Characterization and purification of the discoidin domain-containing protein retinoschisin and its interaction with galactose
Characterization and purification of the discoidin domain-containing protein retinoschisin and its interaction with galactose
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DOI：
10.1021/bi800938g
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发表时间：
2008-09-02
期刊：
影响因子：
2.9
通讯作者：
Molday, Robert S.
中科院分区：
文献类型：
--
作者：
Dyka, Frank M.;Wu, Winco W. H.;Molday, Robert S.
RS 1，也被称为视网膜裂素，是一种细胞外含有盘状结构域的蛋白质，其参与维持脊椎动物视网膜的细胞组织和突触结构。编码RS 1的基因突变导致X连锁视网膜劈裂症，这是一种以视网膜细胞层分裂和视力受损为特征的视网膜变性疾病。为了更好地了解RS 1在视网膜细胞生物学和X连锁视网膜劈裂中的作用，我们研究了野生型和突变型RS 1与各种糖偶联到琼脂糖支持物的相互作用。RS 1有效地结合半乳糖-琼脂糖和较小程度的乳糖-琼脂糖，但不结合琼脂糖、N-乙酰半乳糖胺-琼脂糖、N-乙酰葡糖胺-琼脂糖、甘露糖-琼脂糖或肝素-琼脂糖。阻止二硫键连接的八聚体形成的RS 1半胱氨酸突变体（C59 S/C223 S和C59 S/C223 S/C40 S）表现出很少的（如果有的话）与半乳糖-琼脂糖的结合。致病R141 H突变体结合半乳糖-琼脂糖的水平与野生型RS 1相似，而R141 S突变体导致半乳糖-琼脂糖结合水平显著降低。通过与异丙基-β-D-1-硫代半乳糖苷（IPTG）孵育，可以有效地置换与半乳糖-琼脂糖结合的RS 1。该性质被用作开发有效纯化程序的基础。用阴离子交换和半乳糖亲和层析从表达和分泌RS 1的稳定转化的Sf 21昆虫细胞的培养基中纯化RS 1。这种细胞表达和蛋白纯化方法应证明有用的分离RS 1的详细结构-功能研究。
RS1, also known as retinoschisin, is an extracellular discoidin domain-containing protein that has been implicated in maintaining the cellular organization and synaptic structure of the vertebrate retina. Mutations in the gene encoding RS1 are responsible for X-linked retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers and visual impairment. To better understand the role of RS1 in retinal cell biology and X-linked retinoschisis, we have studied the interaction of wild-type and mutant RS1 with various carbohydrates coupled to agarose supports. RS1 bound efficiently to galactose-agarose and to a lesser extent lactose-agarose, but not agarose, N-acetylgalactosamine-agarose, N-acetylglucosamine-agarose, mannose-agarose, or heparin-agarose. RS1 cysteine mutants (C59S/C223S and C59S/C223S/C40S) which prevent disulfide-linked octamer formation exhibited little if any binding to galactose-agarose. The disease-causing R141H mutant bound galactose-agarose at levels similar to that of wild-type RS1, whereas the R 141 S mutant resulted in a marked reduction in the level of galactose-agarose binding. RS1 bound to galactose-agarose could be effectively displaced by incubation with isopropyl-beta-D-1-thiogalactopyranoside (IPTG). This property was used as a basis to develop an efficient purification procedure. Anion exchange and galactose affinity chromatography was used to purify RS1 from the culture media of stably transformed Sf21 insect cells that express and secrete RS1. This cell expression and protein purification method should prove useful in the isolation of RS1 for detailed structure-function studies.