Mann D M, Romm E, Migliorini M
J. H. Holland Laboratory, Biochemistry Department, American Red Cross, Rockville, Maryland 20855.
J Biol Chem. 1994 Sep 23;269(38):23661-7.
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 manifests iron-dependent and -independent antimicrobial properties in vitro. Since lactoferrin binds to glycosaminoglycans (GAGs) and sulfated polysaccharides can inhibit its clearance in vivo and in vitro, we sought to examine its interaction with the GAGs chondroitin sulfate and heparin. Amino-terminal sequencing of proteolytic fragments of human lactoferrin that were fractionated by GAG chromatography suggested that the amino-terminal 6 kDa of the secreted protein mediates its interaction with GAGs. Synthetic peptides were used to show that the first 33 residues of human lactoferrin can bind well to solid-phase or solution-phase GAGs. The first 33 residues bound fluoresceinamine-labeled heparin with an IC50 (611 nM) which approximated that of the intact protein (124 nM). In contrast, when the first six residues (GRRRRS) were removed from this peptide, it then bound poorly to heparin (IC50 = 49 microM). Our results suggest that the GRRRRS sequence at the amino terminus of human lactoferrin acts synergistically with an RKVR sequence at positions 28-31 to form the predominate functional GAG-binding site of human lactoferrin. Molecular modeling of the crystalline structure of lactoferrin supports a synergistic activity between these two sites since it shows that they juxtapose each other on the surface of the folded protein. Solid docking calculations indicate that they can form a cationic cradle as a binding site for chondroitin sulfate.
乳铁蛋白是一种铁结合蛋白，由黏膜上皮细胞和中性粒细胞合成，并在炎症刺激下由这些细胞释放。它促进中性粒细胞聚集，并在体外表现出铁依赖性和非依赖性抗菌特性。由于乳铁蛋白与糖胺聚糖（GAGs）结合，硫酸化多糖可在体内和体外抑制其清除，因此我们试图研究其与硫酸软骨素和肝素等GAGs的相互作用。通过GAG色谱法分离的人乳铁蛋白蛋白水解片段的氨基末端测序表明，分泌蛋白的氨基末端6 kDa介导其与GAGs的相互作用。合成肽用于表明人乳铁蛋白的前33个残基可以很好地结合固相或溶液相的GAGs。前33个残基与荧光胺标记的肝素结合的IC50（611 nM）接近完整蛋白的IC50（124 nM）。相反，当从该肽中去除前六个残基（GRRRRS）时，它与肝素的结合很差（IC50 = 49 microM）。我们的结果表明，人乳铁蛋白氨基末端的GRRRRS序列与28-31位的RKVR序列协同作用，形成人乳铁蛋白主要的功能性GAG结合位点。乳铁蛋白晶体结构的分子模型支持这两个位点之间的协同活性，因为它表明它们在折叠蛋白的表面上相互并列。固体对接计算表明，它们可以形成一个阳离子支架作为硫酸软骨素的结合位点。
