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Identification of key amino acid residues determining ligand binding specificity, homodimerization and cellular distribution of human galectin-10
Glycobiology ( IF 4.3 ) Pub Date : 2018-09-20 , DOI: 10.1093/glycob/cwy087
Jiyong Su 1 , Chenyang Song 1 , Yunlong Si 1 , Linlin Cui 1 , Tong Yang 1 , Yuying Li 1 , Hao Wang 1 , Guihua Tai 1 , Yifa Zhou 1
Affiliation  

Charcot-Leyden crystal protein/Gal-10, abundantly expressed in eosinophils and basophils, is related to several immune diseases. Recently, crystallographic and biochemical studies showed that Gal-10 cannot bind lactose, because a glutamate residue (Glu33) from another monomer blocks the binding site. Moreover, Gal-10 actually forms a novel dimeric structure compared to other galectins. To investigate the role that Glu33 plays in inhibiting lactose binding, we mutated this residue to glutamine, aspartate, and alanine. The structure of E33A shows that Gal-10 can now bind lactose. In the hemagglutination assay, lactose could inhibit E33A from inducing chicken erythrocyte agglutination. Furthermore, we identified a tryptophan residue (Trp127) at the interface of homodimer that is crucial for Gal-10 dimerization. The variant W127A, which exists as a monomer, exhibited higher hemagglutination activity than wild type Gal-10. The solid phase assay also showed that W127A could bind to lactose-modified sepharose-6B, whereas wild type Gal-10 could not. This indicates that the open carbohydrate-binding site of the W127A monomer can bind to lactose. In addition, the distribution of EGFP-tagged Gal-10 and its variants in HeLa cells was investigated. Because Trp72 is the highly conserved in the ligand binding sites of galectins, we used EGFP-tagged W72A to show that Gal-10 could not be transported into the nucleus, indicating that Trp72 is crucial for Gal-10 transport into that organelle.

中文翻译:

确定关键的氨基酸残基,以确定人galectin-10的配体结合特异性,同型二聚化和细胞分布

嗜酸性粒细胞和嗜碱性粒细胞中大量表达的夏科特莱登晶体蛋白/ Gal-10与几种免疫疾病有关。最近,晶体学和生化研究表明,Gal-10无法结合乳糖,因为来自另一种单体的谷氨酸残基(Glu33)会阻断结合位点。而且,与其他半乳糖凝集素相比,Gal-10实际上形成了一种新型的二聚体结构。为了研究Glu33在抑制乳糖结合中的作用,我们将该残基突变为谷氨酰胺,天冬氨酸和丙氨酸。E33A的结构表明,Gal-10现在可以结合乳糖。在血凝试验中,乳糖可以抑制E33A诱导鸡红细胞凝集。此外,我们在同源二聚体的界面处发现了一个色氨酸残基(Trp127),这对于Gal-10二聚化至关重要。变体W127A,它作为单体存在,比野生型Gal-10表现出更高的血凝活性。固相分析还显示,W127A可以与乳糖修饰的琼脂糖6B结合,而野生型Gal-10则不能。这表明W127A单体的开放的碳水化合物结合位点可以结合乳糖。此外,还研究了带有EGFP标签的Gal-10及其变体在HeLa细胞中的分布。因为Trp72在半乳凝素的配体结合位点中是高度保守的,所以我们使用带有EGFP标签的W72A来显示Gal-10不能转运到细胞核中,这表明Trp72对于Gal-10转运到该细胞器中至关重要。这表明W127A单体的开放的碳水化合物结合位点可以结合乳糖。此外,还研究了带有EGFP标签的Gal-10及其变体在HeLa细胞中的分布。因为Trp72在半乳凝素的配体结合位点中是高度保守的,所以我们使用带有EGFP标签的W72A来显示Gal-10不能转运到细胞核中,这表明Trp72对于Gal-10转运到该细胞器中至关重要。这表明W127A单体的开放的碳水化合物结合位点可以结合乳糖。此外,还研究了带有EGFP标签的Gal-10及其变体在HeLa细胞中的分布。因为Trp72在半乳凝素的配体结合位点中是高度保守的,所以我们使用带有EGFP标签的W72A来显示Gal-10不能转运到细胞核中,这表明Trp72对于Gal-10转运到该细胞器中至关重要。
更新日期:2018-12-13
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