Swamy N, Ghosh S, Schneider G B, Ray R
Bioorganic Chemistry and Structural Biology Group, Vitamin D Laboratory, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Cell Biochem. 2001;81(3):535-46. doi: 10.1002/1097-4644(20010601)81:3<535::aid-jcb1067>3.0.co;2-6.
Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf.
维生素D结合蛋白（DBP）是一种多功能血清蛋白，通过翻译后修饰可转化为维生素D结合蛋白-巨噬细胞激活因子（DBP-maf）。DBP-maf是一种新的细胞因子，它通过激活破骨细胞来介导骨吸收，破骨细胞负责骨的吸收。破骨细胞激活缺陷会导致诸如石骨症等疾病，其特征是骨量过度积累。先前的研究表明，患有石骨症的大鼠中的两个非等位基因突变在DBP转化为DBP-maf的级联反应中具有独立的缺陷。在这些突变体中，通过体内DBP-maf治疗可纠正与石骨症相关的骨骼缺陷。本研究评估了各种形式的DBP-maf（天然型、重组型和结合25-羟基维生素D(3)的形式）对体外破骨细胞功能的影响，以确定该蛋白与骨吸收活性相关的一些结构要求。破骨细胞活性通过评估使用从新生大鼠长骨中分离的破骨细胞在磷酸钙包被的薄膜Ostologic MultiTest载玻片上形成的凹坑来确定。用体外产生的天然DBP-maf孵育破骨细胞会导致破骨细胞呈剂量依赖性、具有统计学意义的激活。无论DBP-maf的维生素D结合位点是否被占据，激活情况都是相似的。对DBP-maf的反应活性水平高于其他已知的破骨细胞功能刺激剂（甲状旁腺激素和1,25(OH)2D3）的最佳剂量所引发的活性水平。此外，另一种有效的巨噬细胞激活因子干扰素-γ对破骨细胞活性没有影响。在大肠杆菌中表达的全长重组DBP的激活形式在体外试验中没有活性。与这一发现相反，杆状病毒表达的重组DBP-maf表现出显著的破骨细胞激活活性。DBP正常转化为DBP-maf需要从该蛋白的第三个结构域选择性去除半乳糖和唾液酸。因此，两种重组形式的DBP-maf的不同作用很可能与糖基化有关；大肠杆菌表达的重组DBP是非糖基化的，而杆状病毒表达的形式是糖基化的。这些数据支持了糖基化对DBP-maf的破骨细胞激活特性的重要作用。
