title: Characterization of the glycosaminoglycan-binding region of lactoferrin.
abstract: Lactoferrin is a prominent component of neutrophil secondary granules and its blood concentration is increased in certain inflammatory diseases. Although the biochemical characterization of lactoferrin as an iron-binding protein has been well described, its physiological role in inflammation remains undefined. We examined the ability of lactoferrin to regulate glycosaminoglycan-accelerated thrombin-serine protease inhibitor (serpin) reactions. Lactoferrin effectively reduced the rate of thrombin-serpin (antithrombin and heparin cofactor II) reactions by three physiological glycosamino-glycans including heparin, heparan sulfate, and dermatan sulfate. An enzyme kinetics analysis showed that lactoferrin did not alter the apparent heparin-thrombin or the heparin-antithrombin dissociation constant values for the heparin-catalyzed thrombin-antithrombin reaction. However, the maximum reaction velocity at saturation with respect to either protein was markedly decreased by lactoferrin. The glycosaminoglycan-binding region of lactoferrin was analyzed following limited proteolysis using Staphylococcus aureus V8 protease. Two lactoferrin fragments with Mr's of approximately 8 and approximately 11 kDa were purified based on their affinity to heparin-Sepharose. Amino acid sequence analysis demonstrated that both peptides were from the N-terminus. Although slightly less capable compared to intact lactoferrin, the lactoferrin peptides effectively neutralized heparin, heparan sulfate, and dermatan sulfate-catalyzed serpin-thrombin inhibition reactions. In addition, lactoferrin N-terminal peptides have approximately the same binding affinity to heparin-Sepharose as that of intact lactoferrin. Inspection of both the N-terminal amino acid sequence and the crystal structure of lactoferrin further supports the conclusion that lactoferrin is a novel glycosaminoglycan binding protein and that the putative glycosaminoglycan-binding site is localized to the N-terminus.
authors.affiliation: Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill 27599.
authors.forename: H F
authors.initials: HF
authors.lastname: Wu
authors.forename: D M
authors.initials: DM
authors.lastname: Monroe
authors.forename: F C
authors.initials: FC
authors.lastname: Church
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry; D011487:Protein Conformation; D016415:Sequence Alignment; D015843:Serpins / Q000187:drug effects / Q000378:metabolism
chemical_list: D006025:Glycosaminoglycans; D015843:Serpins; D007781:Lactoferrin