Tanaka N, Nakada H, Inoue M, Yamashina I
Department of Biotechnology, Faculty of Engineering, Kyto Sangyo, University, Japan.
Eur J Biochem. 1999 Jul;263(1):27-32. doi: 10.1046/j.1432-1327.1999.00401.x.
To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr*-Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.
为了确定抗唾液酸α2-6GalNAcα-丝氨酸/苏氨酸（抗唾液酸化Tn）单克隆抗体MLS 132的表位结构，用胰蛋白酶和嗜热菌蛋白酶组合消化绵羊下颌粘蛋白（OSM），并通过免疫亲和柱色谱和高效液相色谱（HPLC）对消化产物进行分级分离。从胰蛋白酶消化产物中，获得了一个主要的糖肽，命名为T3，作为免疫亲和柱结合级分。在固相放射免疫测定中，发现T3与MLS 132表现出强烈的免疫反应性。用嗜热菌蛋白酶处理后，T3被转化为约50个片段，这在HPLC分级分离中可以看到。其中几个片段具有强烈的免疫反应性，并且具有相同的氨基酸序列，即苯丙氨酸-丝氨酸*-甘氨酸-谷氨酸-苏氨酸*-丝氨酸*-苏氨酸*-苏氨酸*-缬氨酸-异亮氨酸-丝氨酸*-甘氨酸-苏氨酸*-天冬酰胺-缬氨酸，其中星号表示碳水化合物连接位点。在这些糖肽中，一个是完全唾液酸化的，其他的有一个连接有未唾液酸化的GalNAc的丝氨酸或苏氨酸。对这些糖肽中连接的碳水化合物的分析结果使我们推测，由四个唾液酸化Tn抗原组成的簇是MLS 132的基本表位结构。
