CARBOHYDRATE BINDING PROTEIN 35
CARBOHYDRATE BINDING PROTEIN 35
批准号:
6258871
负责人:
JOHN L WANG
金额:
$0.05万
依托单位国家:
美国
项目类别:
财政年份:
1997
资助国家:
美国
项目状态:
已结题
起止时间:
1997-06-01 至 1999-11-30
中文摘要
半乳糖凝集素-3是一种蛋白质(Mr 30,000),最初被鉴定为
基于其半乳糖特异性碳水化合物结合
活动,最近记录为核反应堆的一个必要因素,
前mRNA剪接。 所做的主要观察包括:a)核
来自HeLa细胞的提取物,能够进行剪接,
a)含有半乳糖凝集素-3的无细胞测定; B)耗尽的
半乳糖凝集素-3通过在乳糖-琼脂糖上的亲和吸附变得缺乏
c)乳糖-琼脂糖耗尽的提取物的活性
可以通过添加纯化的重组半乳糖凝集素-3来重建;
和d)以高亲和力结合半乳糖凝集素-3的抑制剂
剪接反应 在细胞内,半乳糖凝集素-3存在于两种细胞中,
细胞质和细胞核。 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
所述多肽被单个磷酸修饰。 磷酸化
(pI 8.2)形式在细胞质和细胞核中发现,而
未修饰的多肽(pI 8.7)仅存在于
原子核 最近,我们开发了一种透化细胞测定法,
监控半乳糖凝集素-3从细胞核的输出 虽然核
残基含有pI 8.2和pI 8.7两种形式,只有
磷酸化(pI 8.2)多肽被输出。 这些结果
表明需要半乳糖凝集素-3的磷酸化
多肽在其从细胞核输出之前。 的基础上
这些结果,我们的研究目标包括:a)
确定鼠半乳糖凝集素-3的磷酸化位点
多肽; B)进行定点突变以衍生
不能被磷酸化的突变多肽(例如,Ser至Ala;
Ser至Asp等);和c)比较细胞核与细胞质
半乳糖凝集素-3在用野生型转染的细胞中的分布,
突变体构建体。 质谱法将用于
在这项研究中确定磷酸化位点。
英文摘要
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.
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PHOSPHORYLATION OF CARBOHYDRATE BINDING PROTEIN 35
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批准号:6248443
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项目类别:
-
资助金额:$0.46万
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财政年份:1997
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负责人:JOHN L WANG
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依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:2179503
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项目类别:
-
资助金额:$20.11万
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财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:6179536
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项目类别:
-
资助金额:$22.22万
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财政年份:1988
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负责人:JOHN L WANG
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依托单位:
CARBOHYDRATE BINDING PROTEIN 35
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批准号:3295375
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项目类别:
-
资助金额:$9.58万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE BINDING PROTEIN 35
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批准号:3295376
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项目类别:
-
资助金额:$9.55万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE BINDING PROTEIN 35
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批准号:3295372
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项目类别:
-
资助金额:$9.01万
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财政年份:1988
-
负责人:JOHN L WANG
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依托单位:
Carbohydrate Binding Protein 35
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批准号:6694083
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项目类别:
-
资助金额:$26.31万
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财政年份:1988
-
负责人:JOHN L WANG
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依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:2684862
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项目类别:
-
资助金额:$20.96万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE BINDING PROTEIN 35
-
批准号:3295377
-
项目类别:
-
资助金额:$9.93万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:2179500
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项目类别:
-
资助金额:$12.38万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE BINDING PROTEIN 35
-
批准号:3295374
-
项目类别:
-
资助金额:$9.38万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
-
批准号:3295373
-
项目类别:
-
资助金额:$11.83万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
Carbohydrate Binding Protein 35
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批准号:6573243
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项目类别:
-
资助金额:$26.01万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:2022174
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项目类别:
-
资助金额:$20.86万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:2179502
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项目类别:
-
资助金额:$19.36万
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财政年份:1988
-
负责人:JOHN L WANG
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依托单位:
CARBOHYDRATE-BINDING PROTEIN 35
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批准号:2900658
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项目类别:
-
资助金额:$21.58万
-
财政年份:1988
-
负责人:JOHN L WANG
-
依托单位:
Carbohydrate Binding Protein 35
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批准号:6838253
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项目类别:
-
资助金额:$26.31万
-
财政年份:1988
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负责人:JOHN L WANG
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依托单位:
Carbohydrate Binding Protein 35
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批准号:7002199
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项目类别:
-
资助金额:$25.69万
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财政年份:1988
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负责人:JOHN L WANG
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依托单位:
CARBOHYDRATE BINDING PROTEIN 35
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批准号:2179501
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项目类别:
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资助金额:$4.6万
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财政年份:1988
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负责人:JOHN L WANG
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依托单位:
ACTION OF LYSOSOMAL ENZYMES ON THE EXTRACELLULAR MATRIX
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批准号:3281031
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项目类别:
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资助金额:$6.62万
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财政年份:1983
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负责人:JOHN L WANG
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依托单位:
海外基金