Wu H F, Monroe D M, Church F C
Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill 27599.
Arch Biochem Biophys. 1995 Feb 20;317(1):85-92. doi: 10.1006/abbi.1995.1139.
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.
乳铁蛋白是中性粒细胞次级颗粒的主要成分，在某些炎症性疾病中其血液浓度会升高。尽管乳铁蛋白作为一种铁结合蛋白的生化特性已得到充分描述，但其在炎症中的生理作用仍不明确。我们研究了乳铁蛋白调节糖胺聚糖加速的凝血酶 - 丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）反应的能力。乳铁蛋白可有效降低三种生理性糖胺聚糖（包括肝素、硫酸乙酰肝素和硫酸皮肤素）催化的凝血酶 - 丝氨酸蛋白酶抑制剂（抗凝血酶和肝素辅因子II）反应速率。酶动力学分析表明，乳铁蛋白不会改变肝素催化的凝血酶 - 抗凝血酶反应中肝素 - 凝血酶或肝素 - 抗凝血酶的表观解离常数值。然而，乳铁蛋白会显著降低两种蛋白质饱和时的最大反应速度。使用金黄色葡萄球菌V8蛋白酶进行有限蛋白酶解后，分析了乳铁蛋白的糖胺聚糖结合区域。基于它们对肝素 - 琼脂糖的亲和力，纯化了两个分子量分别约为8 kDa和约11 kDa的乳铁蛋白片段。氨基酸序列分析表明，这两个肽段均来自N端。尽管与完整的乳铁蛋白相比能力稍弱，但乳铁蛋白肽段可有效中和肝素、硫酸乙酰肝素和硫酸皮肤素催化的丝氨酸蛋白酶抑制剂 - 凝血酶抑制反应。此外，乳铁蛋白N端肽段与肝素 - 琼脂糖的结合亲和力与完整乳铁蛋白大致相同。对乳铁蛋白的N端氨基酸序列和晶体结构的研究进一步支持了以下结论：乳铁蛋白是一种新型的糖胺聚糖结合蛋白，推测的糖胺聚糖结合位点位于N端。
