Dyka Frank M, Wu Winco W H, Pfeifer Tom A, Molday Laurie L, Grigliatti Thomas A, Molday Robert S
Department of Biochemistry and Molecular Biology, Centre for Macular Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
Biochemistry. 2008 Sep 2;47(35):9098-106. doi: 10.1021/bi800938g. Epub 2008 Aug 9.
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 R141S 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.
RS1，也被称为视网膜劈裂蛋白，是一种含细胞外盘状结构域的蛋白质，与维持脊椎动物视网膜的细胞组织和突触结构有关。编码RS1的基因突变会导致X连锁视网膜劈裂症，这是一种视网膜退行性疾病，其特征是视网膜细胞层分裂和视力受损。为了更好地理解RS1在视网膜细胞生物学和X连锁视网膜劈裂症中的作用，我们研究了野生型和突变型RS1与偶联到琼脂糖载体上的各种碳水化合物的相互作用。RS1能有效结合半乳糖琼脂糖，与乳糖琼脂糖的结合程度较低，但不与琼脂糖、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结合。阻止二硫键连接的八聚体形成的RS1半胱氨酸突变体（C59S/C223S和C59S/C223S/C40S）与半乳糖琼脂糖几乎没有结合。致病的R141H突变体与半乳糖琼脂糖的结合水平与野生型RS1相似，而R141S突变体导致半乳糖琼脂糖结合水平显著降低。与异丙基β-D-1-硫代半乳糖苷（IPTG）孵育可有效取代结合在半乳糖琼脂糖上的RS1。这一特性被用作开发高效纯化程序的基础。采用阴离子交换和半乳糖亲和层析从稳定转化的Sf21昆虫细胞的培养基中纯化RS1，这些细胞表达并分泌RS1。这种细胞表达和蛋白质纯化方法在分离RS1以进行详细的结构-功能研究方面应该会很有用。
