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J. Cell Biol.,
Volume 139, Number 3, November 3, 1997 613-623
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002
Calnexin and calreticulin are homologous
molecular chaperones that promote proper folding,
oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum
(ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof,
was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of
the HA molecules was found to occur in ternary HA-
calnexin-calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early
folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans
showed that although the two chaperones share the
same carbohydrate specificity, they display distinct
binding properties; calreticulin binding depends on the
oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating.
Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than
membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was
analyzed, it was observed that removal of stem domain
glycans caused accelerated folding whereas removal of
the top domain glycans (especially the oligosaccharide
attached to Asn81) inhibited folding. In summary, the
data established that individual N-linked glycans in
HA have distinct roles in calnexin/calreticulin binding
and in co- and posttranslational folding.
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