Investigation of lysine side chain interactions of interleukin-8 with heparin and other glycosaminoglycans studied by a methylation-NMR approach

Abstract

Although the interaction between interleukin-8 (IL-8) and glycosaminoglycans (GAGs) is crucial for the mediation of inflammatory effects, little is known about the site specificity of this interaction. Therefore, we studied complexes of IL-8 and heparin (HEP) as well as other GAGs in a multidisciplinary approach, involving site-directed mutagenesis, mass spectrometry, fluorescence and solution NMR spectroscopy as well as computer modeling. The interaction between GAG and IL-8 is largely driven by the amine groups of the lysine and the guanidinium groups of arginine side chains. However, due to fast exchange with the solvent, it is typically not possible to detect NMR signals of those groups. Here, we applied reductive (13)C-methylation of the lysine side chains providing sensitive NMR probes for monitoring directly the sites of GAG interaction in (1)H-(13)C correlation experiments. We focused on the lysine side chains K25, K28, K59, K69 and K72 of IL-8 (1-77), which were reported to be involved in the binding to GAGs. The NMR signals of these residues were assigned in (1)H-(13)C HSQC spectra through the help of site-directed mutagenesis. NMR and fluorescence titration experiments in combination with molecular docking and molecular dynamics simulations were applied to investigate the involvement of each lysine in the binding with HEP and various GAG hexasaccharides. We identified K25, K69 and K72 to be the most relevant binding anchors of IL-8(1-77) for the analyzed GAGs.

Pharmacology of Heparin and Related Drugs

“…Heparin binding site on the C-terminal a-helix and proximal loop (Schlorke et al, 2012;Möbius et al, 2013) …”

“…Examples are studies of hepatocyte growth factor (HGF)/scatter factor (SF), interferon-g, and sclerostin (Deakin et al, 2009;Veverka et al, 2009;Saesen et al, 2013). However, both lysine and arginine, frequently associated with heparin binding sites, have long side chains, and techniques that pick up perturbations to side-chain resonances have been developed and applied to heparin interactions with interleukin (IL)-8 (Möbius et al, 2013) and with the growth factors hepatomaderived growth factor and FGF-2 .…”

Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry

“…413 Conclusively, for all time points and reproducible in all replicates, Table S3). ing [32]. This might also explain why in two of the three allowed models 615 the carbohydrate orientation is different to the NMR based model.…”

“…Chemokine 29 Extracellular matrix 30 3 1 a b s t r a c t 32 The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chon- 33 droitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special 34 interest in the design of artificial extracellular matrices with improved properties concerning GAG inter- 35 action with common regulating proteins.…”

Current structural biology of the heparin interactome

“…The chemokine (cytokine) types most analyzed in the current structural biology of heparin interactome are interleukin-8 (IL-8 or cysteine-amino acid-cysteine ligand (CXCL8)) as the leader of the group [10][11][12], followed by CXCL12 [13,14 ], and then CXCL1 [15 ], CXCL11 [16], interferon-gamma [17], IL-10 [18 ], monocyte chemoattractant protein-1 (CCL2) [12], and the viral macrophage inflammatory protein-II [19].…”