Interleukin-8 binds to syndecan-2 on human endothelial cells

Abstract

Application of reverse transcription–PCR to total RNA prepared from TNF-α (tumour necrosis factor-α)-stimulated HUVECs (human umbilical vein endothelial cells) revealed that the syndecan-2 mRNA was up-regulated by this inflammatory stimulus. By immunoprecipitation using an anti-syndecan-2 antibody on TNF-α-stimulated HUVEC lysates, inflammation-induced interleukin-8 was found to be an interaction partner of this HS (heparan sulphate) proteoglycan, but not of any other syndecan on these cells. The glycosylated [Syn2ect(+HS)] and non-glycosylated [Syn2ect(−HS)] forms of Syn2ect (the syndecan-2 ectodomain) were purified from a stably transfected human cell line and from a bacterial expression system respectively. By CD spectroscopy, Syn2ect was found to adopt an all-β secondary structure. The dissociation constant of Syn2ect(+HS) with respect to interleukin-8 binding was determined by isothermal fluorescence titrations to be 23 nM. Despite its lack of HS chains, Syn2ect(−HS) exhibited significant binding to the chemokine, with a Kd of >1 µM. Thus, in addition to glycosaminoglycan binding, protein–protein contacts might also contribute to the chemokine–proteoglycan interaction.