CARBOHYDRATE BINDING PROTEIN 35

Galectin-3是一种蛋白质(Mr 30,000)，最初发现并 基于其半乳糖特异性碳水化合物结合而提纯 活动，最近被证明是核活动的一个必要因素 前信使核糖核酸剪接。所作的主要观察包括：a)核问题 从HeLa细胞中提取的提取物，能够在 一种含有Galectin-3的无细胞分析；b)耗尽 乳糖-琼脂糖亲和吸附半乳糖凝集素-3成为缺陷 在剪接中；c)乳糖-琼脂糖耗尽提取物的活性 可通过加入纯化的重组Galectin-3进行重组； 和d)与Galectin-3高亲和力结合的糖被抑制 剪接反应。在细胞内，Galectin-3在两个细胞中都存在 细胞质和细胞核。这种蛋白质有两种亚型：a) PI 8.7未修饰的多肽；和b)pI 8.2形式，代表 由单一的磷酸盐修饰的多肽。被磷酸化的 (pI 8.2)形式存在于胞浆和细胞核中，而 未经修饰的多肽(等电点8.7)仅存在于 原子核。最近，我们开发了一种渗透性细胞检测方法来 监测半乳糖凝集素-3从细胞核的输出。而核电 残基包含PI 8.2和PI 8.7两种形式，只有 磷酸化(等电点8.2)多肽正在出口。这些结果 建议需要半乳糖凝集素-3的磷酸化 从细胞核中排出之前的多肽。在…的基础上 这些结果，我们的研究目标包括：a) 建立小鼠半乳糖凝集素-3磷酸化位点(S) 多肽；b)进行定点突变以获得 不能磷酸化的突变多肽(例如，丝氨酸到丙氨酸； Ser到Asp等)；以及c)比较核质和细胞质 半乳糖凝集素-3在野生型和重组型半乳糖凝集素细胞中的分布 突变结构。将使用质谱学方法来 确定这项研究中的磷酸化位置。

Galectin-3 is a protein (Mr 30,000), initially identified and purified on the basis of its galactose-specific carbohydrate-binding activity, recently documented to be a required factor in nuclear pre-mRNA splicing. The key observations made include: a) nuclear extracts derived from HeLa cells, capable of carrying out splicing in a cell-free assay, contain galectin-3; b) nuclear extracts depleted of galectin-3 by affinity adsorption on lactose-agarose become deficient in splicing; c) the activity of the lactose-agarose-depleted extract could be reconstituted by addition of purified recombinant galectin-3; and d) saccharides that bind galectin-3 with high affinity inhibited the splicing reaction. Inside cells, galectin-3 is found in both the cytoplasm and the nucleus. There are two isoforms of the protein: a) a pI 8.7 unmodified polypeptide; and b) a pI 8.2 form, representing the polypeptide modified by a single phosphate. The phosphorylated (pI 8.2) form is found in both the cytosol and the nucleus, whereas the unmodified polypeptide (pI 8.7) is found exclusively in the nucleus. Recently, we have developed a permeabilized cell assay to monitor the export of galectin-3 from the nucleus. While the nuclear residue contained both the pI 8.2 and pI 8.7 forms, only the phosphorylated (pI 8.2) polypeptide was being exported. These results suggest a requirement for the phosphorylation of the galectin-3 polypeptide prior to its export from the nucleus. On the basis of these results, the objectives of our research include: a) to establish the site(s) of phosphorylation of the murine galectin-3 polypeptide; b) to carry out site-directed mutagenesis to derive mutant polypeptides that cannot be phosphorylated (e.g., Ser to Ala; Ser to Asp, etc.); and c) to compare the nuclear versus cytoplasmic distribution of galectin-3 in cells transfected with the wild-type and mutant constructs. Mass spectrometric methods will be used to identify the site of phosphorylation in this study.