Domowicz M, Li H, Hennig A, Henry J, Vertel B M, Schwartz N B
Department of Pediatrics, University of Chicago, Illinois 60637, USA.
Dev Biol. 1995 Oct;171(2):655-64. doi: 10.1006/dbio.1995.1312.
Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross-reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking differences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrecan carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan lacks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relationship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using primers covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base change in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nanomelia, we demonstrated that the same mutation was present in notochord mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochord and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly support the hypothesis that the final structural characteristics of each proteoglycan are determined not only by the core protein but also by tissue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extracellular matrices.
利用单克隆抗体S103L，它能与鸡软骨硫酸软骨素蛋白聚糖（CSPG）核心蛋白富含硫酸软骨素的结构域中的一个表位特异性反应，我们鉴定出了脊索表达的主要CSPG。这种大的脊索CSPG最早在第16阶段通过免疫组织化学检测到，远在软骨形成发生之前，并且在神经嵴活跃迁移期间以及硬骨节分化开始时持续表达。由于脊索和软骨CSPG与S103L抗体都有交叉反应，因此对这两种CSPG进行了广泛的分子和生化分析。在翻译后修饰水平上，脊索和软骨（聚集蛋白聚糖）CSPG存在显著差异。值得注意的是，软骨聚集蛋白聚糖含有大量硫酸角质素（KS）链，而脊索CSPG不含KS。相反，软骨聚集蛋白聚糖缺乏HNK-1表位，而脊索CSPG含有高含量的HNK-1。采用三种不同方法在分子水平上确定这两种CSPG的关系。使用聚集蛋白聚糖探针的Northern印迹分析检测到来自脊索和软骨RNA的大小相同的信息。使用覆盖已知软骨结构整个编码序列98%的引物通过RT-PCR产生的重叠片段，在脊索和软骨中大小相同。利用我们最近的研究，该研究表明聚集蛋白聚糖基因中的单个碱基变化导致鸡聚集蛋白聚糖外显子12中的Glu转换为终止密码子，作为侏儒症缺陷的分子基础，我们证明来自侏儒症小鸡的脊索mRNA中存在相同的突变。这些结果提供了证据，表明鸡聚集蛋白聚糖基因在发育早期就在脊索中表达，并证实鸡脊索和软骨中表达的心蛋白源自同一基因。这些发现有力地支持了这样的假设，即每种蛋白聚糖的最终结构特征不仅由核心蛋白决定，还由组织特异性、发育调控的翻译后机制决定，这些机制在特定细胞外基质需求的背景下起作用。
