Haynesworth S E, Carrino D A, Caplan A I
Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106.
Connect Tissue Res. 1991;25(3-4):311-20. doi: 10.3109/03008209109029166.
Chondrocytes of different ages synthesize proteoglycans which have structural differences in both the chondroitin sulfate and keratan sulfate glycosaminoglycans. In order to ascertain whether age-dependent differences also occur in the core protein, the chick limb bud mesenchymal cell culture system was utilized to analyze newly synthesized proteoglycan core protein from undifferentiated mesenchymal cells (day 1 and 2), newly differentiated cartilage (day 4), mature cartilage (day 8), and senescent cartilage (day 16). The core protein synthesized at various times was identified by radiolabeling with [3H]leucine and [35S]sulfate immediately prior to extraction and purification. The sizes of the various core protein preparations were compared by electrophoresis on a 3% polyacrylamide gel after partial deglycosylation with chondroitinase AC and keratanase. The proteoglycans from day 4, 8, and 16 cultures each give rise to a single band of approximately 475,000 daltons. The proteoglycans from day 1 and 2 cultures also give rise to the 475,000 dalton band, but each contains several other components which produce a smear of high molecular weight material on the gel. The monomer proteoglycans were incubated with cyanogen bromide and the resultant peptides separated by electrophoresis on a 5-17.5% polyacrylamide gel. The peptide displays of core proteins synthesized on days 4, 8 and 16 are virtually identical in terms of the number and electrophoretic distribution of the core protein peptides. In contrast, proteoglycan core proteins from day 1 and day 2 cultures give rise to peptide displays which resemble those from older cultures in some respects but have distinct features as well. The absence of structural variation in the newly synthesized proteoglycan core proteins from cartilage of different ages suggests that the age-related changes in the structure of the intact proteoglycans result from differences in the glycosaminoglycan biosynthetic machinery rather than alterations in the acceptor molecule (i.e., the core protein).
不同年龄的软骨细胞合成的蛋白聚糖，其硫酸软骨素和硫酸角质素糖胺聚糖在结构上存在差异。为了确定核心蛋白中是否也存在年龄依赖性差异，利用鸡胚肢芽间充质细胞培养系统，分析了未分化间充质细胞（第1天和第2天）、新分化软骨（第4天）、成熟软骨（第8天）和衰老软骨（第16天）新合成的蛋白聚糖核心蛋白。在提取和纯化之前，通过用[3H]亮氨酸和[35S]硫酸盐进行放射性标记，鉴定在不同时间合成的核心蛋白。在用软骨素酶AC和角质素酶进行部分去糖基化后，通过在3%聚丙烯酰胺凝胶上电泳比较各种核心蛋白制剂的大小。来自第4、8和16天培养物的蛋白聚糖各自产生一条约475,000道尔顿的条带。来自第1和第2天培养物的蛋白聚糖也产生475,000道尔顿的条带，但每种都含有其他几种成分，这些成分在凝胶上产生高分子量物质的拖尾现象。将单体蛋白聚糖与溴化氰孵育，所得肽段在5 - 17.5%聚丙烯酰胺凝胶上进行电泳分离。就核心蛋白肽段的数量和电泳分布而言，第4、8和16天合成的核心蛋白的肽段图谱几乎相同。相比之下，第1和第2天培养物的蛋白聚糖核心蛋白产生的肽段图谱在某些方面类似于 older cultures，但也有明显特征。来自不同年龄软骨的新合成蛋白聚糖核心蛋白缺乏结构变异，这表明完整蛋白聚糖结构中与年龄相关的变化是由糖胺聚糖生物合成机制的差异而非受体分子（即核心蛋白）的改变引起的。
