Matsuura H, Takio K, Titani K, Greene T, Levery S B, Salyan M E, Hakomori S
Program of Biochemical Oncology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98119.
J Biol Chem. 1988 Mar 5;263(7):3314-22.
Previously, monoclonal antibody FDC-6 was established, which defines a structure specific for fibronectins isolated from fetal and malignant cells and tissues. The presence of the FDC-6-defined structure at type III connecting segment (III CS) is characteristic of oncofetal fibronectin (onf-FN), and its absence is characteristic of normal fibronectin (nor-FN) (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). Hepatoma fibronectin was sequentially digested by various proteases, followed by subsequent chromatography on an FDC-6 affinity column and reverse-phase columns at each step of digestion. A single strongly active glycosylhexapeptide (glycopeptide 1) and an inactive glycosylpentapeptide (glycopeptide 3) were isolated from glycopeptide A containing 35 amino acid residues. The minimum essential structure required for the FDC-6 activity was found to be a hexapeptide sequence Val-Thr-His-Pro-Gly-Tyr having NeuAc alpha 2----3Gal beta 1----3GalNAc or its core (Gal beta 1----3GalNAc or GalNAc) linked at threonine. Various synthetic peptides including the Val-Thr-His-Pro-Gly-Tyr sequence and a glycopeptide having the Val-Thr-His-Pro-Gly pentapeptide with the same glycosylation at threonine were all inactive. Elimination of sialic acid slightly increased the activity, and subsequent elimination of galactose did not alter the activity; however, removal of the Gal beta 1----3GalNAc residue by endo-alpha-N-acetylgalactosaminidase from desialylated glycopeptide A resulted in total inactivation of the reactivity with FDC-6 antibody. Thus, a single glycosylation at a defined threonine residue of the III CS region may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. This finding opens the possibility that a number of other oncofetal epitopes consist of a peptide and a common O-linked carbohydrate and that the combination produces a conformation specific to cancer or to a stage of development.
先前已制备出单克隆抗体FDC-6，它可识别从胎儿及恶性细胞和组织中分离出的纤连蛋白所特有的一种结构。III型连接段（III CS）存在FDC-6所定义的结构是癌胚纤连蛋白（onf-FN）的特征，而其缺失则是正常纤连蛋白（nor-FN）的特征（松浦浩和羽田森司（1985年）《美国国家科学院院刊》82卷，6517 - 6521页）。用各种蛋白酶对肝癌纤连蛋白进行顺序消化，然后在每次消化步骤后依次在FDC-6亲和柱和反相柱上进行层析。从含有35个氨基酸残基的糖肽A中分离出一个单一的强活性糖基六肽（糖肽1）和一个无活性的糖基五肽（糖肽3）。发现FDC-6活性所需的最小基本结构是一个六肽序列Val-Thr-His-Pro-Gly-Tyr，其苏氨酸处连接有NeuAcα2----3Galβ1----3GalNAc或其核心（Galβ1----3GalNAc或GalNAc）。包括Val-Thr-His-Pro-Gly-Tyr序列的各种合成肽以及在苏氨酸处具有相同糖基化的含有Val-Thr-His-Pro-Gly五肽的糖肽均无活性（。去除唾液酸会使活性略有增加，随后去除半乳糖并不会改变活性；然而，用内切α-N-乙酰半乳糖胺酶从去唾液酸化的糖肽A中去除Galβ1----3GalNAc残基会导致与FDC-6抗体的反应性完全丧失。因此，III CS区域特定苏氨酸残基处的单一糖基化可能会诱导肽的构象变化，从而形成FDC-6抗体识别的特定癌胚表位。这一发现揭示了一种可能性，即许多其他癌胚表位由一个肽和一个共同的O-连接碳水化合物组成，并且这种组合产生了特定于癌症或发育阶段的构象。
