Eukaryotic expression of recombinant biglycan - Post-translational processing and the importance of secondary structure for biological activity
Eukaryotic expression of recombinant biglycan - Post-translational processing and the importance of secondary structure for biological activity
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DOI：
10.1074/jbc.271.32.19571
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发表时间：
1996-08-09
影响因子：
4.8
通讯作者：
McQuillan, DJ
中科院分区：
文献类型：
--
作者：
Hocking, AM;Strugnell, RA;McQuillan, DJ
双糖链蛋白聚糖是一种小的硫酸软骨素蛋白聚糖，存在于许多组织中，在结构上与核心蛋白聚糖、纤维调节蛋白和鲁米聚糖相关。双糖链蛋白聚糖的生物学功能知之甚少，尽管一些研究表明与其他细胞外基质成分相互作用，我们已经开始使用痘苗病毒/T7噬菌体表达系统通过瞬时真核表达来研究双糖链蛋白聚糖的结构和功能域，重组痘苗病毒，vBGN4编码成熟双糖链蛋白聚糖核心蛋白作为受T7噬菌体启动子控制的多组氨酸融合蛋白在HT-1080细胞中表达，并且UMR106 细胞，通过分析在[S-35]硫酸盐、[H-3]葡萄糖胺和[S-35]甲硫氨酸存在下标记的分子来确定这些细胞分泌的重组双糖链蛋白聚糖的结构。在非变性条件下，通过咪唑梯度洗脱分离双糖链的糖形式，并包含：被分子量类似于 34 kDa（HT-1080 细胞）或类似于 40 kDa（UMR106 细胞）的两条硫酸软骨素链取代的大蛋白聚糖形式；被两条硫酸软骨素链取代的小蛋白聚糖形式，中值分子量类似于 28 kDa； HT-1080 细胞分泌的重组双糖链约 70% 被硫酸软骨素链取代，而 UMR106 细胞表达的双糖链约 50% 被硫酸软骨素链取代。硫酸软骨素链，在 HT-1080 和 UMR106 细胞中感染 vBGN4 导致每 24 小时产生约 10 mg 双糖链蛋白聚糖/10(9) 个细胞，天然重组双糖链蛋白聚糖显示出与 V 型胶原蛋白和补体蛋白 Clq 结合，但是，当重组双糖链蛋白聚糖的二级结构因暴露于 4 hi 盐酸胍而被破坏时，与 V 型胶原蛋白的亲和力显着降低减少。这些数据证明了二级结构对于这种小蛋白多糖的功能的重要性。
Biglycan is a small chondroitin sulfate proteoglycan found in many tissues and is structurally related to decorin, fibromodulin, and lumican. The biological function of biglycan is poorly understood, although several studies have indicated interaction with other extracellular matrix components, We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vaccinia virus/T7 bacteriophage expression system, A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistidine fusion protein under control of the T7 phage promoter was expressed in HT-1080 cells and UMR106 cells, The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules labeled in the presence of [S-35]sulfate, [H-3]glucosamine, and [S-35]methionine. Glycoforms of biglycan were separated by imidazole gradient elution, under non-denaturing conditions, and comprised: a large proteoglycan form substituted with two chondroitin sulfate chains of molecular mass similar to 34 kDa (HT-1080 cells) or similar to 40 kDa (UMR106 cells); a small proteoglycan form substituted with two chondroitin sulfate chains with a median molecular mass similar to 28 kDa; and a core protein form secreted devoid of glycosaminoglycan chains, All the glycoforms were substituted with two N-linked oligosaccharides, and the disaccharide composition of the two glycosaminoglycan populations were identical, Approximately 70% of the recombinant biglycan secreted by HT-1080 cells was substituted with chondroitin sulfate chains, whereas about 50% of the biglycan expressed by UMR106 cells was substituted with chondroitin sulfate chains, Infection with vBGN4 in both HT-1080 and UMR106 cells resulted in the production of approximately 10 mg of biglycan/10(9) cells per 24 h, The native recombinant biglycan was shown to bind to collagen type V and the complement protein, Clq, However, when the secondary structure of recombinant biglycan was disrupted by exposure to 4 hi guanidine hydrochloride, the affinity for collagen type V was dramatically reduced. These data demonstrate the importance of secondary structure to the function of this small proteoglycan.