The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin

doi:10.1083/jcb.139.3.613

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© The Rockefeller University Press, 0021-9525/1997//613 $5.00
The Journal of Cell Biology, Volume 139, Number 3, , 1997 613-623

Article

The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin

Daniel N. Hebert, Jian-Xin Zhang, Wei Chen, Brigitte Foellmer, and Ari Helenius

Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002

Calnexin and calreticulin are homologous molecular chaperonesthat promote proper folding, oligomeric assembly, and qualitycontrol of newly synthesized glycoproteins in the endoplasmicreticulum (ER). Both are lectins that bind to substrate glycoproteinsthat have monoglucosylated N-linked oligosaccharides. Theirbinding to newly translated influenza virus hemagglutinin (HA),and various mutants thereof, was analyzed in microsomes afterin vitro translation and expression in live CHO cells. A largefraction of the HA molecules was found to occur in ternaryHA– calnexin–calreticulin complexes. In contrastto calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with differentnumbers and locations of N-linked glycans showed that althoughthe two chaperones share the same carbohydrate specificity,they display distinct binding properties; calreticulin bindingdepends on the oligosaccharides in the more rapidly foldingtop/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as asoluble anchor-free protein rather than membrane bound. Whenthe co- and posttranslational folding and trimerization ofglycosylation mutants was analyzed, it was observed that removalof stem domain glycans caused accelerated folding whereas removalof the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the dataestablished that individual N-linked glycans in HA have distinctroles in calnexin/calreticulin binding and in co- and posttranslationalfolding.

Abbreviations used in this paper: GSSG, oxidized glutathione; HA, hemagglutinin; NEM, N-ethylmaleimide; NHA, NH₂-terminal peptide of HA; 2-D, two-dimensional.

We would like to acknowledge R. Gilmore (University of Massachusetts Medical Center), I. Braakman (University of Amsterdam, Amsterdam, The Netherlands), and T. Marquardt (University of Meunster,Muenster, Germany) for providing the canine pancreas microsomesand assistance with some of the glycosylation and truncationmutants, respectively. We would also like to thank N. Ayad(Yale University, New Haven, CT) and E.S. Trombetta (Yale University)for helpful discussions.

Address all correspondence to Ari Helenius, Department of CellBiology, Yale Univeristy School of Medicine, P.O. Box 208002,New Haven, CT 06520-8002. Tel.: (203) 785-4313. Fax: (203)737-1756. E-mail: ari_helenius @qm.yale.edu

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