DELINEATION OF THE GLYCOSAMINOGLYCAN-BINDING SITE IN THE HUMAN INFLAMMATORY RESPONSE PROTEIN LACTOFERRIN

Lactoferrin is an iron-binding protein which is synthesized by mucosal epithelium and neutrophils and released by these cells in response to inflammatory stimuli. It promotes neutrophil aggregation and manifest s iron-dependent and -independent antimicrobial properties in vitro. S ince lactoferrin binds to glycosaminoglycans (GAGs) and sulfated polys accharides can inhibit its clearance in vivo and in vitro, we sought t o examine its interaction with the GAGs chondroitin sulfate and hepari n. Amino-terminal sequencing of proteolytic fragments of human lactofe rrin that were fractionated by GAG chromatography suggested that the a minoterminal 6 kDa of the secreted protein mediates its interaction wi th GAGs. Synthetic peptides were used to show that the first 33 residu es of human lactoferrin can bind well to solid-phase or solution-phase GAGs. The first 33 residues bound fluoresceinamine-labeled heparin wi th an IC50 (611 nM) which approximated that of the intact protein (124 nM). In contrast, when the first six residues (GRRRRS) were removed f rom this peptide, it then bound poorly to heparin (IC50 = 49 mu M). Ou r results suggest that the GRRRRS sequence at the amino terminus of hu man lactoferrin acts synergistically with an RKVR sequence at position s 28-31 to form the predominate functional GAG-binding site of human l actoferrin. Molecular modeling of the crystalline structure of lactofe rrin supports a synergistic activity between these two sites since it shows that they juxtapose each other on the surface of the folded prot ein. Solid docking calculations indicate that they can form a cationic cradle as a binding site for chondroitin sulfate.