Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, dept. 24.6.48, DK-2200 N Copenhagen, Denmark.
Glycobiology. 2012 Apr;22(4):529-42. doi: 10.1093/glycob/cwr178. Epub 2011 Dec 5.
CD175 or Tn antigen is a carbohydrate moiety of N-acetylgalactosamine (GalNAc)α1-O- linked to the residue of amino acid serine or threonine in a polypeptide chain. Despite the chemical simplicity of the Tn antigen, its antigenic structure is considered to be complex and the clear determinants of Tn antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal GalNAc residue of the Tn antigen irrespective of the peptide context or with low selectivity to the glycoproteins. In contrast, IgG mAbs recognized the Tn antigen in the context of a specific peptide motif. Particularly, JA3 mAb reacted to the GSPP or GSPAPP, and JA5 mAb recognized specifically the GSP motif (glycosylation sites are underlined). The major O-glycan carrier proteins CD43 and CD162 and isoforms of CD45 expressed on Jurkat cells were precipitated by anti-Tn mAbs with different affinities. In summary, our data suggest that Tn antigen-Ab binding capacity is determined by the peptide context of the Tn antigen, antigenic specificity of the Ab and class of the immunoglobulin. The newly generated anti-Tn IgG mAbs with the strong specificity to glycoprotein CD43 can be particularly interesting for the application in leukemia diagnostics and therapy.
CD175 或 Tn 抗原是 N-乙酰半乳糖胺 (GalNAc)α1-O-连接到多肽链中丝氨酸或苏氨酸残基上的碳水化合物部分。尽管 Tn 抗原的化学结构简单，但它的抗原结构被认为是复杂的，并且 Tn 抗原性的明确决定因素仍知之甚少。因此，产生了广泛的抗 Tn 单克隆抗体 (mAb)。为了进一步研究 Tn 抗原的性质和复杂性，我们针对 Tn 阳性的人类 Jurkat T 细胞产生了七种不同的 IgM 和 IgG 类抗 Tn mAb，这是由于 T-合成酶活性低和特定伴侣 Cosmc 突变所致。抗 Tn mAb 与一系列合成糖、糖肽和 O-糖蛋白的结合分析揭示了抗 Tn mAb 特异性的意外差异。IgM mAb 结合 Tn 抗原的末端 GalNAc 残基，无论肽结构如何，其结合特异性都较低，或者对糖蛋白的结合特异性较低。相比之下，IgG mAb 在特定肽基序的背景下识别 Tn 抗原。特别是，JA3 mAb 与 GSPP 或 GSPAPP 反应，而 JA5 mAb 特异性识别 GSP 基序（糖基化位点用下划线表示）。Jurkat 细胞上表达的主要 O-聚糖载体蛋白 CD43 和 CD162 以及 CD45 同工型被抗 Tn mAb 以不同的亲和力沉淀。总之，我们的数据表明，Tn 抗原-Ab 结合能力取决于 Tn 抗原的肽结构、Ab 的抗原特异性和免疫球蛋白的类别。具有强特异性的新型抗 Tn IgG mAb 对 CD43 糖蛋白在白血病诊断和治疗中的应用特别有趣。
