Plaas A H, Wong-Palms S, Roughley P J, Midura R J, Hascall V C
Shriners Hospital for Children, Tampa Unit, Tampa, Florida 33612, USA.
J Biol Chem. 1997 Aug 15;272(33):20603-10. doi: 10.1074/jbc.272.33.20603.
Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcAbeta1,3GalNAc4S and GlcAbeta1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were approximately 50% 4,6-disulfated in aggrecans from adults (22-72 years of age). Second, the terminal disaccharide GlcAbeta1,3GalNAc4S was on approximately 7% of chains on aggrecan from fetal through 15 years of age, but on only approximately 3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAbeta1,3GalNAc6S, approximately 9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(-) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.
从胎儿至72岁个体的正常人膝关节软骨中分离出的聚集蛋白聚糖硫酸软骨素样本，用硫酸软骨素裂解酶进行消化。除内部不饱和二糖产物外，通过基于荧光的阴离子交换高效液相色谱法分析产物，以分离和定量非还原末端结构。主要的末端结构是单糖GalNAc4S和GalNAc4,6S，因为它们存在于所有链的85 - 90%上。其余的链以二糖GlcAbeta1,3GalNAc4S和GlcAbeta1,3GalNAc6S终止。在从生长软骨到成人关节软骨的转变过程中，这些末端的相对丰度出现了显著变化。首先，在胎儿至15岁的聚集蛋白聚糖中，末端GalNAc残基几乎完全是4 - 硫酸化的，但在成人（22 - 72岁）的聚集蛋白聚糖中约50%是4,6 - 二硫酸化的。其次，末端二糖GlcAbeta1,3GalNAc4S在胎儿至15岁的聚集蛋白聚糖的约7%的链上存在，但在成人聚集蛋白聚糖的仅约3%的链上存在。相比之下，以GlcAbeta1,3GalNAc6S终止的链的比例约为9%，从胎儿到72岁保持不变。这种末端二糖被认为可被广泛使用的单克隆抗体3B3识别。然而，对该结构的化学定量以及对胎儿和成人聚集蛋白聚糖的固相3B3(-)免疫测定表明，末端二糖的含量不一定与蛋白聚糖的免疫反应性相关，因为链密度和在固相上的呈现是3B3识别链末端的关键因素。从聚集蛋白聚糖硫酸软骨素链的所有非还原末端的化学分析获得的定量结果揭示了在生长软骨去除后形成成人关节软骨时细胞活动改变时发生的链终止的重要变化。结合硫酸软骨素蛋白聚糖生物合成途径中产生链的非还原末端的特定酶促步骤及其在组织发育和病理学中的调节来讨论这些发现。
