Funderburgh J L, Funderburgh M L, Mann M M, Conrad G W
Division of Biology, Kansas State University, Manhattan 66506.
J Biol Chem. 1991 Aug 5;266(22):14226-31.
Recent work demonstrates isoforms of bovine corneal keratan sulfate proteoglycan containing structurally unique core proteins of 25 and 37 kDa (Funderburgh, J., and Conrad, G. (1990) J. Biol. Chem. 265, 8297-8303). In the current study, two forms (37A and 37B) of the 37-kDa protein were separated by ion-exchange chromatography after removal of keratan sulfate with endo-beta-galactosidase. Keratan sulfate linkage sites in core proteins were labeled with UDP-[3H]galactose using galactosyltransferase. Labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by tryptic digestion and reversed-phase chromatography. The 37A protein has three keratan sulfate-linkage sites, and the 37B and 25-kDa proteins each contain one linkage site. Reversed-phase tryptic maps of the three proteins differed in total peptide profile and in glycosylated peptides labeled with periodate-[3H]-NaBH4. Tryptic mapping of the two 37-kDa isoforms after deglycosylation showed differences in total tryptic peptides, in peptides labeled with [14C]iodoacetic acid, and in peptides recognized by antibodies to a mixture of the 37-kDa cores. Antibody to a synthetic peptide with N-terminal sequence obtained from mixed 37-kDa cores reacted exclusively with the 37B isoform. These results show that bovine corneal keratan sulfate proteoglycan has three different core proteins each with distinct glycosylation and unique primary structure.
最近的研究表明，牛角膜硫酸角质素蛋白聚糖的亚型含有结构独特的25 kDa和37 kDa核心蛋白（芬德伯格，J.，和康拉德，G.（1990年）《生物化学杂志》265，8297 - 8303）。在当前研究中，用内切β - 半乳糖苷酶去除硫酸角质素后，通过离子交换色谱法分离出了37 kDa蛋白的两种形式（37A和37B）。使用半乳糖基转移酶用UDP - [³H]半乳糖标记核心蛋白中的硫酸角质素连接位点。标记的蛋白通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离，并通过胰蛋白酶消化和反相色谱法进行分析。37A蛋白有三个硫酸角质素连接位点，37B和25 kDa蛋白各自含有一个连接位点。这三种蛋白的反相胰蛋白酶图谱在总肽谱以及用高碘酸盐 - [³H] - NaBH₄标记的糖基化肽方面存在差异。对两种37 kDa亚型去糖基化后的胰蛋白酶图谱分析显示，在总胰蛋白酶肽、用[¹⁴C]碘乙酸标记的肽以及被针对37 kDa核心混合物的抗体识别的肽方面存在差异。针对从混合的37 kDa核心获得的具有N端序列的合成肽的抗体仅与37B亚型反应。这些结果表明，牛角膜硫酸角质素蛋白聚糖有三种不同的核心蛋白，每种都具有独特的糖基化和独特的一级结构。
