Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells
Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells
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DOI：
10.1074/jbc.m007022200
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发表时间：
2000-12-29
影响因子：
4.8
通讯作者：
Mosher, DF
中科院分区：
文献类型：
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作者：
Misenheimer, TM;Huwiler, KG;Mosher, DF
血小板反应蛋白 1 (TSP1) 是一种同源三聚体糖蛋白，由通过二硫桥连接的 150 kDa 亚基组成。血小板反应蛋白 1 的前胶原模块与抗血管生成活性有关。原胶原模块存在于许多细胞外蛋白质中，并且可以通过 10 个具有特征间距的半胱氨酸来识别。我们表达并研究了人 TSP1 的前胶原模块 (C)，无论是其本身还是在相邻寡聚序列 (o) 和 N 末端模块 (N) 的背景下。将编码序列与 N 端信号序列和 C 端多组氨酸标签一起引入杆状病毒中。通过镍螯合色谱法从受感染昆虫细胞的条件培养基中纯化蛋白质。 NoC 是一种二硫键三聚体，很容易在优先蛋白水解位点裂解，产生单体 N 和三聚 oC。这些是全长 TSP1 的已知属性。质谱表明C是N-糖基化的，并且C的所有10个半胱氨酸残基都是二硫键。通过平衡超速离心，C在生理盐溶液中为单体。圆二色性、本征荧光和差示扫描量热实验表明C的稳定性由二硫化物决定。通过碘化物荧光猝灭和溶剂扰动评估，C 的两个色氨酸处于极性暴露环境中。 oC 远紫外圆二色光谱可以建模为 C 和卷曲螺旋低聚域的总和。结果表明，重组 C 自主折叠成其天然结构，并且 TSP1 中模块的三聚化不会扰乱其结构。
Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal poly-histidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography. NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C is N-glycosylated, and all 10 cysteine residues of C are in disulfides. By equilibrium ultracentrifugation, C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.