Swamy N, Addo J, Vskokovic M R, Ray R
Vitamin D Laboratory, Department of Medicine, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118, USA.
Arch Biochem Biophys. 2000 Jan 15;373(2):471-8. doi: 10.1006/abbi.1999.1537.
The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ¿25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ¿1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ¿(14)C25-OH-D(3)-11-BE and ¿(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ¿(14)C25-OH-D(3)-6-BE and ¿(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ¿(14)C25-OH-D(3)-3-BE or ¿(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.
维生素D结合蛋白（DBP）具有多种生理特性，包括维生素D（3）及其代谢产物的器官特异性运输，这表现为它能够以高亲和力结合维生素D甾醇。在本研究中，我们使用了含有溴乙酸烷基化探针的25-羟基维生素D（3）（25-OH-D（3））和1,25-二羟基维生素D（3）（1,25（OH）（2）D（3））的亲和标记类似物，对人DBP的维生素D甾醇结合口袋进行了探测，该探针位于母体甾醇的C-3（A环）、C-6（三烯）、C-11（C环）和C-19（环外亚甲基）处。与DBP的竞争性结合试验表明，与25-OH-D（3）相比，1,25（OH）（2）-D（3）-11-BE、25-OH-D（3）-3-BE和25-OH-D（3）-6-BE的结合分别下降了约22倍、68倍和2000倍，而25-OH-D（3）-19-BE和25-OH-D（3）的DBP结合亲和力没有显著差异。令人惊讶的是，尽管（14）C25-OH-D（3）-11-BE和（14）C1,25（OH）（2）-D（3）-19-BE具有高亲和力的DBP结合能力，但它们未能标记DBP，这表明在其溴乙酸部分附近不存在任何亲核氨基酸以形成共价键，而这些类似物位于结合口袋内。相反，（14）C25-OH-D（3）-6-BE和（14）C25-OH-D（3）-3-BE特异性地标记了DBP。用（14）C25-OH-D（3）-3-BE或（14）C25-OH-D（3）-6-BE标记的DBP经BNPS-粪臭素消化产生了两种肽（分子量分别为17,400和33,840），放射性分别与N端和C端肽相关，这增加了同一维生素D甾醇结合口袋的不同区域或DBP的不同结构域可能被这些类似物标记的可能性。两种试剂对DBP重组结构域I（1-203）的成功亲和标记表明，同一维生素D结合口袋（结构域I）的不同区域被标记。这些亲和类似物可能有助于“绘制”维生素D甾醇结合口袋并建立功能模型。
