Ikeda S, Kuroki M, Haruno M, Oikawa S, Nakazato H, Kosaki G, Matsuoka Y
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Mol Immunol. 1992 Feb;29(2):229-40. doi: 10.1016/0161-5890(92)90104-6.
Antigenic epitopes of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) were analysed in relation to their domain structures [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M for CEA and domains N, I (A1-B1), and M for NCA]. We reconstructed cDNAs for CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M (CEA-whole), NCA-N, NCA-N-I and NCA-N-I-M (NCA-whole), which were expressed in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. Their mol. wts judged from Western blotting were 17,000-26,000 for CEA-N, 70,000 for CEA-N-I, 150,000 for CEA-N-I-II, 165,000 for s-CEA-whole which was spontaneously released from cells into culture medium, 180,000 for p-CEA-whole which was solubilized with phosphatidylinositol specific phospholipase C (PI-PLC) from cells, 18,000-25,000 for NCA-N, 63,000 for NCA-N-I, and 96,000 for p-NCA-whole which was solubilized with PI-PLC from cells. The divergence of the observed mol. wts from those calculated from cDNA sequences seems to indicate that these recombinant proteins are highly N-glycosylated. By enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 25 distinct anti-CEA monoclonal antibodies (MAbs), each representative of 25 different subgroups within five groups (Groups 1-5) previously classified by us in terms of the reactivity with CEA and CEA-related antigens. Twenty-one MAbs previously shown to react with different protein epitopes of the CEA molecule allow to define six groups (A-F) of epitopes according to their expression by different domains of the CEA and NCA molecules. Among four epitopes common to CEA and NCA, two were found to be present on domain N (Group A) and two on domain I (Group B). Among 15 epitopes absent from NCA but expressed by CEA and normal fecal antigens (NFAs), four were on domain N (Group C), five on domain I (Group D) and six on domain II (Group E). Two epitopes were previously described as "CEA distinctive", because they were recognized by MAbs reacting with CEA but not with the NFAs. These two epitopes (Group F) were found to be expressed by p-CEA-whole but not by s-CEA-whole. The latter results suggest that the Group F epitopes are located on a part of the domain III close to the anchoring device of the CEA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
分析了癌胚抗原（CEA）和非特异性交叉反应抗原（NCA）的抗原表位与其结构域结构的关系[CEA的结构域N、I（A1 - B1）、II（A2 - B2）、III（A3 - B3）和M以及NCA的结构域N、I（A1 - B1）和M]。我们构建了CEA - N、CEA - N - I、CEA - N - I - II、CEA - N - I - II - III - M（CEA全长）、NCA - N、NCA - N - I和NCA - N - I - M（NCA全长）的cDNA，并在中国仓鼠卵巢（CHO）细胞中表达。重组蛋白通过免疫吸附和凝胶过滤进行纯化。通过蛋白质印迹法判断，其分子量对于CEA - N为17,000 - 26,000，CEA - N - I为70,000，CEA - N - I - II为150,000，s - CEA全长（从细胞自发释放到培养基中的）为165,000，p - CEA全长（用磷脂酰肌醇特异性磷脂酶C（PI - PLC）从细胞中溶解的）为180,000，NCA - N为18,000 - 25,000，NCA - N - I为63,000，p - NCA全长（用PI - PLC从细胞中溶解的）为96,000。观察到的分子量与根据cDNA序列计算出的分子量存在差异，这似乎表明这些重组蛋白高度N - 糖基化。通过酶免疫测定法，用25种不同的抗CEA单克隆抗体（MAb）检测纯化的重组蛋白的免疫反应性，这些单克隆抗体分别代表我们之前根据与CEA和CEA相关抗原的反应性划分的五组（1 - 5组）中的25个不同亚组。先前显示与CEA分子的不同蛋白质表位反应的21种单克隆抗体，根据它们在CEA和NCA分子不同结构域的表达情况，可定义为六个表位组（A - F）。在CEA和NCA共有的四个表位中，发现两个位于结构域N（A组），两个位于结构域I（B组）。在NCA中不存在但由CEA和正常粪便抗原（NFA）表达的15个表位中，四个位于结构域N（C组），五个位于结构域I（D组），六个位于结构域II（E组）。两个表位先前被描述为“CEA独特表位”，因为它们被与CEA反应但不与NFA反应的单克隆抗体识别。发现这两个表位（F组）由p - CEA全长表达，而不由s - CEA全长表达。后一结果表明F组表位位于结构域III靠近CEA分子锚定装置的部分。（摘要截断于400字）
