van Hoek A N, Wiener M C, Verbavatz J M, Brown D, Lipniunas P H, Townsend R R, Verkman A S
Department of Medicine, University of California, San Francisco 94143.
Biochemistry. 1995 Feb 21;34(7):2212-9. doi: 10.1021/bi00007a015.
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)
CHIP28以糖基化和非糖基化形式天然存在。本研究的目的是确定糖基化在CHIP28结构和功能中的作用。开发了一种基于苯硼酸 - 琼脂糖（PBA）亲和色谱的新纯化方法来分离CHIP28。在从红细胞中纯化的天然CHIP28中，约50%的CHIP28分子被糖基化；每摩尔糖基化的CHIP28含有5.4 kDa的单糖，由2摩尔岩藻糖、8摩尔半乳糖、1摩尔氨基半乳糖、13摩尔氨基葡萄糖、3摩尔甘露糖和1摩尔唾液酸组成。每种单糖的比例以及对内切β - 半乳糖苷酶的敏感性表明CHIP28含有多乳糖胺基寡糖。当溶解在辛基β - D - 葡萄糖苷（OG）中时，糖基化和非糖基化的CHIP28保持紧密结合，并且不能通过常规色谱方法分离。为了去除糖部分，CHIP28用PNGase F进行酶促去糖基化，并通过Q - Sepharose阴离子交换和刺桐凝集素色谱进行纯化。高效尺寸排阻色谱显示天然CHIP28以明显的二聚体形式洗脱，而去糖基化的CHIP28以明显的单体形式洗脱。在重组蛋白脂质体中，去糖基化的CHIP28的单通道水渗透率（pf）为3.1×10⁻¹⁴ cm³/s（10℃），与天然CHIP28的3.2×10⁻¹⁴ cm³/s没有差异。在OG中天然和去糖基化的CHIP28的圆二色性分别显示45%和48%的α - 螺旋；色氨酸内在荧光显示糖基化对色氨酸环境没有影响。旋转阴影冷冻断裂电子显微镜表明天然和去糖基化的CHIP28在重组蛋白脂质体中组装成四聚体。（摘要截短于250字）
