Biological significance, expression and characteristics of cancer associated carbohydrate antigen

用人结肠癌细胞(LS180细胞)免疫人结肠癌细胞，建立了抗TN抗体的小鼠单抗MLS128。MLS128与LS180细胞及其asialo形式的粘蛋白糖肽以及颌下腺粘蛋白(OSM)和asialo OSM有相同程度的结合。为了确定抗TN单抗的表位结构，用不同的酶消化asialo OSM，并用免疫亲和层析和高效液相色谱分离纯化。经胰酶消化，得到糖肽GP-I和5个糖肽GP-1-5，分别作为免疫亲和柱的结合组分和未结合组分。固相放射免疫分析表明，GP-I呈强免疫反应。在V8酶作用下，GP-I被转化为两个糖肽，一个为弱反应性，一个为中等反应性，从热裂解酶中分离出最小的片段GP-II，它与GP-I具有较强的免疫反应性。GP-II对应于GP-I的一部分，其序列为Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly，其中星号表示GalNAc残基连接的氨基酸。其他抗TN的单抗NCC-LU-35和CA3239对这些糖肽的反应性与MLS128基本相同。糖肽(GP-1-5)含有GalNAc-Thr/Ser、GaInAc-Ser/Thr-(GalNAc)-Ser/Thr等结构，具有较低的免疫反应性。这些结果表明，含有簇结构的糖肽Ser*-Thr*-Thr*是抗Tn抗体识别的表位的重要组成部分。我们还发现Tn抗原表达于T淋巴细胞株Jurkat的白唾液蛋白上，并且Tn抗原比GalNAc-Ser/Thr更复杂。

A murine monoclonal antibody, MLS128, that was assigned to an anti-Tn antibody has been established by immunizing with human colonic cancer cells (LS180cell). MLS128 bound to mucin glycopeptides from LS180 cells and their asialo forms to the same extent as well as to submaxillary mucin (OSM) and asialo OSM.To determine the epitopic structure for an anti-Tn monoclonal antibody, MLS128, asialo OSM was digested with various proteases, and the digests were fractionated by immunoaffinity chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassay, it was found that GP-I was strongly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity.From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an GalNAc residue is attached. Other anti Tn monoclonal antibodies, NCC-LU-35 and CA3239, showed essentially the same reactivity to these glycopeptides as MLS128 did. The glycopeptides (GP-1-5), which exibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Thr/Ser, and GaINAc-Ser/Thr-(GalNAc)-Ser/Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies. We also found that Tn antigen is expressed on leukosialin of T-lymphoid cell line, Jurkat and the Tn antigen is more complex than GalNAc-Ser/Thr.