Role of individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of influenza C virus hemagglutinin-esterase protein
K. Sugahara et al., Role of individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of influenza C virus hemagglutinin-esterase protein, VIROLOGY, 285(1), 2001, pp. 153-164
The hemagglutinin-esterase (HE) glycoprotein of influenza C virus is compos
ed of three domains: a stem domain active in membrane fusion (5), an acetyl
esterase domain (E), and a receptor-binding domain (R). The protein contain
s eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603
) in the F domain, three (positions 61, 131, and 144) in the E domain, and
one (position 189) in the R domain. Here, we investigated the role of the i
ndividual oligosaccharide chains in antigenic properties, intracellular tra
nsport, and biological activities of the HE protein by eliminating each of
the glycosylation sites by site-specific mutagenesis. Comparison of electro
phoretic mobility between the wild-type and the mutant proteins showed that
while seven of the glycosylation sites are used, one (position 131) is not
. Analysis of reactivity of the mutants with anti-H E monoclonal antibodies
demonstrated that glycosylation at position 144 is essential for the forma
tion of conformation-dependent epitopes. it was also evident that glycosyla
tion at the two sites in the F domain (positions 26 and 603), in addition t
o that in the E domain (position 144), is required for the HE molecule to b
e transported from the endoplasmic reticulum and that mutant HEs lacking on
e of these three sites failed to undergo the trimer assembly. Removal of an
oligosaccharide chain at position 144 or 189 resulted in a decrease in the
esterase activity. By contrast, two mutants lacking an oligosaccharide cha
in at position 26 or 603, which were defective not only in cell surface exp
ression but in trimerization, possessed full-enzyme activity, suggesting th
at the HE monomers present within the cell have acetylesterase activity. Fu
sion activity of cells expressing each of mutant HEs was found to be compar
able with the ability of the protein to be transported to the cell surface,
suggesting that there is no specific oligosaccharide chain that plays a cr
itical role in promoting membrane fusion. (C) 2001 Academic Press.