Funderburgh J L, Funderburgh M L, Mann M M, Conrad G W
Division of Biology, Kansas State University, Manhattan 66506.
J Biol Chem. 1991 Dec 25;266(36):24773-7.
A glycoprotein reactive with antibodies against corneal keratan sulfate proteoglycan (KSPG) was purified 300-fold from extracts of bovine aorta using DEAE ion-exchange, gel-filtration, hydrophobic interaction, and reverse-phase chromatographic separations. The intact glycoprotein was 70-80 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Deglycosylation with endo-beta-galactosidase and N-glycanase reduced the size to 48 and 37 kDa, respectively, similar to the large isoforms of corneal KSPG. N-terminal amino acid sequence of the arterial KSPG was identical with lumican, the 37B isoform of corneal KSPG, and the arterial KSPG reacted with an antibody to synthetic peptide duplicating this sequence. Arterial KSPG and corneal lumican displayed identical tryptic maps. Arterial lumican contains fucose and mannose in amounts similar to corneal KSPG, but galactose, glucosamine, and sulfate were reduced compared to KSPG from cornea. Treatment of arterial lumican with endo-beta-galactosidase released 8-9 mol of glucosamine and galactose per mol of protein as oligosaccharides. These eluted as neutral, nonsulfated oligosaccharides on high pH anion-exchange chromatography. The size of arterial lumican was not altered by glycosidases having specificity for sulfated keratan sulfate, nor was the charge of the lumican molecule altered by digestion with endo-beta-galactosidase. These data show arterial lumican to be a glycoprotein containing unsulfated lactosaminoglycan chains. Abundance of low sulfate lumican in many tissues indicates that this protein occurs predominantly as a glycoprotein rather than as the more widely studied, highly sulfated proteoglycan present in the cornea.
一种与抗角膜硫酸角质素蛋白聚糖（KSPG）抗体反应的糖蛋白，使用DEAE离子交换、凝胶过滤、疏水相互作用和反相色谱分离法，从牛主动脉提取物中纯化了300倍。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，完整的糖蛋白分子量为70-80 kDa。用内切β-半乳糖苷酶和N-聚糖酶去糖基化后，其大小分别降至48 kDa和37 kDa，类似于角膜KSPG的大同工型。动脉KSPG的N端氨基酸序列与角膜KSPG的37B同工型、角膜蛋白聚糖相同，并且动脉KSPG与针对复制该序列的合成肽的抗体发生反应。动脉KSPG和角膜蛋白聚糖显示出相同的胰蛋白酶图谱。动脉蛋白聚糖所含岩藻糖和甘露糖的量与角膜KSPG相似，但与角膜KSPG相比，半乳糖、氨基葡萄糖和硫酸盐的含量减少。用内切β-半乳糖苷酶处理动脉蛋白聚糖，每摩尔蛋白质可释放出8-9摩尔的氨基葡萄糖和半乳糖作为寡糖。在高pH阴离子交换色谱上，这些寡糖以中性、非硫酸化寡糖的形式洗脱。对硫酸角质素具有特异性的糖苷酶不会改变动脉蛋白聚糖的大小，用内切β-半乳糖苷酶消化也不会改变蛋白聚糖分子的电荷。这些数据表明动脉蛋白聚糖是一种含有非硫酸化乳糖胺聚糖链的糖蛋白。许多组织中低硫酸化蛋白聚糖的丰富表明，这种蛋白质主要以糖蛋白的形式存在，而不是以角膜中研究较多的高硫酸化蛋白聚糖的形式存在。
