Uchimura Kenji
Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan,
Methods Mol Biol. 2015;1229:389-400. doi: 10.1007/978-1-4939-1714-3_30.
Keratan sulfate is a glycosaminoglycan that has been investigated in the cornea and skeletal tissues for decades. Endoglycosidases and monoclonal antibodies specific for keratan sulfate have been developed. These materials have facilitated the analysis of keratan sulfate biosynthesis and structures. Likewise, they have expedited study of the biological roles of keratan sulfate in vitro and in vivo. It has been shown that keratan sulfate is also expressed in the central nervous system and functions as a regulator of neuronal regeneration/sprouting. Here, we describe methods to determine the enzymatic activity of GlcNAc6ST, which is involved in keratan sulfate biosynthesis, and to extract and prepare ocular keratan sulfate for a disaccharide composition analysis. Immunohistochemistry for an anti-keratan sulfate epitope in the brain is also described.
硫酸角质素是一种糖胺聚糖，数十年来一直在角膜和骨骼组织中进行研究。已经开发出了针对硫酸角质素的内切糖苷酶和单克隆抗体。这些材料促进了硫酸角质素生物合成和结构的分析。同样，它们也加快了对硫酸角质素在体外和体内生物学作用的研究。研究表明，硫酸角质素也在中枢神经系统中表达，并作为神经元再生/发芽的调节剂发挥作用。在这里，我们描述了测定参与硫酸角质素生物合成的GlcNAc6ST酶活性的方法，以及提取和制备用于二糖组成分析的眼硫酸角质素的方法。还描述了针对大脑中抗硫酸角质素表位的免疫组织化学方法。
