Recombinant decorin glycoforms - Purification and structure
Recombinant decorin glycoforms - Purification and structure
复制标题
DOI：
10.1074/jbc.271.32.19578
复制
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：
Ramamurthy, P;Hocking, AM;McQuillan, DJ
使用牛痘病毒/T7噬菌体表达系统，通过编码T7 RNA聚合酶的vTF7-3和编码与多组氨酸-胰岛素信号序列融合蛋白盒融合的核心蛋白聚糖核心蛋白的vDCN1共感染，在HT-1080细胞中表达人核心蛋白聚糖。使用牛痘病毒/T7噬菌体系统的过度表达导致每24小时分泌约30mg核心蛋白聚糖/10(9)细胞，这使得能够在天然条件下纯化和分离多种糖型。在[S-35]甲硫氨酸或[H-3]葡萄糖胺和[S-35]硫酸盐的混合物存在下培养细胞，通过金属亲和层析纯化重组糖蛋白，将分泌的核心蛋白聚糖分为两类，蛋白聚糖形式和核心蛋白形式。约25%的重组蛋白作为不含糖胺聚糖链的核心蛋白分泌到培养基中。核心蛋白聚糖核心蛋白被解析为两种形式（类似于 49 kDa 和类似于 53 kDa），其 N 连接寡糖取代程度不同（分别为 2 个和 3 个 N 连接寡糖）。当通过 SDS 聚丙烯酰胺凝胶电泳分析时，重组蛋白聚糖和核心蛋白的去糖基化导致单条带迁移，表观分子量类似于 43 kDa。天然核心蛋白聚糖蛋白聚糖的远紫外圆二色性光谱在 218 nm 处显示最小值，与主要为 β-折叠的二级结构一致。从关节软骨中提取的牛核心蛋白聚糖和经过类似处理的重组核心蛋白聚糖的圆二色光谱显示，最小值为 205 nm，表明二级结构的损失。证明了核心蛋白聚糖蛋白聚糖和核心蛋白对胶原蛋白样分子的亲和力，其中补体成分 C1q 对核心蛋白聚糖表现出最显着的亲和力，尽管也观察到对 I 型和 V 型胶原蛋白的粘附。纯化的重组蛋白中保留的广泛二级结构可能对核心蛋白聚糖的生物学功能很重要。
The vaccinia virus/T7 bacteriophage expression system was used to express human decorin in HT-1080 cells by co-infection with vTF7-3, encoding T7 RNA polymerase, and vDCN1, encoding the decorin core protein fused to a polyhistidine-insulin signal sequence fusion-protein cassette. Overexpression using the vaccinia virus/T7 phage system resulted in secretion of approximately 30 mg of decorin/10(9) cells per 24 h which enabled purification and separation of multiple glycoforms under native conditions. Cells were cultured in the presence of [S-35]methionine or a mixture of [H-3]glucosamine and [S-35]sulfate, and recombinant glycoprotein purified by metal affinity chromatography which resolved the secreted decorin into two classes, a proteoglycan form and a core protein form, About 25% of the recombinant protein was secreted into the culture medium as core protein devoid of glycosaminoglycan chains. The decorin core protein was resolved into two forms (similar to 49 and similar to 53 kDa) that differed in the extent of N-linked oligosaccharide substitution (2 and 3 N-linked oligosaccharides, respectively), Deglycosylation of the recombinant proteoglycans and core proteins resulted in a single band migrating with an apparent molecular mass similar to 43 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Far-UV circular dichroism spectra of native decorin proteoglycan showed a minima at 218 nm, consistent with a secondary structure that is predominantly beta-sheet. Circular dichroism spectra of bovine decorin extracted from articular cartilage and recombinant decorin similarly treated revealed a minima of 205 nm indicating a loss of secondary structure, The affinity of decorin proteoglycan and core protein for collagen-like molecules was demonstrated, with the complement component C1q exhibiting the most striking affinity for decorin, although adherence to collagen types I and V was also observed, The extensive secondary structure maintained in the purified recombinant protein is likely to be important for the biological function of decorin.