Hefta L J, Chen F S, Ronk M, Sauter S L, Sarin V, Oikawa S, Nakazato H, Hefta S, Shively J E
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Cancer Res. 1992 Oct 15;52(20):5647-55.
Carcinoembryonic antigen (CEA) is a member of the immunoglobulin gene superfamily with one predicted variable domain-like region (N domain; 108 amino acids) and three sets of constant domain-like regions (A1B1, A2B2, and A3B3; 92 amino acids for A domains and 86 amino acids for B domains). In addition, CEA possesses two signal peptides, one at the amino terminus and one at the carboxyl terminus. Both are removed during posttranslational processing, with the one at the carboxyl terminus being replaced by a glycosylphosphatidylinositol (GPI) moiety. We have previously expressed the full length complementary DNA clone for CEA in Chinese hamster ovary cells and murine L cells, demonstrating proper processing of nascent polypeptide chains to mature, fully glycosylated CEA including the GPI anchor. Using the same full length CEA complementary DNA clone and the polymerase chain reaction, we have now constructed expression clones for secreted versions of the N domain, the A3B3 domain, and the A3 and B3 subdomains. The clones were expressed in HeLa cells using the beta-actin promoter. A stop codon was introduced at the end of the A3B3 and the A3 and B3 domains to allow secretion instead of retention on plasma membranes with the GPI anchor. Expressed products were purified to homogeneity by affinity chromatography using monoclonal antibodies specific for each domain and by reversed phase high pressure liquid chromatography. Purified domains were characterized by Western blotting, antibody binding and inhibition studies, amino-terminal sequence and amino acid analyses, and laser desorption/time of flight mass spectrometry. These analyses revealed that the monomeric N domain is of size 15,990, with a glycosylation mass of about 4100, in good agreement with two N-linked glycosyl units of about mass 2100. There is some evidence that the N domain forms dimers. The N domain reacted with antibodies specific for this domain with an affinity similar to that of intact CEA. The A3B3 domain had a mass of 34,462, with a glycosylation mass of 14,900, in good agreement with seven N-linked glycosylation sites of average mass 2100. The A3B3 domain reacted only with antibodies specific for this domain, with a slightly lower affinity than that of native CEA. The amino-terminal sequences of the N domain and A3B3 domain proteins demonstrated proper processing of the signal peptide.(ABSTRACT TRUNCATED AT 400 WORDS)
癌胚抗原（CEA）是免疫球蛋白基因超家族的成员，有一个预测的可变结构域样区域（N结构域；108个氨基酸）和三组恒定结构域样区域（A1B1、A2B2和A3B3；A结构域92个氨基酸，B结构域86个氨基酸）。此外，CEA有两个信号肽，一个在氨基末端，一个在羧基末端。两者在翻译后加工过程中均被去除，羧基末端的信号肽被糖基磷脂酰肌醇（GPI）部分取代。我们之前已在中国仓鼠卵巢细胞和小鼠L细胞中表达了CEA的全长互补DNA克隆，证明新生多肽链能正确加工成成熟的、完全糖基化的CEA，包括GPI锚定。利用相同的全长CEA互补DNA克隆和聚合酶链反应，我们现在构建了N结构域、A3B3结构域以及A3和B3亚结构域分泌型的表达克隆。这些克隆利用β-肌动蛋白启动子在HeLa细胞中表达。在A3B3以及A3和B3结构域末端引入了一个终止密码子，以使产物分泌而不是通过GPI锚定保留在质膜上。通过使用针对每个结构域的单克隆抗体进行亲和层析以及反相高压液相色谱，将表达产物纯化至同质。通过蛋白质印迹、抗体结合和抑制研究、氨基末端序列和氨基酸分析以及激光解吸/飞行时间质谱对纯化的结构域进行表征。这些分析表明，单体N结构域大小为15990，糖基化质量约为4100，与两个质量约为2100的N-连接糖基单元高度一致。有一些证据表明N结构域形成二聚体。N结构域与针对该结构域的特异性抗体反应，亲和力与完整CEA相似。A3B3结构域质量为34462，糖基化质量为14900，与七个平均质量为2100的N-连接糖基化位点高度一致。A3B3结构域仅与针对该结构域的特异性抗体反应，亲和力略低于天然CEA。N结构域和A3B3结构域蛋白的氨基末端序列表明信号肽加工正确。（摘要截短至400字）
