Cloning and sequencing of cDNA encoding mouse cytohesin-1.
Cloning and sequencing of cDNA encoding mouse cytohesin-1.
复制标题
编码小鼠细胞粘连素 1 的 cDNA 的克隆和测序。
DOI：
10.1007/s002510050444
复制
发表时间：
1998
期刊：
影响因子：
3.2
通讯作者：
Feeney,AJ
中科院分区：
文献类型：
--
作者：
O'Rourke,AM;Escuro,G;Feeney,AJ
淋巴细胞功能相关抗原-1（LFA-1; aL β 2; CD 11 a/CD 18）是细胞表面粘附受体的整联蛋白家族的成员，并与广泛表达的细胞间粘附分子-1、-2和-3（ICAM-1、-2、-3）结合。LFA-1-ICAM相互作用在T淋巴细胞免疫应答中起关键作用，介导与抗原呈递细胞和靶细胞的牢固粘附，并另外为T细胞活化提供共刺激信号（Diamond and Springer 1994; Gahmberg 1997）。具有CD 18基因缺陷的个体缺乏功能性细胞表面β 2链并患有白细胞粘附缺陷，这是一种以复发性病原性感染为特征的潜在致命疾病，这一事实强调了LFA-1-ICAM相互作用对参与免疫应答的细胞的重要性（安德森and Springer 1987）。T细胞LFA-1的亲合力通过抗原特异性T细胞受体和CD 28分子结合后产生的细胞内信号以及几种趋化因子瞬时增加，但控制LFA-1粘附状态的细胞内途径尚不清楚（Springer 1994; Stewart and Hogg 1996）。最近，细胞粘连素-1被鉴定为人T细胞中LFA-1的潜在调节分子（Kolanus等，1996）。细胞粘连蛋白-1是一种含有Sec 7结构域和普列克底物蛋白同源（PH）结构域的细胞质蛋白，由先前在自然杀伤和细胞毒性T细胞中鉴定的cDNA编码（Liu和Pohajdak 1992）。在人T细胞白血病细胞系Jurkat中，全长胞粘连蛋白-1或分离的Sec 7结构域的过表达引起LFA-1的组成性激活;相反，PH结构域的过表达抑制TCR激活的LFA-1与ICAM-1的粘附（Kolanus et al. 1996）。这些发现强烈地表明，细胞粘连素-1通过LFA-1在T细胞粘附的生理调节中起作用。为了鉴定细胞粘连素-1的小鼠同源物，我们从来自6周龄B10淋巴结的体外活化的T细胞中分离RNA。D2/nSnJ雄性小鼠。用oligo-dT引物制备第一链cDNA，然后用基于人细胞粘连素-1 cDNA序列的引物扩增（Liu和Pohajdak 1992; Kolanus等1996）。5 b引物：5 b-GGGGAATTCGCCACCATGGAGGAGGACGACAGCTACGTT-3b; 3b引物：5 b-GGGGCGGCCGCTTTAGTGTCGCTTCGTGGAGGAGACCTT-3b。聚合酶链反应（PCR）条件为：940 ℃ 4分钟的一个循环，94 ℃ 1分钟、55 ℃ 1分钟、72 ℃ 1分钟的35个循环，以及72 ℃ 10分钟的最终延伸。将1200个碱基对（bp）的预测全长PCR产物连接到TA克隆载体（Invitrogen，卡尔斯巴德，CA）中，制备微量制备物，并通过使用测序酶试剂盒（阿默舍姆Corp.，伊利诺伊州阿灵顿高地）如前所述（Feeney和Thuerauf 1989），或通过使用跨越整个cDNA序列的测序引物用ABI测序仪自动测序。cDNA包含1197 bp的完整编码序列，并显示与人cytohesin-1 cDNA序列的高度相似性（图1）。小鼠核苷酸序列的检查表明与人cytohesin-1具有89.6%的同一性。
Lymphocyte function-associated antigen-1 (LFA-1; aLß2; CD11a/CD18) is a member of the integrin family of cell surface adhesion receptors, and binds to the widely-expressed intercellular adhesion molecules-1,-2, and-3 (ICAM-1,-2,-3). LFA-1-ICAM interactions play critical roles in T-lymphocyte immune responses, mediating firm adhesion to antigen-presenting cells and target cells and additionally providing co-stimulatory signals for T-cell activation (Diamond and Springer 1994; Gahmberg 1997). The importance of LFA-1-ICAM interactions to cells involved in an immune response is emphasized by the fact that individuals with a CD18 gene defect lack functional cell surface ß2 chains and suffer from leukocyte adhesion deficiency, a potentially fatal disease characterized by recurrent pathogenic infections (Anderson and Springer 1987). The avidity of T-cell LFA-1 is transiently increased by intracellular signals generated following engagement of the antigen-specific T-cell receptor and CD28 molecules, as well as by several chemokines, but the intracellular pathways that control the LFA-1 adhesive state are not well understood (Springer 1994; Stewart and Hogg 1996). Cytohesin-1 has recently been identified as a potential regulatory molecule for LFA-1 in human T cells (Kolanus et al. 1996). Cytohesin-1 is a cytoplasmic protein containing a Sec7 domain and a pleckstrin homology (PH) domain, and is encoded by cDNA previously identified in natural killer and cytotoxic T cells (Liu and Pohajdak 1992). In the human T-cell leukemia cell line Jurkat, overexpression of full-length cytohesin-1 or the isolated Sec7 domain gave rise to constitutive activation of LFA-1; conversely, over-expression of the PH domain-inhibited TCR-activated LFA-1 adhesion to ICAM-1 (Kolanus et al. 1996). These findings strongly suggest that cytohesin-1 plays a role in the physiological regulation of T-cell adhesion through LFA-1.To identify a mouse homologue of cytohesin-1, we isolated RNA from in vitro-activated T cells derived from lymph nodes of 6-week-old B10. D2/nSnJ male mice. First-strand cDNA was made with an oligo-dT primer and was then amplified with primers based on the human cytohesin-1 cDNA sequence (Liu and Pohajdak 1992; Kolanus et al. 1996). 5b primer: 5b-GGGGAATTCGCCACCATGGAGGAGGACGACAGCTACGTT-3b; 3b primer: 5b-GGGGCGGCCGCTTTAGTGTCGCTTCGTGGAGGAGACCTT-3b. Polymerase chain reaction (PCR) conditions were: one cycle of 940 7C for 4 min, 35 cycles of 94 7C for 1 min, 55 7C for 1 min, 72 7C for 1 min, and a final extension at 72 7C for 10 min. Predicted full-length PCR products of 1200 base pairs (bp) were ligated into a TA cloning vector (Invitrogen, Carlsbad, Calif.), mini-preps were produced, and seven cDNA clones derived from two independent PCR reactions were sequenced in both directions either by dideoxy sequencing using a Sequenase kit (Amersham Corp., Arlington Heights, Ill.) as previously described (Feeney and Thuerauf 1989), or by automated sequencing with an ABI sequencer using sequencing primers spanning the entire cDNA sequence. cDNAs contained the complete coding sequence of 1197 bp and showed high similarity to the human cytohesin-1 cDNA sequence (Fig. 1). Examination of the mouse nucleotide sequence indicated 89.6% identity with human cytohesin-1.