A general method for site specific fluorescent labeling of recombinant chemokines.
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.
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
Tetsuya
T
Kawamura
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America ; Biomedical Sciences Graduate Program, School of Medicine, University of California San Diego, La Jolla, California, United States of America.
Bryan
B
Stephens
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
Ling
L
Qin
Marine Drug Research Institute, Huaihai Institute of Technology, Lianyungang, China ; Veterans Affairs San Diego Healthcare System, San Diego, California, United States of America ; Department of Medicine, Division of Pulmonary and Critical Care, University of California San Diego, La Jolla, California, United States of America.
Xin
X
Yin
Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America.
Michael R
MR
Dores
Biomedical Sciences Graduate Program, School of Medicine, University of California San Diego, La Jolla, California, United States of America ; Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America.
Thomas H
TH
Smith
Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America.
Neil
N
Grimsey
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
Ruben
R
Abagyan
Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America.
Joann
J
Trejo
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
Irina
I
Kufareva
Veterans Affairs San Diego Healthcare System, San Diego, California, United States of America ; Department of Medicine, Division of Pulmonary and Critical Care, University of California San Diego, La Jolla, California, United States of America.
Mark M
MM
Fuster
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
Catherina L
CL
Salanga
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
Tracy M
TM
Handel
D002460:Cell Line; D018932:Chemokine CCL2 / Q000737:chemistry / Q000378:metabolism; D054421:Chemokine CCL21 / Q000737:chemistry / Q000378:metabolism; D054377:Chemokine CXCL12 / Q000737:chemistry / Q000378:metabolism; D018925:Chemokines / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D054390:Receptors, CCR2 / Q000737:chemistry / Q000378:metabolism; D054400:Receptors, CCR7 / Q000737:chemistry / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
C515334:CCR2 protein, human; C515370:CCR7 protein, human; C527276:CXCR4 protein, human; D018932:Chemokine CCL2; D054421:Chemokine CCL21; D054377:Chemokine CXCL12; D018925:Chemokines; D054390:Receptors, CCR2; D054400:Receptors, CCR7; D019718:Receptors, CXCR4; D011994:Recombinant Proteins