Arginine 25 and Arginine 28 of lactoferrin are critical for effective heparin neutralization in blood.
The interaction of lactoferrin with endogenous heparin-like molecules modulates glycosaminoglycan-mediated biological processes. We performed site-specific mutagenesis and expressed recombinant lactoferrin and lactoferrin mutants by the baculovirus insect cell expression system. Five basic residues at the lactoferrin N terminus; Arg 5, Arg 25, Arg 28, Lys 29, and Arg 31, were individually replaced by alanines. Heparin chromatography on fast-performance liquid chromatography system showed that the NaCl concentrations corresponding to the peak of each eluted recombinant protein from the column were 665, 620, 540, 550, 630, or 650 mM for wild-type recombinant lactoferrin, Arg 5, Arg 25, Arg 28, Lys 29, or Arg 31 recombinant lactoferrin mutant, respectively. We compared the ability of each mutated lactoferrin derivative to neutralize glycosaminoglycans in the thrombin serpin inhibition assays. In comparison to wild-type recombinant lactoferrin, all the mutants showed decreased ability to neutralize glycosaminoglycan in a dose-dependent manner. The mutations of lactoferrin at Arg 25 and Arg 28 demonstrated the most striking decrease in lactoferrin's ability to neutralize various glycosaminoglycans in both enzymatic and plasma clotting-based experiments. Therefore, our results suggest that Arg 25 and Arg 28 are the critical basic residues at the lactoferrin N terminus responsible for heparin binding. The other basic residues on the N terminus, Arg 5, Lys 29, and Arg 31, also contribute to heparin binding by presenting an additional cationic motif.
Wu
Haifeng M
HM
Department of Pathology, Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA. wu-6@medctr.osu.edu
Church
Frank C
FC
D020816:Amino Acid Motifs; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D007781:Lactoferrin / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D012685:Sepharose / Q000737:chemistry; D013997:Time Factors
D006025:Glycosaminoglycans; D006493:Heparin; D012685:Sepharose; D001120:Arginine; D007781:Lactoferrin; D008239:Lysine