Masson D, Kreis T E
European Molecular Biology Laboratory, Heidelberg, Germany.
J Cell Biol. 1993 Oct;123(2):357-71. doi: 10.1083/jcb.123.2.357.
A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.
通过用针对HeLa细胞微管结合蛋白产生的抗血清筛选HeLa细胞cDNA表达文库，鉴定出一种分子量为115,000的新型微管相关蛋白（MAP）。制备了单克隆抗体和亲和纯化的多克隆抗体，用于对该MAP进行进一步表征。它与目前已表征的分子量相似的微管结合蛋白不同，其对微管的结合不依赖核苷酸，且沉降行为不同。由于它主要在上皮来源的细胞（Caco-2、HeLa、MDCK）中表达，在成纤维细胞中很少表达（人皮肤、A72）或无法检测到（Vero），我们将其命名为E-MAP-115（115kD的上皮MAP）。在HeLa细胞中，E-MAP-115优先与核周微管的亚结构域或亚群相关联。在Caco-2细胞中，当它们极化并形成水泡时，E-MAP-115的标记增加。E-MAP-115的分子表征表明它是一种新型蛋白质，与其他已知蛋白质没有明显的同源性。根据其序列预测的二级结构表明有两个结构域，由富含脯氨酸和丙氨酸的假定铰链区（PAPA区）连接。E-MAP-115有两个带高电荷的区域，预测具有α-螺旋结构，一个在NH2末端结构域呈碱性，pI为10.9，另一个在分子酸性COOH末端一半紧邻PAPA区呈中性，pI为7.6。使用体外微管结合试验和体内突变多肽的表达，已将一个新的微管结合位点定位到NH2末端结构域的碱性α-螺旋区域。通过用E-MAP-115该结构域的cDNA转染缺乏该蛋白显著水平的成纤维细胞，使其过表达，可使微管对诺考达唑稳定。我们得出结论，E-MAP-115是一种微管稳定蛋白，可能在上皮细胞极化和分化过程中微管重组期间发挥重要作用。
