Purification and structure-function analysis of native, PNGase F-treated, and endo-beta-galactosidase-treated CHIP28 water channels.
Purification and structure-function analysis of native, PNGase F-treated, and endo-beta-galactosidase-treated CHIP28 water channels.
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DOI：
10.1021/bi00007a015
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发表时间：
1995-02
期刊：
影响因子：
2.9
通讯作者：
A. Hoek;Michael C. Wiener;J. Verbavatz;Dennis Brown;Peter H. Lipniunas;Raymond R. Townsend;A. Verkman
中科院分区：
文献类型：
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作者：
A. Hoek;Michael C. Wiener;J. Verbavatz;Dennis Brown;Peter H. Lipniunas;Raymond R. Townsend;A. Verkman
CHIP28以糖基化和非糖基化形式自然存在。本研究的目的是确定糖基化在CHIP28结构和功能中的作用。建立了苯基硼酸-琼脂糖亲和层析分离CHIP28的新方法。在红细胞纯化的原生CHIP28中，大约50%的CHIP28分子被糖基化；每摩尔糖基化CHIP28含有5.4 kDa的单糖，由2 mol的Fuc、8 mol的Gal、1 mol的GalN、13 mol的GlcN、3 mol的Man和1 mol的Neu5Ac组成。各单糖的比例和对内切- β -半乳糖苷酶的敏感性表明，CHIP28含有聚乳糖胺基低聚糖。糖基化和非糖基化的CHIP28在辛烷基β - d -葡萄糖苷（OG）中溶解时仍然紧密相关，并且不能通过常规色谱方法分离。为了去除糖部分，CHIP28通过PNGase F酶解去糖基化，并通过Q-Sepharose阴离子交换和赤藓凝集素层析纯化。高效粒径排除色谱法显示原生CHIP28洗脱为明显的二聚体，而去糖基化的CHIP28洗脱为明显的单体。在重组的蛋白脂质体中，去糖基化的CHIP28具有3.1 x 10(-14) cm3/s（10℃）的单通道水渗透性（pf），与原生CHIP28的3.2 x 10(-14) cm3/s没有区别。OG中原生CHIP28和去糖基化CHIP28的圆二色性分别显示45%和48%的α -螺旋；本征色氨酸荧光显示糖基化对色氨酸环境没有影响。带有旋转阴影的冷冻断裂电镜显示，原生和去糖基化的CHIP28在重组的蛋白脂质体中以四聚体的形式组装。（摘要删节250字）
CHIP28 occurs naturally in glycosylated and nonglycosylated forms. The purpose of this study was to determine the role of glycosylation in CHIP28 structure and function. A new purification procedure based on phenylboronic acid-agarose (PBA) affinity chromatography was developed to isolate CHIP28. In purified native CHIP28 from erythrocytes, approximately 50% of CHIP28 molecules were glycosylated; each mole of glycosylated CHIP28 contained 5.4 kDa of monosaccharides consisting of 2 mol of Fuc, 8 mol of Gal, 1 mol of GalN, 13 mol of GlcN, 3 mol of Man, and 1 mol of Neu5Ac. The proportions of each monosaccharide and the sensitivity to endo-beta-galactosidase indicated that CHIP28 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP28 remained tightly associated when solubilized in octyl beta-D-glucoside (OG) and could not be separated by conventional chromatographic procedures. To remove the sugar moiety, CHIP28 was enzymatically deglycosylated by PNGase F and purified by Q-Sepharose anion-exchange and Erythrina cristagalli lectin chromatography. High-performance size-exclusion chromatography revealed that native CHIP28 eluted as an apparent dimer, whereas deglycosylated CHIP28 eluted as an apparent monomer. In reconstituted proteoliposomes, deglycosylated CHIP28 had a single channel water permeability (pf) of 3.1 x 10(-14) cm3/s (10 degrees C), not different from that of 3.2 x 10(-14) cm3/s for native CHIP28. Circular dichroism of native and deglycosylated CHIP28 in OG revealed 45% and 48% alpha-helix, respectively; intrinsic tryptophan fluorescence showed no effects of glycosylation on tryptophan environment. Freeze-fracture electron microscopy with rotary shadowing indicated that native and deglycosylated CHIP28 assembled as tetramers in reconstituted proteoliposomes.(ABSTRACT TRUNCATED AT 250 WORDS)