Nakada H, Inoue M, Numata Y, Tanaka N, Funakoshi I, Fukui S, Mellors A, Yamashina I
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Japan.
Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2495-9. doi: 10.1073/pnas.90.6.2495.
Glycophorin A was digested with glycoprotease (Pasteurella haemolytica) and the digest was fractionated by a combination of high-pressure column chromatographies to produce the glycopeptides GPA-1 to GPA-6. Sequence analysis of the glycopeptides revealed that two serine residues (Ser-14 and Ser-15) are not glycosylated, Thr-17 and Ser-19 being glycosylated instead, in disagreement with the accepted structure. The glycopeptides thus obtained were treated with sialidase and beta-galactosidase. The Tn antigenicity, as assayed by the binding to a monoclonal anti-Tn antibody (MLS 128), was found exclusively in the glycopeptides including three (cluster I) or four (cluster II) consecutive residues of GalNAc-Ser/Thr, whereas the glycopeptide (GPA-2) containing two nonconsecutive GalNAc-Ser/Thr residues had practically no Tn antigenicity. The immunoreactivities of GPA-1 and GPA-3, containing both clusters I and II, and GPA-4, containing cluster II, were 63% (calcd. 67%), 81% (calcd. 86%), and 50% (calcd. 50%), respectively, of the immunoreactivity of GPA-5 or GPA-6, containing cluster I (the average being taken as the basis), based on the reactivity per GalNAc residue. These results indicate that clusters I and II react with the antibody to the same extent. The structure consisting of three consecutive glycosylated Ser/Thr residues may be essential for Tn antigenicity in the light of previous results for ovine submaxillary mucin.
血型糖蛋白A用糖蛋白酶（溶血巴斯德菌）进行消化，消化产物通过高压柱色谱组合进行分级分离，得到糖肽GPA - 1至GPA - 6。对这些糖肽的序列分析表明，两个丝氨酸残基（Ser - 14和Ser - 15）未被糖基化，取而代之的是Thr - 17和Ser - 19被糖基化，这与公认的结构不一致。由此获得的糖肽用唾液酸酶和β - 半乳糖苷酶处理。通过与单克隆抗Tn抗体（MLS 128）结合测定的Tn抗原性，仅在包含三个（簇I）或四个（簇II）连续GalNAc - Ser/Thr残基的糖肽中发现，而含有两个不连续GalNAc - Ser/Thr残基的糖肽（GPA - 2）几乎没有Tn抗原性。包含簇I和簇II的GPA - 1和GPA - 3以及包含簇II的GPA - 4的免疫反应性，分别为基于每个GalNAc残基的反应性，以包含簇I的GPA - 5或GPA - 6的免疫反应性（取平均值作为基础）的63%（计算值67%）、81%（计算值86%）和50%（计算值50%）。这些结果表明簇I和簇II与抗体的反应程度相同。根据先前对羊颌下粘蛋白的研究结果，由三个连续糖基化的Ser/Thr残基组成的结构可能对Tn抗原性至关重要。
