Effects of Tunicamycin Mannosamine and Other Inh

Authors: JR Farley P Magnusson

Publish Date: 2004/10/14

Volume: 76, Issue: 1, Pages: 63-74

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Abstract

Skeletal alkaline phosphatase sALP is a glycoprotein—~20 carbohydrate by weight with five presumptive sites for Nlinked glycosylation as well as a carboxyterminal site for attachment of the glycolipid structure glycosylphosphatidylinositol GPI which anchors sALP to the outer surface of osteoblasts The current studies were intended to characterize the effects of inhibiting glycosylation and glycosylprocessing on the synthesis plasma membrane attachment cellular–extracellular distribution and reaction kinetics of sALP in human osteosarcoma SaOS2 cells sALP synthesis glycosylation and GPIanchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis sialic acid content ie wheat germ agglutinin precipitation and insolubility ie temperaturedependent phaseseparation respectively sALP reaction kinetics were characterized by analysis of dosedependent initial velocity data with a phosphoryl substrate The results of these studies revealed that the inhibition of either Nlinked glycosylation or oligosaccharide synthesis for GPIanchor addition could affect the synthesis and the distribution of sALP but not the kinetics of the phosphatase reaction Tunicamycin—which blocks Nlinked glycosylation by inhibiting core oligosaccharide synthesis—decreased cell layer protein and the total amount of sALP in the cells while increasing the relative level of sALP in the cellconditioned culture medium CM ie the amount of sALP released These effects were attributed to dose and timedependent decreases in sALP synthesis and Nlinked glycosylation and an increase in apoptotic cell death P 0001 for each In contrast to the effects of tunicamycin on Nlinked glycosylation the effects of mannosamine which inhibits GPIanchor glycosylation/formation included 1 an increase in cell layer protein 2 decreases in sALP specific activity in the cells and in the CM and 3 increases in the percentages of both anchorless and wheat germ agglutinin WGAsoluble sALP in the medium but not in the cells P 0005 for each These effects of mannosamine were presumably a consequence of inhibiting the insertion/attachment of sALP to the outside of the plasma membrane surface Neither mannosammine nor tunicamycin had any effect on the reaction kinetics of sALP or on the apparent affinity the value of KM for the phosphoryl substrateThe authors are thankful for the technical assistance of Aaron Eden and the administrative assistance of the Office of Research Administration of the VA Loma Linda Healthcare System These studies were supported by the Veterans Administration Merit Review funded research and the Swedish Research Council

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