Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

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 function^1,2. 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 lig....

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.......

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 study^29,31. Additionally, in vitro methods (cell culture) should be used to confirm the interaction of the putative bi.......

Materials

Name	Company	Catalog Number	Comments
2-D Quant Kit	Cytiva	80648356
Amine Coupling Kit	Cytiva	BR100050
Biacore evaluation software	Biacore S200	Version 1.1
Biacore Sensor chip CM5	Cytiva	BR100530
Bis tris propane	Sigma	B6755-25G	20 mM
BL21 DE3 competent cells	Thermo Scientific	EC0114
CD spectrophotometer	Jasco	J-815 Spectropolarimeter
Glycine HCL	Fisher Bioreagents	BP381-1
GraphPad Prism			Model fitting, data analysis
LB broth	Fisher Bioreagents	BP1426-500
n-dodecyl-β-d-maltoside (DDM)	EMD Millipore	324355-1GM	2-20 mM
pET28a His-tag Kanamycin-resistant expression vector	Addgene	69864-3
Plasmid purification kit	Qiagen	27106
Rho1D4 MagBeads	CubeBiotech	33299
Slide-A_Lyzer 10K dialysis cassette	Thermo Scientific	66810
Tween20	Fisher Bioreagents	BP337-500	0.05%
UV vis Spectrophotometer	Agilent	Cary 60 UV-Vis
Peptide name	Peptide sequence	HPLC-purity	Mass 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