Ellis J A, Luzio J P
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, United Kingdom.
J Biol Chem. 1995 Sep 1;270(35):20717-23. doi: 10.1074/jbc.270.35.20717.
Antisera raised against detergent-extracted membrane fractions from the human intestinal epithelial cell line Caco-2 were used to screen a human colon cDNA library in a bacteriophage expression vector. This led to the identification, molecular cloning, and sequencing of a novel plasma membrane protein (p137) which was present in approximately equal amounts on the basolateral and apical surfaces of the cell. The pattern of extraction of p137 from membranes by Triton X-114 and its release from membranes after incubation with phosphatidylinositol-specific phospholipase C were consistent with it being a glycosylphosphatidylinositol-anchored membrane protein. Using antibodies raised against bacterial fusion proteins, it was shown that p137 was present on the cell surface as a reducible homodimer of 137 kDa subunits. There was constitutive release of p137 into the culture medium as a non-reducible 280-kDa entity. Pulse-chase experiments showed that newly synthesized p137 appeared at the basolateral side of a Caco-2 cell layer before appearing at the apical domain. Domain-specific surface biotinylation of Caco-2 cells at 4 degrees C, followed by chasing at 37 degrees C, demonstrated that p137 is capable of transcytosing in both directions across Caco-2 cells. The unusual plasma membrane domain distribution of this glycosylphosphatidylinositol-linked protein and its transcytosis characteristics demonstrate the existence of a previously uncharacterized apical to basolateral transcytotic pathway in Caco-2 cells.
用人肠道上皮细胞系Caco - 2经去污剂提取的膜组分制备的抗血清，用于筛选噬菌体表达载体中的人结肠cDNA文库。这导致鉴定、分子克隆和测序了一种新型质膜蛋白（p137），该蛋白在细胞的基底外侧和顶端表面的含量大致相等。Triton X - 114从膜中提取p137的模式及其与磷脂酰肌醇特异性磷脂酶C孵育后从膜中释放的情况，与它是一种糖基磷脂酰肌醇锚定膜蛋白一致。使用针对细菌融合蛋白产生的抗体表明，p137在细胞表面以137 kDa亚基的可还原同二聚体形式存在。p137以不可还原的280 kDa实体形式组成性释放到培养基中。脉冲追踪实验表明，新合成的p137在出现在顶端区域之前先出现在Caco - 2细胞层的基底外侧。在4℃对Caco - 2细胞进行结构域特异性表面生物素化，然后在37℃进行追踪，结果表明p137能够在Caco - 2细胞中双向跨细胞转运。这种糖基磷脂酰肌醇连接蛋白不寻常的质膜结构域分布及其跨细胞转运特性，证明了Caco - 2细胞中存在一种以前未被表征的从顶端到基底外侧的跨细胞转运途径。
