Role of sialic acid in the mobility of membrane proteins containing O-linked oligosaccharides on polyacrylamide gel electrophoresis in sodium dodecyl sulfate.
The major sialoglycoprotein of the human red-cell membrane, glycophorin A, contains 15 O-glycosidically linked oligosaccharides and one N-glycosidic oligosaccharide. The protein shows a decreased mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate after neuraminidase treatment of the non-denatured protein. The molecular mechanism behind this phenomenon has been elucidated. Neuraminidase treatment of glycophorin A in intact cells or after solubilization in buffers containing Triton X-100 resulted in conversion of the predominant tetrasaccharide N-acetylneuraminosyl alpha 2-3galactosyl beta 1-3(N-acetylneuraminosyl alpha 2-6)-N-acetylgalactosamine to the trisaccharide galactosyl beta 1-3(N-acetylneuraminosyl alpha 2-6)-N-acetylgalactosamine and the disaccharide galactosyl beta 1-3-N-acetylgalactosamine. After denaturation with sodium dodecyl sulfate, Vibrio cholerae neuraminidase also liberated the N-acetylgalactosamine-bound sialic acids. Such treatment resulted in increased electrophoretic mobility. The results show that distal sialic acids linked to galactose are readily available to neuraminidase, and that their negative charge gives an increased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In contrast, most of the N-acetylgalactosamine-linked sialic acids of glycophorin A are not liberated by neuraminidase without denaturation of the substrate. Like sialic acids of complex-type oligosaccharides the decreased electrophoretic mobility caused by them is exclusively due to their mass while no significant contribution by the charge was seen.
D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006021:Glycophorins / Q000032:analysis; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D009844:Oligosaccharides / Q000302:isolation & purification*; D012794:Sialic Acids; D012967:Sodium Dodecyl Sulfate
D006021:Glycophorins; D006023:Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D012794:Sialic Acids; D012967:Sodium Dodecyl Sulfate