Purification and Structure-Function Analysis of Native, PNGase F-Treated, and Endo-β-galactosidase-Treated CHIP28 Water Channels

A. N. van Hoek, Michael C. Wiener, Jean Marc Verbavatz, Dennis Brown, Peter H. Lipniunas, R. Reid Townsend, A. S. Verkman

Research output: Contribution to journalArticlepeer-review

70 Scopus citations

Abstract

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, ~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-β-galactosidase indicated that CHIP28 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP28 remained tightly associated when solubilized in octyl β-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. Highperformance 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 (pt) of 3.1 x 10-14 cm3/s (10 °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% a-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. The results establish a procedure to purify deglycosylated CHIP28 in functional form and indicate that glycosylation is required neither for the water transport function nor for the tetrameric assembly in membranes.

Original languageEnglish
Pages (from-to)2212-2219
Number of pages8
JournalBiochemistry
Volume34
Issue number7
DOIs
StatePublished - Feb 1995

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