Castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin

Abstract

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits alpha- and beta-glucosidase in fibroblast extracts [Saul, R., Chambers, J. P., Molyneux, R. J., & Elbein, A. D. (1983) Arch. Biochem. Biophys. 221, 593-597]. In the present study, castanospermine also proved to be a potent inhibitor of glycoprotein processing by virtue of the fact that it inhibits glucosidase I. Thus, when influenza virus was raised in the presence of castanospermine, at 10 micrograms/mL or higher, 80-90% of the viral glycopeptides were susceptible to the action of endoglucosaminidase H, whereas in the normal virus 70% of the glycopeptides are resistant to this enzyme. The major oligosaccharide released by endoglucosaminidase H from castanospermine-grown virus migrated like a hexose10GlcNac on a calibrated Bio-Gel P-4 column. This oligosaccharide was characterized as a Glc 3 Man 7 GlcNAc on the basis of various enzymatic treatments, as well as by methylation analysis of the [2-3H]-mannose-labeled or [6-3H]galactose-labeled oligosaccharide. The presence of three glucose residues in the oligosaccharide was also confirmed by periodate oxidation studies of the [6-3H]galactose-labeled hexose10GlcNAc. Castanospermine did not inhibit the incorporation of [3H]leucine or [14C]alanine into protein in MDCK cells at levels as high as 50 micrograms/mL. In addition, influenza virus produced in the presence of this alkaloid were fully infective and apparently produced in similar amounts to that of control cells, as determined by plaque counts. Castanospermine did, however, cause considerable changes in cell surface properties, since MDCK cells grown in 10 micrograms/mL castanospermine were able to bind twice as much [3H]concanavalin A as were control cells.

Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins

“…Here we show that the glucosylated N-linked glycoproteins that accumulate in the ER are insensitive to autophagic sequestration, despite continued autophagy in glucosidase-inhibited cells [21,22]. Similar results were observed in castanospermine (CST)-treated Chinese hamster ovary (CHO) cells and in the glucosidase I-deficient CHO cell line Lec23 [23].…”

Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins

“…4). Treatment of cells expressing the glycosylation mutants or wild-type env proteins with Cs, an inhibitor of u-glucosidase I (Pan et al, 1983;Saul et al, 1983), resulted in a slight decrease in the electrophoretic mobility of gpl60 and gp 120, but no band corresponding to gp135 was found by S D S -P A G E (Fig. 4a).…”

Cell division modulates prion accumulation in cultured cells

“…The unifying pathological mechanism of neurodegenerative proteinopathies is the misfolding and aggregation of normally benign soluble proteins. Prion diseases result from the misfolding of the ␣-helixrich cellular prion protein, termed PrP C , into the ␤-sheet-rich disease-causing infectious isoform, termed PrP Sc (2)(3)(4). PrP C and PrP Sc are characterized by their differing aggregation properties, resistance to proteolysis, and ability to become solubilized in detergents (5,6).…”