Sheehan J K, Ratcliffe A, Oates K, Hardingham T E
Department of Biological Sciences, University of Lancaster, Bailrigg, U.K.
Biochem J. 1987 Oct 15;247(2):267-76. doi: 10.1042/bj2470267.
Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.
以苄基二甲基烷基氯化铵作为铺展剂，通过电子显微镜对猪喉软骨中的蛋白聚糖单体进行了检查。蛋白聚糖呈现为具有串珠状结构的伸展分子，代表硫酸软骨素链围绕蛋白质核心折叠。通常在一端有一条细的丝状尾巴。通过与特异性抗体孵育，随后用蛋白A-金（直径4纳米）来定位蛋白聚糖分子内的亚结构。使用抗（结合区域）血清后、蛋白A-金（通常一到三个颗粒）结合在丝状区域的末端。一小部分标记分子（10 - 15%）在两端都显示有金颗粒。一种对硫酸角质素表位特异的单克隆抗体（MZ15）将富含硫酸角质素 的区域定位在了蛋白聚糖的一端，但在蛋白质核心的伸展部分未观察到金颗粒。蛋白聚糖先用软骨素AC裂解酶消化，这种分布也不会改变。用另一种针对硫酸角质素的单克隆抗体（5-D-4）定位，导致一部分（约20%）蛋白聚糖单体的铺展行为发生变化，这些单体失去了串珠状结构，硫酸软骨素链从蛋白质核心伸出。在这些分子中蛋白A-金沿着蛋白质核心的长度定位抗体（5-D-4），而在那些呈串珠状外观的分子中，它只在一端标记。用任何一种单克隆抗体标记都是特异的，因为它会被外源添加的硫酸角质素抑制。所实现的差异定位可能反映了蛋白聚糖群体中涉及硫酸角质素及其所附着的蛋白质核心的结构差异。结果表明，通过这种技术，在使用特异性单克隆或多克隆抗体后，可通过蛋白A-金标记来识别蛋白聚糖分子内的亚结构。
