Abstract
Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11–12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4°C, and co-internalization with their receptors at 37°C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.
Highlights
Chemokine-mediated cell migration is a complex process involving many dynamic steps including transport of chemokines across cells, presentation of chemokines on cell surface glycosaminoglycans (GAGs) and extracellular matrix components, binding of chemokines to their G protein-coupled receptors (GPCRs), and scavenging and transport of chemokines by atypical chemokine receptors [1,2,3,4]
As an alternative approach we investigated the use of recently described genetically encoded peptide tags that can be labeled with phosphopantetheinyl transferase enzymes (PPTases) [19,20]
The site-specific protein-labeling method developed by Yin and coworkers utilizes the phosphopantetheinyl transferase (PPTase) reaction, in which a genetically encoded peptide tag is modified with the phosphopantetheinyl (Ppant) arm of coenzyme A, and, by extension, any molecule that can be coupled to the sulfhydryl group of CoA [19,20]
Summary
Chemokine-mediated cell migration is a complex process involving many dynamic steps including transport of chemokines across cells, presentation of chemokines on cell surface glycosaminoglycans (GAGs) and extracellular matrix components, binding of chemokines to their G protein-coupled receptors (GPCRs), and scavenging and transport of chemokines by atypical chemokine receptors [1,2,3,4]. Binding to receptors typically involves monomeric forms of chemokines. In order to understand the complex interactions and dynamic mechanisms involved in chemokine biology and to track their spatial and temporal dependence, fluorescence and bioluminescent imaging methods have become important tools. Fluorescent chemokines have been used to detect binding interactions with receptors [14]
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