homology with STRA629,30,31. RBP4 is proposed to bind to residues S294, Y295, and L296 on RBPR2, an amino acid-binding domain partially conserved between RBPR2 and STRA6 as well29,30,31. From these studies, vitamin A membrane receptors such as STRA6 and RBPR2, which contain one or more extracellular binding residues/domains, are proposed to interact with circulatory RBP4-ROL. Membrane receptors, therefore, play an important role in receptor binding to circulatory RBP4 for ROL internalization into target tissues, such as the liver and eye.

In the first part of this study, we utilized Surface Plasmon Resonance (SPR) to investigate the interaction of two vitamin A membrane receptors (RBPR2 and STRA6) with their physiological ligand RBP431. The binding affinities and association/dissociation kinetics of protein to ligand complexes can be measured in real time by using SPR. This methodology aimed to provide critical kinetic, structural, and biochemical information on RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency31,32. As mentioned above, circulatory ROL is internalized into the RPE via STRA6 to generate the chromophore 11-cis retinal, which binds to opsin to generate the retinylidene protein, rhodopsin, in photoreceptors33. We used spectrophotometry methodologies to quantify the GPCR-opsin and its ligand 11-cis retinal complex in murine retinal lysates, which is critical for understanding mechanisms of reduced retinylidene protein, rhodopsin, in Retinitis Pigmentosa or Leber Congenital Amaurosis ocular pathological states34. In general, these protocols can be applied to study in vitro the physiological consequences of mutant RBP4, STRA6, or RBPR2 in influencing systemic and ocular vitamin A homeostasis or the impact of mutant rhodopsin or retinoid cycle proteins on visual function35,36.

Protocol

1. Surface plasmon resonance (SPR) methodology

  1. Preparation and purification of mouse RBP4 protein
    1. Design primers to generate full-length mouse RBP4 (msRBP4) cDNA (NCBI Reference Sequence: NM_001159487.1). Clone the msRBP4 cDNA into a pET28a His-tag Kanamycin-resistant expression vector and express in BL-21 DE3 cells (Table of Materials).
    2. Transform the BL-21 DE3 competent cells (according to the manufacturer's protocol) with ligated msRBP4-pET28a vector and then plate onto Luria-Bertani (LB) broth Agar plates containing Kanamycin 50 µg/mL30,37. Incubate the plates overnight at 37 °C in an incubator and screen for positive colonies the next day.
    3. Pick a single colony and incubate overnight at 37 °C in 5 mL of LB broth containing Kanamycin 50 µg/mL.
    4. For secondary culture in 500 mL of LB broth containing Kanamycin 50 µg/mL, use 1% volume of primary culture (from step 1.1.3.) and incubate at 37 °C for 3-4 h; check the optical density using a spectrophotometer. Ideally, proceed if the OD value is between 0.6 and 1 to induce protein expression with 0.5 mM IPTG at 24 °C overnight.
    5. Use multiple IPTG concentrations of 0.1-1 mM and 16-30 °C temperature range to optimize msRBP4 expression and stability.
    6. Centrifuge the culture (from step 1.1.5.) at 16,000 × g for 10 min, discard the supernatant, and keep the pellet on ice.
    7. Homogenize the pellet with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Triton X-100, 10% Glycerol, 0.5 mM PMSF, and Protease Inhibitors [pH 8.0]). Sonicate the lysate for 15 s ON and 30 s OFF cycle using a homogenizer with a 250 Watt output and 40-50% 20 kHz output frequency. Repeat 5x.
    8. Remove the cell debris and unlysed cells by centrifugation at 16,000 × g for 20 min. Pass the supernatant of the lysate through a buffer-equilibrated nickel NTA column.
    9. Wash the column with lysis buffer (from step 1.1.8) containing 30 mM imidazole.
    10. In fractions, elute the bound msRBP4 protein using buffer containing 250 mM imidazole. Run the eluted fractions and visually monitor the protein quantity with a 12% SDS polyacrylamide gel, stained with Coomassie Brilliant Blue R-250 in methanol:water:acetic acid (45:45:10) solution and de-stain with solution without Coomassie stain methanol:water:acetic acid (45:45:10).
    11. Pool the unused fractions and dialyze the pooled fractions with a 10K dialysis cassette of 10 kDa MWCO.
    12. Evaluate the quality of the dialyzed fraction on a 12% SDS-PAGE gel and quantify the protein concentration.
      NOTE: Apo-RBP4 protein was used in the SPR analysis. For generating holo-RBP4 (RBP4-retinol), refer to detailed protocols published elsewhere36,37.
  2. Initial validation of RBPR2 and STRA6 peptides with RBP4 for structure and interaction by Tryptophan fluorescence assay and circular dichroism (CD) spectroscopy
    1. Tryptophan fluorescence assay
      1. Use the online protein composition analysis tool (https://web.expasy.org/protparam/) and paste the amino acid sequence in one-letter code to assess the structural composition of recombinant RBP4 protein and the individual peptides of RBPR2 and STRA6 to determine the tryptophan amino acid residues in the individual peptides and protein (Table of Materials).
      2. Click on the compute parameters button and evaluate the amino acid composition for tryptophan (Trp) residues.
        NOTE: Tryptophan residues must be present in the individual peptides or proteins being analyzed.
      3. Dilute the individual RBPR2 and STRA6 peptides in various micromolar concentrations and incubate individually with 3 µg msRBP4 in a 96-well plate at room temperature for 5 min. Excite the mix at 290 nm and scan the emission from 300 nm to 400 nm wavelength.
      4. Normalize the data with the Blank (buffer only, no peptide and no protein) and peptide control condition (no protein) and plot.
        NOTE: The tryptophan fluorescence increment upon exposure to a low to high peptide concentration suggests a positive interaction between the two individual peptides and RBP4 protein.
    2. Circular dichroism (CD) spectroscopy
      1. Dilute the individual peptides and recombinant RBP4 protein in 1x PBS buffer, pH 7.0. Use various concentrations of RBPR2 and STRA6 peptides and RBP4 protein from 0.1 to 10 μg/mL for a quality check, as described below.
      2. Add individual diluted peptides and protein in a 0.1 cm path length cuvette and measure the CD absorbance/ellipticity using a CD spectrophotometer (195-250 nm). Use the below formula to calculate the mean residue ellipticity (θ).
        [v] = (S × mRw)/(10cl)
        S represents the CD signal in mθ; mRw represents the mean residue mass; c represents the concentration of the protein in mg/mL; and l represents the path length in cm.
      3. Calculate the percent change in molecule structure using BeStSel Secondary Structure Analysis to Protein Fold Prediction by CD Spectroscopy (https://bestsel.elte.hu).
        NOTE: The secondary structure indicates the proper folding and conserved confirmations crucial for the interaction study and the outcome of the interactions. Inspect the predicted structure of the domain and proceed further with the interaction study.

2. SPR analysis to determine the binding affinities and kinetic parameters of vitamin A membrane receptors (RBPR2 and STRA6) with their physiological ligand RBP4

  1. For the immobilization of RBP4 on CM5 Chip surface setup, we use the SPR sensor chip, which carries a carboxymethyl dextran linker extended to a size of ~100 nm covalently attached on a gold surface. In the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) converts the carboxyl groups on the chip's surface to N-Hydroxysuccinimide esters, which spontaneously react with the amine groups of amino acids and proteins, forcing the ligand to bond covalently onto the chip's surface (Table of Materials).
    1. Immobilize the purified msRBP4 protein on the Sensor chip (Figure 1 and Figure 2). In the software Run menu, choose the Wizard and proceed with the immobilization option. Dilute the ligand (msRBP4 protein obtained from step 1.1) in immobilization buffer (10 mM Sodium acetate buffer, pH 5.0) to 10 µg/mL (4 µL of ligand + 396 µL of buffer) and aliquot the coupling reagents in vials. Open the Immobilization Setup wizard window from the software control, select CM5 Chip, and option Immobilize flow cell 2 and target level of 1,200 Response Unit (RU) for achieving Rmax of 30 RU in the kinetic study and Wash solution: Ethanolamine. Enter the name RBP4 and click Next.
      NOTE: Optional: System Preparations dialog box for the Prime before run option is not required if priming with running buffer PBST (phosphate-buffered saline solution with a 0.05% Tween 20 detergent solution) was performed previously.
    2. In the Rack position dialog box, click Eject Rack. Take the rack and place the vials with assigned volumes for each reagent: 156 µL of msRBP4, 177 µL of ethanolamine, 89 µL of EDC, 89 µL of NHS, and an empty vial. Insert the rack with sample vials and click OK and Next.
    3. In the Prepare Run Protocol dialog, click Start and Save.
  2. RBPR2 and STRA6 peptides kinetic assay setup
    1. Serially dilute the mouse RBPR2, mutant RBPR2, and STRA6 peptides (chemically synthesized previously31 and listed in Table of Materials) in running buffer in a range of 0.8-26.6 μM.
    2. Open the control software and select Kinetics/Affinity wizard. Click File | Open template and select Kinetics/Affinity. Input the injection sequence dialog flow path 2-1 (Flow cell 2 as active and Flow cell 1 as reference), check the Chip Type CM5, and click Next. In the setup dialog, check the Run startup cycles, enter Solution: buffer and cycles: and click Next.
    3. In the injection parameters dialog, enter the values for sample parameters: contact time: 120 s, flowrate 30 μL/min, Dissociation time 300 s; for regeneration parameters: enter the Regeneration solution name: Glycine-HCl (pH 2.5), contact time: 30 s, flowrate: 30 μL/min and click Next.
    4. In the samples dialog, enter the name of the peptides, molecular weight, and concentrations and click next. In the Rack position dialog, assign the diluted peptides as individual ~120 µL samples of various concentrations 1 to 100 µM and ~230 µL of Buffer, ~120 µL of Buffer, and ~1,000 µL of Glycine, pH 2.5, to the positions on the Rack. Click Eject Rack and insert all the vials. Click Next, and in the Prepare Run Protocol dialog, click Start, enter a file name, and Save (Figure 2).
    5. In the software, click the Start menu and select Evaluation. Choose open the file in the software, and from the dropdown selection menu, choose Fc2-1 to analyze the Reference cell Blank-subtracted Active sensorgram for the association and dissociation phases of the msRBP4 and mutant msRBPR2 and msSTRA6 peptides.
    6. Click on the dropdown Kinetics/Affinity button and choose Surface bound; to open the Select Curves dialog, check include column in the table and click Next. Select Data dialog to show blank-subtracted sensorgrams; click Kinetics and Affinity, keep the default parameters, and click Fit option for curve fitting. Inspect the Report tab for equilibrium dissociation constant Kd and Ka or Kon association rate and Kd or Koff dissociation rate. Save the Kinetics data sensorgram in .txt tab delimited format and use the values to plot graphs (Figure 3).
    7. To determine the affinity kinetics constant Kd (ligand concentration that binds to half the receptors at equilibrium), copy and paste the data columns in the .txt format file generated from the Evaluation to XY plot used in the software. Click Analyze and Nonlinear regression (curve fit), check the data column, and click Ok. In the parameter dialog, click Binding saturation and One site Specific binding fitting model with the formula of:
      Y= (Bmax*X)/(Kd + X)
      Where Bmax is the maximum number of binding sites (Response Unit), X is the concentration of the peptides, and Y is the binding (Response unit) (Figure 3).

3. Spectrophotometry methodology to quantify the GPCR-11- cis retinal protein complex (retinylidene protein rhodopsin) in retinal lysates

  1. Transport the predetermined group of mice consisting of adult WT-C57BL/6J mice to the dark room.
  2. Dark adapt the mice overnight for 12-16 h, and under red light, place the mouse in a euthanasia chamber and open the CO2 gas valve on the regulator to 5 L/min for ~5 min. Ensure death by applying pressure on the neck and tail pulled backward (cervical dislocation).
  3. Enucleate the eye globes and homogenize the eyes in an ice-cold buffer containing 20 mM bis-tris propane (1,3-bis (tris(hydroxymethyl)methylamino)propane; BTP) pH 7.4, 150 mM NaCl, 1 mM EDTA and with protease inhibitor (Table of Materials).
  4. Centrifuge the homogenate for 15 min at 16000 x g at 4 °C, discard the supernatant containing water soluble cytosolic proteins. Re-suspend and solubilize the pelleted membrane and membrane proteins in buffer containing 20 mM n-dodecyl-β-d-maltoside (DDM), 20 mM bis-tris propane (BTP) pH 7.4, 100 mM NaCl, 1 mM EDTA and with protease inhibitor for 1 h at 4 °C on a rotating platform.
  5. Centrifuge the resolubilized membrane fraction for 1 h at 16000 x g, 4 °C. Mix the supernatant with 30 μL of Rho 1D4 antibody beads and incubate for 1 h at 4 °C on a rotating platform.
  6. Using a magnetic stand, separate the pulldown using beads, discard the supernatant, and wash the beads with buffer 20 mM bis-tris propane (BTP) pH 7.4, 500 mM NaCl, and 2 mM DDM.
  7. Elute the rhodopsin protein by incubating for 5-10 min at room temperature with buffer 20 mM bis-tris propane (BTP) pH 7.4, 100 mM NaCl, 2 mM DDM containing 0.2 mg/mL 1D4 peptide (-TETSQVAPA) (Figure 4 and Figure 5).
  8. Analyze the elute for absorbance from 200 nm to 800 nm using a rectangular, sub-micro, quartz, 2 mm aperture, 10 mm path length, 50 μL cuvette in a fast scan setting with aspectrophotometer.
    NOTE: Use only high-quality Quartz cuvettes (Figure 4).
  9. To validate and determine quality, check and expose the elute to bright light for 1 min, then repeat step 3.8.
  10. Subtract the Blank absorbance and calculate the ratio of the analyzed absorbance spectra with absorbance at 500 nm for 11-cis retinal bound opsin, 380 nm for light-exposed all-trans retinal bound opsin, and 280 nm for ligand-free Apo-Opsin protein (Figure 6). The free opsin with no ligand retinal gives a baseline absorbance at 500 nm.
  11. To quantify rhodopsin concentration, use Beer-Lambert's Law:
    A=ε·C·l or C=A/ε·l
    Where A is the absorbance, ε the molar extinction coefficient (rhodopsin ε500 = 40,600 M−1 cm−1 for free Apo Opsin ε280 = 81,200 M−1 cm−1), C is the concentration, and l path length.
  12. For quantifying liganded-Opsin in percentage, use a ratio of A280/A500.
  13. Plot the Blank-subtracted absorbance spectra and calculate the free opsin value32.
  14. Calculate the concentration of rhodopsin using the extinction coefficient ε500 = 40600 M−1 cm−1. The concentration of ligand-free opsin is calculated with Beer-Lambert Law using the extinction coefficient ε280 = 81,200 M−1 cm−1.

Results

Quantitative methods are described to study protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands. The recombinant mouse RBP4 should be expressed in E. coli and the purified protein used as a conjugated ligand on a SPR Chip. The chemically synthesized RBPR2, STRA6, and mutant S294A RBPR2 "SYL motif RBP4 interacting extracellular site" of a ~40 amino acid peptide is used as analyte at various concentrations to measure the kinetics o...

Discussion

Critical steps in the protocol
SPR Methodology
In silico modeling and docking analysis: The predicted structure of RBPR2 (https://alphafold.ebi.ac.uk/entry/Q9DBN1) and STRA6, and the known structure for msRBP4 PDB database (RSCB PDB ID: 2wqa), should be used for docking study29,31. Additionally, in vitro methods (cell culture) should be used to confirm the interaction of the putative bi...

Disclosures

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Acknowledgements

The authors thank Dr. Beata Jastrzebska, Ph.D. (Department of Pharmacology, Case Western Reserve University, OH) for her advice on the rhodopsin absorbance protocol. This work was supported by an NIH-NEI grant (EY030889 and 3R01EY030889-03S1) and, in part, by the University of Minnesota start-up funds to G.P.L.

Materials

NameCompanyCatalog NumberComments
2-D Quant KitCytiva80648356
Amine Coupling KitCytivaBR100050
Biacore evaluation softwareBiacore S200Version 1.1
Biacore Sensor chip CM5CytivaBR100530
Bis tris propaneSigmaB6755-25G20 mM
BL21 DE3 competent cellsThermo ScientificEC0114
CD spectrophotometerJascoJ-815 Spectropolarimeter
Glycine HCLFisher BioreagentsBP381-1
GraphPad Prism Model fitting, data analysis
LB brothFisher BioreagentsBP1426-500
n-dodecyl-β-d-maltoside (DDM)EMD Millipore324355-1GM2-20 mM
pET28a His-tag Kanamycin-resistant expression vector Addgene69864-3
Plasmid purification kitQiagen27106
Rho1D4 MagBeadsCubeBiotech33299
Slide-A_Lyzer 10K dialysis cassetteThermo Scientific66810
Tween20Fisher BioreagentsBP337-5000.05%
UV vis SpectrophotometerAgilent Cary 60 UV-Vis
Peptide namePeptide sequenceHPLC-purityMass Spec
Mouse Rbpr2 (42)HVRDKLDMFEDKLESYLTHM
NETGTLTPIILQVKELISVTKG		92.14%Conforms
Mouse Stra6 (40)SVVPTVQKVRAGINTDVSYL
LAGFGIVLSEDRQEVVELVK		90.84%Conforms
Mouse Rbpr2 mutant S294A (42)HVRDKLDMFEDKLEAYLTHM
NETGTLTPIILQVKELISVTKG		0.92%Conforms

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