Characteristics of the sulfation of N-linked oligosaccharides in vesicles from Dictyostelium discoideum: in vitro sulfation of lysosomal enzymes.
Lysosomal enzymes from Dictyostelium discoideum contain unusual sulfated N-linked oligosaccharides, whose synthesis has been well studied in vivo. However, little is known about the properties of the pertinent sulfotransferases. To study these transferases, we have prepared a cell-free system which transfers 35SO4 from 3'-phosphoadenosine 5'-phosphosulfate to either endogenous or exogenous acceptors. We found that the 35SO4 was released from macromolecules by protein N-glycanase F to yield a mixture of anionic oligosaccharides with 1-6 negative charges. Some of the labeled molecules contained acid-stable methyl phosphodiesters but none contained phosphomoesters or acid-labile diesters. The sulfate was found in molecules with the acid stability characteristic of esters of primary alcohols. In all these ways, the products resembled those generated in vivo. We also demonstrated that a membrane-associated form of beta-hexosaminidase and the precursor of alpha-mannosidase were among the products. In addition, glycoproteins prepared from a sulfation-deficient mutant strain could act as exogenous acceptors in permeabilized vesicles.
Department of Microbiology, St Louis University School of Medicine, Missouri 63104.
C H
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Lacoste
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Freeze
J A
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Jones
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Kaplan
D002852:Chromatography, Ion Exchange; D003902:Detergents; D004023:Dictyostelium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D017830:Octoxynol; D009844:Oligosaccharides / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism*
D003902:Detergents; D009844:Oligosaccharides; D013431:Sulfates; D011092:Polyethylene Glycols; D017830:Octoxynol; D015238:Sulfotransferases