Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties

• Published in: The Journal of biological chemistry Vol. 267; no. 17; pp. 11940 - 11948
• Main Authors: CASEY, J. R, PIRRAGLIA, C. A, REITHMEIER, R. A. F
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-06-1992
• Subjects: Analytical, structural and metabolic biochemistry; Anion Exchange Protein 1, Erythrocyte - chemistry; Anion Exchange Protein 1, Erythrocyte - metabolism; band 3 protein; Binding and carrier proteins; Biological and medical sciences; Biological Transport; Chromatography, Gel; Chromatography, High Pressure Liquid; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; enzymes; Erythrocyte Membrane - metabolism; erythrocytes; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - metabolism; Glycosylation; Humans; Lectins - metabolism; man; membranes; Protein Conformation; Proteins; Serine Endopeptidases - metabolism; Spectrophotometry, Ultraviolet; Trypsin - metabolism; Human; Hydrodynamic properties; Band 3 protein; Solubility; Quaternary structure; Red blood cell; Ion transport; Glycosylation; Property structure relationship; Secondary structure; Carrier protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)49787-1

The structural and functional roles of the single asparagine (N)-linked oligosaccharide chain of Band 3 (AE1), the anion transport
protein of the human erythrocyte membrane, were examined. Purified Band 3 (M(r) = 95,000) in 0.1% octaethylene glycol mono
n-dodecyl ether (C12E8) detergent solution was deglycosylated using N-glycosidase F. This treatment sharpened the protein
band on sodium dodecyl sulfate gel electrophoresis and decreased its apparent molecular weight by 5,000. The purified membrane
domain could be deglycosylated under similar conditions, causing a shift from a broad band centered at 55 kDa to a sharp 46-kDa
band. Band 3 was shown to bind tomato lectin, and loss of lectin binding on blots provided a sensitive assay for deglycosylation.
Carbohydrate analysis revealed that greater than 80% of the oligosaccharide could be removed from Band 3 by N-glycosidase
F digestion. The deglycosylated protein maintained its dimeric structure and level of detergent binding but had a smaller
Stokes radius (RS = 72 A) than native Band 3 (RS = 75 A). The Stokes radius of the membrane domain (RS = 60 A) also decreased
upon deglycosylation (RS = 58 A). Circular dichroism studies showed that deglycosylation did not change the secondary structure
of Band 3 or the membrane domain. The sensitivity of Band 3 or the membrane domain to proteolytic digestion by trypsin or
proteinase K was also unaffected by deglycosylation. The deglycosylated protein aggregated more rapidly and was much more
readily precipitable by ammonium sulfate. The deglycosylated protein bound the anion transport inhibitor 4-benzamido-4'-amino-stilbene-2,2'-disulfonate
with the same affinity (Kd = 1 microM) as the native protein. Transport studies using reconstituted Band 3 and resealed ghosts
showed that deglycosylated Band 3 retained its ability to transport anions. We conclude that removal of the oligosaccharide
chain from Band 3 and any resultant structural changes had no effect on the transport function of this protein.