Study of the biological effects of the glycosylation of the chemokine CCL2 and its receptor CCR2

Posttranslational modifications such as limited proteolytic truncation, nitration and citrullination have been shown to affect the biological activity of chemokines. CCL2 is a major attractant and activator of monocytes and one of the only chemokines for which natural glycosylation has been reported. Recently, also chemokine receptors that belong to the G protein-coupled receptor family were shown to be affected by glycosylation. However, almost no studies are available on the biological effects of CCL2 or CCR2 (the CCL2 receptor) glycosylation. With joint project with the University of Copenhagen, we wish to combine our expertise in chemokine ligand biochemistry and biology with their expertise in GPCR technology and glycobiology. We will define 1) whether CCL2 or CCR2 glycosylation is cell type specific, 2) identify the sugar structures, 3) develop methods to determine specific glycoforms of CCL2 and CCR2, 4) develop methods to quantify CCL2 glycoforms in cell cultures and patient samples, 5) study biochemical and biological effects of ligand and/or receptor glycosylation. Biochemical and biological assays will include receptor binding and glycosaminoglycan binding studies, stability assays, receptor signalling tests, cell migration assays in vitro and in vivo, etc. This project will enhance our understanding of the role of glycosylation in CCL2/CCR2 biology and will provide means to study this CCL2/CCR2 biology in patient samples.