Dissociation of the carbohydrate-binding and splicing activities of galectin-1.
Dissociation of the carbohydrate-binding and splicing activities of galectin-1.
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DOI：
10.1016/j.abb.2008.07.003
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发表时间：
2008-10-01
影响因子：
3.9
通讯作者：
Wang, John L.
中科院分区：
文献类型：
--
作者：
Voss, Patricia G.;Gray, Richard M.;Dickey, Seth W.;Wang, Weizhong;Park, Jung W.;Kasai, Ken-ichi;Hirabayashi, Jun;Patterson, Ronald J.;Wang, John L.
半乳糖凝集素-1（Gal 1）和半乳糖凝集素-3（Gal 3）是在细胞核中发现的碳水化合物结合蛋白家族的两个成员，参与在无细胞系统中测定的前mRNA剪接。当HeLa细胞的核提取物（NE）进行吸附的融合蛋白含有谷胱甘肽S-转移酶（GST）和Gal 3，一般的转录因子II-I（TFII-I）被鉴定为特异性结合的多肽之一，通过质谱。半乳糖凝集素的乳糖和其他糖配体抑制TFII-I的GST-Gal 3下拉，而非结合性碳水化合物不能产生相同的效果。使用GST-Gal 1也获得了类似的结果。Gal 1的定点突变体表达并纯化为GST融合蛋白，在三种测定中与野生型（WT）进行比较：（a）与脱唾液酸胎球蛋白-琼脂糖凝胶的结合，作为碳水化合物结合活性的测量;（B）从NE下拉TFII-I;和（c）在耗尽半乳糖凝集素的NE中重组剪接，作为体外剪接活性的测试。GST-Gal 1（N46 D）与脱唾液酸胎球蛋白-琼脂糖凝胶的结合小于GST-Gal 1（WT）观察到的结合的10%，表明突变体缺乏碳水化合物结合活性。相比之下，GST-Gal 1（WT）和GST-Gal 1（N46 D）在TFII-I的下拉和半乳糖凝集素耗尽的NE中的剪接活性的重建中同样有效。此外，虽然野生型蛋白质的剪接活性可以被糖配体抑制，但碳水化合物结合缺陷突变体对这种抑制不敏感。总之，所有的结果表明，Gal 1的碳水化合物结合和剪接活性可以被解离，因此，糖结合本身不是剪接活性所必需的。
Galectin-1 (Gal1) and galectin-3 (Gal3) are two members of a family of carbohydrate-binding proteins that are found in the nucleus and that participate in pre-mRNA splicing assayed in a cell-free system. When nuclear extracts (NE) of HeLa cells were subjected to adsorption on a fusion protein containing glutathione S-transferase (GST) and Gal3, the general transcription factor II-I (TFII-I) was identified by mass spectrometry as one of the polypeptides specifically bound. Lactose and other saccharide ligands of the galectins inhibited GST-Gal3 pull-down of TFII-I while non-binding carbohydrates failed to yield the same effect. Similar results were also obtained using GST-Gal1. Site-directed mutants of Gal1, expressed and purified as GST fusion proteins, were compared with the wild-type (WT) in three assays: (a) binding to asialofetuin-Sepharose as a measure of the carbohydrate-binding activity; (b) pull-down of TFII-I from NE; and (c) reconstitution of splicing in NE depleted of galectins as a test of the in vitro splicing activity. The binding of GST-Gal1(N46D) to asialofetuin-Sepharose was less than 10% of that observed for GST-Gal1(WT), indicating that the mutant was deficient in carbohydrate-binding activity. In contrast, both GST-Gal1(WT) and GST-Gal1(N46D) were equally efficient in pull-down of TFII-I and in reconstitution of splicing activity in the galectin-depleted NE. Moreover, while the splicing activity of the wild-type protein can be inhibited by saccharide ligands, the carbohydrate-binding deficient mutant was insensitive to such inhibition. Together, all of the results suggest that the carbohydrate-binding and the splicing activities of Gal1 can be dissociated and therefore, saccharide-binding, per se, is not required for the splicing activity.
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DOI：
10.1083/jcb.81.3.528
发表时间：
1979-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Briles EB;Gregory W;Fletcher P;Kornfeld S
通讯作者：
Kornfeld S
DOI：
10.1016/0167-4889(96)00031-6
发表时间：
1996-06-13
影响因子：
5.1
作者：
Adams, L;Scott, GK;Weinberg, CS
通讯作者：
Weinberg, CS
影响因子：
4
作者：
Neugebauer, KM
通讯作者：
Neugebauer, KM
影响因子：
3.9
作者：
Butcher, SE;Brow, DA
通讯作者：
Brow, DA
影响因子：
2.9
作者：
MERRIL, CR;DUNAU, ML;GOLDMAN, D
通讯作者：
GOLDMAN, D