CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties

I Scurci,K.B Akondi,I Pinheiro,M Paolini-Bertrand,A Borgeat,F Cerini,O Hartley

doi:10.1016/j.bbagen.2020.129753

Abstract

BackgroundChemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. MethodsWe investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. ResultsTwo commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. ConclusionsCCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. General significanceWe provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Highlights

The chemokine receptor CCR5 is a G protein-coupled receptor ex pressed on a subset of leukocytes whose main physiological role is in the recruitment of effector cells to sites of inflammation [1–3]
We provide further evidence in support of the existence of these subpopulations, but in contrast to earlier studies, which im plicated either local membrane lipid composition [22], receptor oligo merization state [29,47] or receptor G-protein coupling state [26,30] in their formation, our results demonstrate that heterogeneity at the level of cell surface CCR5 sulfation plays an important role in defining the binding properties of both native chemokines and certain anti-CCR5 monoclonal antibodies (mAbs)
Our results are consistent with earlier observations that CCR5 sulfation affects binding of native chemokines [33,34,45]

Summary

Introduction

The chemokine receptor CCR5 is a G protein-coupled receptor ex pressed on a subset of leukocytes whose main physiological role is in the recruitment of effector cells to sites of inflammation [1–3]. Structural studies indicate that the increased anti-HIV potency of 5P12-RANTES is due to an improved molecular fit between its modified N-terminal region and the transmembrane domain of CCR5 [20]. The increased anti-HIV potency of 5P12-RANTES has been explained in terms of its capacity, unlike native CCL5 from which it was derived, to bind with high af finity to different CCR5 subpopulations [26]. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. General significance: We provide the first experimental evidence for sulfation heterogeneity of chemokine re ceptors and its impact on ligand binding, a phenomenon that is important both for the understanding of che mokine cell biology and for the development of drugs that target chemokine receptors

Methods

Results

Conclusion