Hutchens T W, Henry J F, Yip T T
U.S. Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030.
Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):2994-8. doi: 10.1073/pnas.88.8.2994.
Two forms of lactoferrin, an intact lactoferrin and a "nicked" but apparently intact (i.e., 78-kDa) form, have been isolated from the urine of preterm infants fed human milk. These two forms of lactoferrin, demonstrated to be entirely of maternal origin, were copurified using affinity columns of immobilized single-stranded DNA-agarose. The relative concentrations of the intact lactoferrin and the "nicked" lactoferrin were determined after denaturation and separation by reverse-phase HPLC. N-terminal sequence analyses showed that the intact 78-kDa form had lost two residues from its N terminus. The nicked 78-kDa form was composed of only two fragments; one fragment was identified as the N terminus of the N-lobe (residues 3-283). The other fragment started with Ser-284 and included the alpha-helical structures at the C terminus of the N-lobe, as well as the entire C-lobe. Although no disulfide bonds connect these two fragments, they were tightly associated in vivo and were not separated in vitro except under denaturing conditions. Limited in vitro digestion of human milk lactoferrin with trypsin produced a nicked, but stable (78-kDa), form of DNA-binding lactoferrin nearly indistinguishable from the isolated urinary lactoferrin, except for the absence of one additional arginine residue at the N terminus of the N-lobe. Residues involved in the stable molecular interaction between fragments were evaluated using data obtained from the high-resolution crystal structure of hololactoferrin. Two features, entirely within the N-lobe, account for the lack of fragment dissociation after cleavage at residue 283 in vivo: an extensive interface at the hinge region behind the iron-binding cleft and an "anchor" sequence traversing the remainder of the N-lobe at 90 degrees relative to the fragment interface. These results document the remarkably limited degradation of absorbed lactoferrin in vivo and suggest that iron-binding activity, receptor-binding properties, and postulated immune cell regulatory functions remain intact.
从喂食母乳的早产儿尿液中分离出了两种乳铁蛋白形式，一种是完整的乳铁蛋白，另一种是“有切口”但显然完整（即78 kDa）的形式。这两种乳铁蛋白形式被证明完全来自母体，使用固定化单链DNA - 琼脂糖亲和柱进行共纯化。通过反相高效液相色谱变性和分离后，测定了完整乳铁蛋白和“有切口”乳铁蛋白的相对浓度。N端序列分析表明，完整的78 kDa形式在其N端失去了两个残基。有切口的78 kDa形式仅由两个片段组成；一个片段被鉴定为N叶的N端（残基3 - 283）。另一个片段从Ser - 284开始，包括N叶C端的α - 螺旋结构以及整个C叶。尽管没有二硫键连接这两个片段，但它们在体内紧密相连，除了在变性条件下，在体外不会分离。用胰蛋白酶对人乳乳铁蛋白进行有限的体外消化产生了一种有切口但稳定（78 kDa）的DNA结合乳铁蛋白形式，除了N叶N端缺少一个额外的精氨酸残基外，与分离出的尿液乳铁蛋白几乎无法区分。利用全乳铁蛋白高分辨率晶体结构获得的数据评估了片段之间稳定分子相互作用所涉及的残基。完全在N叶内的两个特征解释了体内283位残基切割后片段不分离的原因：铁结合裂隙后方铰链区的广泛界面以及相对于片段界面以90度穿过N叶其余部分的“锚定”序列。这些结果证明了体内吸收的乳铁蛋白降解非常有限，并表明铁结合活性、受体结合特性以及假定的免疫细胞调节功能保持完整。
