Residue 75 of Interleukin‐8 is Crucial for its Interactions with Glycosaminoglycans

Abstract

The interactions between regulatory proteins such as interleukin-8 (IL-8) and glycosaminoglycans are of great interest both for the general understanding of regulatory processes in biology and for the development of implant coatings and innovative materials that suppress undesired immune responses and improve wound healing. In previous work, a number of residues of IL-8 that interact strongly with several glycosaminoglycans (GAGs) have been identified. In particular, the negatively charged Glu75 was reported to be involved in interactions with charged GAGs. To improve understanding of the role of this residue, we generated a selectively (15)N-labeled E75K variant of IL-8(1-77) by expressed protein ligation. NMR and fluorescence spectroscopy in combination with molecular modeling were applied to evaluate the particular role of residue 75 in interactions with GAGs. Remarkably, more residues in the variant responded to GAG binding than in the wild-type. For the first time, we identified amino acids 34 to 36 as additional residues in the loop region of IL-8(1-77) that participate in the interactions with GAGs. These findings indicate that the N terminus of the E75K variant is more important as a second binding site for GAGs than that of the wild-type IL-8(1-77).

The effect of interleukin‐8 truncations on its interactions with glycosaminoglycans

“…The pTXB1plasmid was used as an expression vector for all variants of interleukin‐8 [IL‐8(1‐54), IL‐8(1‐70), IL‐8(6‐77), IL‐8(12‐77)] and were expressed as Mxe intein and chitin binding domain (CBD) fusion proteins to fully exploit the IMPACT™‐system (New England Biolabs, Germany) . Protein expression, purification and isolation were done as previously described . Briefly, proteins were expressed in Escherichia coli ER2566 cells upon induction with 1 mM IPTG for 6 hour (220 rpm, 37°C).…”

“…In order to hydrolyze the DTT‐protein thioesters, solution was adjusted to pH 9.5 and incubated over night at 4°C. For the generation of the IL‐8(1‐72) and IL‐8(1‐63) expressed protein ligation (EPL) was applied and performed as previously described . All IL‐8 variants were refolded by stepwise dialysis and purified by preparative RP‐HPLC using a gradient of 10%‐60% acetonitrile/0.08% TFA in 0.1% TFA over 55 minutes.…”

Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes

“…# CRL-1651) by determining the inositol phosphate (IP) accumulation after pNPY stimulation, as described (42,43), using the method established by Berridge (44,45). Cells were treated with increasing concentrations of pNPY, from 10 pM to 1 µM.…”

Recent advances in employing molecular modelling to determine the specificity of glycan-binding proteins

“…Nevertheless, the relative lack of experimental structural data for these systems, combined with a growing awareness of their biological significance, has led to a number of innovative approaches to GAG docking. Recent examples include advances in the prediction of GAG binding sites [38,39], improvement in pose prediction via inclusion of specific bound waters [40], co-complex generation via threading and superimposition [41], mimetic discovery [42], as well as combining spectroscopic data with GAG modelling [43,44]. Very recently, an online utility for the automatic generation of 3D structures for GAGs has appeared (www.glycam.org/gag, [25]) that should find application in GAG modelling.…”