Inoue M, Takahashi S, Yamashina I, Kaibori M, Okumura T, Kamiyama Y, Vichier-Guerre S, Cantacuzène D, Nakada H
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Japan.
Cancer Res. 2001 Feb 1;61(3):950-6.
A synthetic peptide corresponding to the human MUC2 tandem repeat unit was glycosylated in vitro using UDP-GalNAc and extracts of colonic adenocarcinoma and paired normal mucosa, followed by fractionation of the products by reverse phase high-performance liquid chromatography. Several peaks of glycopeptides with different numbers of GalNAc residues attached were detected. It is notable that the adenocarcinoma extract was capable of glycosylating peptides to a much greater extent than was normal mucosa. The levels of mRNA for N-acetylgalactosaminyltransferases-1, -2, and -3 were determined by reverse transcription-PCR. Only N-acetylgalactosaminyltransferase-3 mRNA was expressed at a higher level in the adenocarcinoma than in the normal tissue. When the MUC2 tandem repeat peptide was glycosylated with a mixture of the normal mucosa extract and recombinant N-acetylgalactosaminyltransferase-3, larger amounts of glycopeptides with higher contents of GalNAc residues were produced. The MUC2 tandem repeat peptides glycosylated extensively by recombinant N-acetylgalactosaminyltransferase-1, -2, or -3 were prepared and characterized. Substitution at each Thr residue, as revealed by Edman degradation sequencing, in conjunction with evidence obtained on mass spectrometry indicated a heterogeneous pattern of site-specific glycosylation within the MUC2 tandem repeat. It was found that maximum numbers of 6, 8, and 11 GalNAc residues were incorporated by N-acetylgalactosaminyltransferases-1, -2, and -3, respectively, and that only N-acetylgalactosaminyltransferase-3 could completely glycosylate both consecutive sequences composed of three and five Thr residues in the MUC2 tandem repeat unit. These results suggest that O-glycosylation of the clustered Thr residues is a selective process controlled by N-acetylgalactosaminyltransferase-3 in the synthesis of clustered carbohydrate antigens.
使用UDP-GalNAc以及结肠腺癌和配对正常黏膜提取物，在体外对与人MUC2串联重复单元对应的合成肽进行糖基化，随后通过反相高效液相色谱对产物进行分级分离。检测到了几个连接有不同数量GalNAc残基的糖肽峰。值得注意的是，腺癌提取物对肽进行糖基化的程度比正常黏膜大得多。通过逆转录-PCR测定N-乙酰半乳糖胺基转移酶-1、-2和-3的mRNA水平。仅N-乙酰半乳糖胺基转移酶-3的mRNA在腺癌中的表达水平高于正常组织。当用正常黏膜提取物和重组N-乙酰半乳糖胺基转移酶-3的混合物对MUC2串联重复肽进行糖基化时，产生了大量含有更高GalNAc残基含量的糖肽。制备并表征了由重组N-乙酰半乳糖胺基转移酶-1、-2或-3广泛糖基化的MUC2串联重复肽。如埃德曼降解测序所揭示的，每个苏氨酸残基的取代，结合质谱获得的证据，表明MUC2串联重复内存在位点特异性糖基化的异质性模式。发现N-乙酰半乳糖胺基转移酶-1、-2和-3分别最多掺入了6、8和11个GalNAc残基，并且只有N-乙酰半乳糖胺基转移酶-3能够完全糖基化MUC2串联重复单元中由三个和五个苏氨酸残基组成的连续序列。这些结果表明，在簇状碳水化合物抗原的合成中，簇状苏氨酸残基的O-糖基化是一个由N-乙酰半乳糖胺基转移酶-3控制的选择性过程。
