Innovative Research Initiatives, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama, Japan.
Glycobiology. 2012 Feb;22(2):181-95. doi: 10.1093/glycob/cwr118. Epub 2011 Aug 31.
Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3'-sialylated, 6'-sulfated LNnT [Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3'-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives [SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3'-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3'-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb.
人血清 Krebs von den Lugen-6 (KL-6) 抗原是一种 MUC1 糖蛋白 (KL-6/MUC1)，可被抗 KL-6 单克隆抗体 (KL-6/mAb) 识别，已被用作间质性肺炎的诊断标志物。KL-6/mAb 被认为识别 MUC1 的特定糖肽序列，但表位的精确糖结构尚不清楚。在这项研究中，我们确定了 KL-6/MUC1 的碳水化合物结构，以寻找 KL-6/mAb 的碳水化合物表位。KL-6/MUC1 用人乳腺癌 YMB-S 细胞的培养基通过 KL-6/mAb 亲和层析进行纯化；O-连接糖链结构通过纸电泳、几种凝集素柱层析、唾液酸酶消化和甲醇解结合确定。KL-6/MUC1 含有核心 1 和扩展核心 1 糖，其中一个或两个唾液酸/硫酸盐残基修饰。基于这些结构，通过表面等离子体共振比较了与抗 KL-6/mAb 结合的几种合成糖。随后，通过酶促合成并分析与 KL-6/mAb 偶联亲和柱的结合来比较相关放射性标记的寡糖。3'-唾液酸化、6'-硫酸化 LNnT [Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc]、3'-唾液酸化、6-硫酸化核心 1 [Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc]和二硫酸化核心 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc 对 KL-6/mAb 具有显著亲和力，而 3'-硫酸化核心 1 衍生物 [SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] 和 3'-唾液酸化核心 1 与 KL-6/mAb 弱相互作用。这些结果表明，KL-6/mAb 的可能碳水化合物表位不仅涉及 3'-唾液酸化核心 1，还涉及带有硫酸和唾液酸残基的新型核心 1 和扩展核心 1。表位表达随着 Gal6ST（Gal 6-O-磺基转移酶）基因的抑制或过表达而发生变化，表明 Gal6ST 参与了 KL-6/mAb 独特表位的生物合成。
