Legrand D, Mazurier J, Colavizza D, Montreuil J, Spik G
Laboratoire de Chimie Biologique, C.N.R.S. no. 111, Université des Sciences et Techniques de Lille Flandres-Artois, Villeneuve d'Ascq, France.
Biochem J. 1990 Mar 1;266(2):575-81.
The recent determination by X-ray diffraction of the tridimensional structure of human lactotransferrin has underlined the presence of two lobes, each composed of two domains, I and II, as well as the involvement of five ligands in the binding of iron. Only one of the ligands (Asp-61) is located in domain I (residues 1-90 and 252-320), while the others [two tyrosine, one histidine and one (bi)carbonate ion linked to an arginine residue] belong to domain II (residues 91-251). On the basis of these data and of our previous results concerning the isolation of the 30 kDa N-tryptic fragment (residues 4-281) and the 20 kDa N2-glycopeptide (N-terminal domain II; residues 91-253) from human and bovine lactotransferrins, we have compared the iron-binding properties of these two fragments. The results demonstrate that Asp-61, which is missing from domain II, does not take part in the stability upon protonation of the iron complex of both human and bovine lactotransferrins. Furthermore, by comparing the iron-binding properties of human and bovine lactotransferrins to those of isolated 30 kDa N-tryptic and 50 kDa C-tryptic fragments and of the reassociated N,C-tryptic complex of both proteins, it has been shown that the non-covalent interactions which occurred between the two lobes of lactotransferrins and in the reassociated N,C-tryptic complex can explain in part the high affinity of lactotransferrins for iron. Finally, deglycosylation experiments on the 30 kDa N-tryptic fragment and N-terminal domain II from human and bovine lactotransferrins demonstrate that full removal of the glycan moiety leads to the loss of iron-binding capacity and so underlines the importance of the glycan moiety in the stability upon protonation of the N-terminal iron-binding site of both lactotransferrins.
最近通过X射线衍射确定的人乳铁蛋白三维结构突出显示了其存在两个叶，每个叶由两个结构域I和II组成，以及五个配体参与铁的结合。只有一个配体（Asp-61）位于结构域I（残基1-90和252-320）中，而其他配体[两个酪氨酸、一个组氨酸和一个与精氨酸残基相连的（双）碳酸根离子]属于结构域II（残基91-251）。基于这些数据以及我们之前关于从人乳铁蛋白和牛乳铁蛋白中分离出30 kDa N-胰蛋白酶片段（残基4-281）和20 kDa N2-糖肽（N端结构域II；残基91-253）的结果，我们比较了这两个片段的铁结合特性。结果表明，结构域II中缺失的Asp-61不参与人乳铁蛋白和牛乳铁蛋白铁复合物质子化后的稳定性。此外，通过比较人乳铁蛋白和牛乳铁蛋白与分离的30 kDa N-胰蛋白酶和50 kDa C-胰蛋白酶片段以及两种蛋白质重新结合的N、C-胰蛋白酶复合物的铁结合特性，已表明乳铁蛋白两个叶之间以及重新结合的N、C-胰蛋白酶复合物中发生的非共价相互作用可以部分解释乳铁蛋白对铁的高亲和力。最后，对人乳铁蛋白和牛乳铁蛋白的30 kDa N-胰蛋白酶片段和N端结构域II进行的去糖基化实验表明，聚糖部分的完全去除会导致铁结合能力的丧失，因此突出了聚糖部分在两种乳铁蛋白N端铁结合位点质子化稳定性中的重要性。
