Influenza Hemagglutinin

Influenza virus hemagglutinin (HA) is a type I transmembrane glycoprotein that is well characterized in terms of structure, function and intracellular transport. HA appears on virus particles as homotrimers. Each monomer (84 kDa) has six intrachain disulfide bonds and seven N-linked glycans in the N-terminal ectodomain (514 amino acids residues), a 26-amino acid transmembrane domain and a 10 residue cytosolic tail.

Upon synthesis by membrane-bound ribosomes, the ectodomain of HA is translocated across the ER membrane where it is welcomed by an array of resident ER chaperones and folding enzymes. The ectodomain already begins to fold co-translationally, but folding and disulfide bond formation go on after chain termination. During folding, HA interacts with two lectin-like chaperones: calnexin and calreticulin. They assist the protein in folding and homotrimer formation.

By pulse-chase experiments we monitor disulfide bond formation of HA. During the folding process, three different forms of HA can be distinguished by nonreducingc SDS-PAGE: two folding intermediates, IT1 and IT2, and the native form (NT) of the protein. Recently, the analysis of different cysteine mutants of HA showed that folding and disulfide bond formation in fact coincide. Disulfide bonds form in a strict hierarchy from the top of the hairpin towards the bottom, close to the transmembrane domain. We also monitor folding of HA by the appearance of antigenic epitopes (epitopes A, B, E, F1 and F2) and the transient association with ER resident proteins; again by pulse-chase analysis.

Braakman I, Hoover-Litty H, Wagner KR & Helenius A Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol. 1991;114:401-11. pdf

Currently, we investigate whether nonnative intrachain disulfide bonds are essential intermediates during the folding of newly synthesized proteins. To understand the role of nonnative disulfide bonds during folding one of the research line in our lab use cysteine mutants of HA and pulse-chase experiments to get insight on this point.

The use of pulse-chase semi in vitro experiments allow us to remove and to add different components of the ER and to do a fast identification of crucial folding factors needed for the correct folding of proteins in the lumen of the ER. For this purpose we use HA as a model protein.

• The HA folding crew
  Ineke Braakman
• Guus van Zadelhoff
• Mariano  Stornaiuolo (alumnus)
  Ari Ora (alumnus)
  Marije Liscaljet (alumna)
  Ana Azuaga (alumna)
  Claudia Maggioni (alumna)