Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Glycoconj J. 2010 Feb;27(2):267-76. doi: 10.1007/s10719-009-9275-4.
O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITCPTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.
O-糖基化的粘蛋白是由 UDPGalNAc 的启动：多肽 N-乙酰半乳糖胺基转移酶（ppGalNAc-Ts）附着在粘蛋白核心多肽的丝氨酸或苏氨酸残基上的 N-乙酰-D-半乳糖胺（GalNAc）。目前还不清楚在每个细胞中多个 ppGalNAc-Ts 如何调节 GalNAc 的附着。在本研究中，通过竞争 RT-PCR 方法比较了表达水平的小鼠 ppGalNAc-Ts（mGalNAc-Ts）、T1、T2、T3、T4、T6 和 T7 之间在小鼠结肠腺癌结肠 38 细胞和选择用于其转移潜力的变体 SL4 细胞。SL4 细胞中 mGalNAc-T1、T2 和 T7 的表达水平略高于结肠 38 细胞，而 SL4 细胞中 mGalNAc-T3 的表达水平为结肠 38 细胞的 1.5%。使用这些细胞的微粒体部分作为酶源，将 GalNAc 残基通过酶促掺入到 FITCPTTTPITTTTK 肽中，并对其数量及其位置进行分离和特征分析。当使用结肠 38 细胞和 SL4 细胞的微粒体部分时，附着的 GalNAc 残基的最大数量分别为 6 和 4。当用针对 mGalNAc-T3 的多克隆抗体处理结肠 38 细胞的微粒体部分时，掺入的 GalNAc 残基的最大数量为 4。这些结果强烈表明结肠 38 细胞中的 mGalNAc-T3 参与了向该肽转移额外的 GalNAc 残基。
