THE NUMBER AND LOCATION OF GLYCANS ON INFLUENZA HEMAGGLUTININ DETERMINE FOLDING AND ASSOCIATION WITH CALNEXIN AND CALRETICULIN

Calnexin and calreticulin are homologous molecular chaperones that pro mote 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 monoglucosylat ed N-linked oligosaccharides. Their binding to newly translated influe nza virus hemagglutinin (HA), and various mutants thereof, was analyze d in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in tern ary HA-calnexin-calreticulin complexes. In contrast to calnexin, calre ticulin was found to bind primarily to early folding intermediates. An alysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohy drate specificity, they display distinct binding properties; calreticu lin binding depends on the oligosaccharides in the more rapidly foldin g top/hinge domain of HA whereas calnexin is less discriminating. Caln exin's binding was reduced if the HA was expressed as a soluble anchor -free protein rather than membrane bound. When the co- and posttransla tional folding and trimerization of glycosylation mutants was analyzed , it was observed that removal of stem domain glycans caused accelerat ed folding whereas removal of the top domain glycans (especially the o ligosaccharide attached to Asn81) inhibited folding. In summary, the d ata established that individual N-linked glycans in HA have distinct r oles in calnexin/calreticulin binding and in co- and posttranslational folding.