We studied the effect of the
plant alkaloid castanospermine on the biosynthesis and secretion of human
hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the
alkaloid, were labelled with [2-3H]
mannose and then the labelled
glycopeptides were prepared by
Pronase digestion. This material was analysed by gel filtration on
Bio-Gel P-4 before and
after treatment with
endo-beta-N-acetylglucosaminidase H.
Castanospermine caused an accumulation of high-
mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]
galactose and was only partially susceptible to
alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the
alkaloid inhibits
glucosidase I in the human
hepatoma cells. Analysis of total
glycoproteins secreted by the cells into the medium revealed the presence of only complex
oligosaccharides in both control and treated cultures, and the amount of the
oligosaccharides labelled with radioactive
mannose,
galactose or
N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total
protein labelled with [35S]
methionine and precipitated from the medium with
trichloroacetic acid was inhibited by up to 40% in the presence of
castanospermine. Pulse-chase studies utilizing [35S]
methionine labelling were performed to study the effect of the
alkaloid on secretion of individual
plasma proteins. Immunoprecipitation at different chase times with monospecific
antisera showed that
castanospermine markedly decreased the secretion rates of
alpha 1-antitrypsin,
caeruloplasmin and, to a lesser extent, that of
antithrombin-III. Secretions of
apolipoprotein E, a
glycoprotein containing only O-linked
oligosaccharide(s), and
albumin, a non-
glycosylated protein, were not affected by the
drug. It is suggested that
castanospermine inhibits secretion of at least some
glycoproteins containing N-linked
oligosaccharides, owing to the inhibition of
oligosaccharide processing.