Biological significance, expression and characteristics of cancer associated carbohydrate antigen

用人结肠癌细胞（LS 180）免疫小鼠，获得了一株抗Tn抗体的鼠源性单克隆抗体MLS 128。MLS 128结合到来自LS 180细胞的粘蛋白糖肽和它们的去唾液酸形式以相同的程度，以及结合到颌下粘蛋白（OSM）和去唾液酸OSM。为了确定抗Tn单克隆抗体的表位结构，MLS 128，去唾液酸OSM用各种蛋白酶消化，并通过免疫亲和色谱和高效液相色谱法分级分离。从胰蛋白酶消化，糖肽，GP-1，和其他五个糖肽，GP-1-5，分别作为免疫亲和柱的结合和未结合级分。固相放射免疫分析表明，GP-Ⅰ呈强免疫反应性。在用V8蛋白酶处理时，GP-Ⅰ被转化为两种糖肽，一种反应性差，另一种反应性中等，从嗜热菌蛋白酶消化物中分离出最小的片段，GP-Ⅱ，它与GP-Ⅰ一样具有强烈的免疫反应性。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，S表示连接有GalNAc残基的氨基酸。其他抗Tn单克隆抗体NCC-LU-35和CA 3239对这些糖肽的反应性与MLS 128基本相同。糖肽（GP-1-5）含有多种含GalNAc的结构，如GalNAc-Thr/Ser和GalNAc-Ser/Thr-（GalNAc）-Ser/Thr。这些结果表明，包括簇结构Ser*-Thr*-Thr* 的糖肽是抗Tn抗体识别的表位的必要部分。我们还发现Tn抗原在T淋巴细胞系Jurkat的白唾液酸上表达，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.