Abstract
Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native conditions, is necessary. Herein is the first report of a tetrameric assembly of the human chemokine CCL11, which was shown bound to the GAG Arixtra™. Isothermal titration calorimetry data indicated that CCL11 interacts with Arixtra, and ion mobility mass spectrometry (IM-MS) was used to identify ions corresponding to the CCL11 tetrameric species bound to Arixtra. Collisional cross sections (CCS) of the CCL11 tetramer-Arixtra noncovalent complex were compared to theoretical CCS values calculated using a preliminary structure of the complex deduced using X-ray crystallography. Experimental CCS values were in agreement with theoretical values, strengthening the IM-MS evidence for the formation of the noncovalent complex. Tandem mass spectrometry data of the complex indicated that the tetramer-GAG complex dissociates into a monomer and a trimer-GAG species, suggesting that two CC-like dimers are bridged by Arixtra. As development of chemokine inhibitors is of utmost importance to treatment of medical inflammatory conditions, these results provide vital insights into chemokine-GAG interactions.
Highlights
Chemokines are secreted signaling proteins that are involved in biological processes such as inflammation, lymphocyte homing, and development
In order to confirm the interaction between CCL11 and Arixtra and measure the binding constant (KD) arising from this interaction, isothermal titration calorimetry was performed by titrating Arixtra into a CCL11 solution
The experimentally-determined n value for Arixtra of ~0.7 for CCL2 and CCL11 binding may reflect binding across a variety of chemokine oligomeric states
Summary
Chemokines are secreted signaling proteins that are involved in biological processes such as inflammation, lymphocyte homing, and development. Chemotactic gradients are formed as secreted chemokines diffuse away from the site of release before binding to cell-surface glycosaminoglycans (GAGs) [7] The importance of this interaction is demonstrated by the observation that while mutant chemokines with decreased GAG binding activity can still signal receptors in vitro, these mutants fail to recruit leukocytes in vivo [8]. These results show that CCL11 forms a previously unreported homotetramer species, but this tetrameric species binds to Arixtra These results support previous models of GAG-dependent chemokine oligomerization [8,10,30] and suggest that it may be possible to utilize synthetic GAG molecules to modulate chemokine function in vivo
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