Purification and structure-function analysis of native, PNGase F-treated, and endo-beta-galactosidase-treated CHIP28 water channels.
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)
van Hoek
A N
AN
Department of Medicine, University of California, San Francisco 94143.
Wiener
M C
MC
Verbavatz
J M
JM
Brown
D
D
Lipniunas
P H
PH
Townsend
R R
RR
Verkman
A S
AS
D000581:Amidohydrolases / Q000494:pharmacology; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004910:Erythrocyte Membrane / Q000737:chemistry; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry* / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D009005:Monosaccharides / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000378:metabolism
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D007473:Ion Channels; D008562:Membrane Glycoproteins; D009005:Monosaccharides; D051398:Aquaporin 1; D006596:Hexosaminidases; D001616:beta-Galactosidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase