Spin labelling of sialic acid and galactose residues on lymphocyte plasma membrane: effects of lectins on oligosaccharide dynamics.
To investigate the behaviour of glycoprotein and glycolipid receptors at the lymphocyte cell surface, a spin label probe has been introduced into either sialic acid or galactose residues on lymphocyte plasma membrane, using specific activation of sugars with periodate or galactose oxidase, followed by reductive amination. The extent of membrane labelling could be controlled by varying the mole ratios of reactants used. Chloroform-methanol extraction of the labelled membranes showed that approximately 17% of the label is bound to glycolipids. A large fraction of the spin label could be released from both sialic acid and galactose-labelled membrane by treatment with pronase, indicating attachment to membrane proteins. Rotational correlation times (tau c) for both labelled sialic acid and galactose residues were in the range 10-13 X 10(-10) sec, indicating a reduction in sugar headgroup mobility at the membrane surface. Isolated lymphocyte membrane glycoproteins spin labelled on galactose residues and reassembled into phospholipid bilayer vesicles showed similar motional characteristics. Prolonged incubation of conc. suspensions of labelled membrane resulted in cleavage of the sialic acid-bound (but not the galactose-bound) label. Binding of several lectins to labelled plasma membrane produced significant immobilization of cell surface oligosaccharides while others had no effect. This differential restriction in oligosaccharide motion following lectin binding appears to be at least partly related to the sugar specificity of the lectin. Binding of wheat germ agglutinin and Ricinus communis agglutinin to sialic acid and galactose-labelled membrane respectively produced a dramatic decrease in oligosaccharide mobility which was reversible on addition of the appropriate sugar inhibitor. The concn dependence of lectin-induced spin label immobilization suggested a cooperative interaction between the lectins and their oligosaccharide receptors. Binding of lectins to the lymphocyte cell surface thus seems to have distinct effects on the dynamic state of glycoproteins and glycolipids within the glycocalyx.
Sharom
F J
FJ
Ross
T E
TE
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005690:Galactose / Q000031:analogs & derivatives / Q000378:metabolism*; D037102:Lectins / Q000494:pharmacology*; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D013552:Swine
D037102:Lectins; D009844:Oligosaccharides; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic; D012794:Sialic Acids; C032193:glycoprotein receptor GPIb-IX; C016735:sialooligosaccharides; D019158:N-Acetylneuraminic Acid; D005690:Galactose