Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.

Abstract

We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae. The cDNA sequences encoding variant forms of influenza HA were expressed in S. cerevisiae. The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated. In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S. cerevisiae. The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery. The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S. cerevisiae compared with the signal-minus, nonglycosylated HA molecules. Although both the anchor-minus HA (HA500) and HAl (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different. During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium. Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans. Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.

Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

“…The full length and partial H1N1 HA protein has been previously expressed in bacterial system [ 46 ]. There is a report on lower level of expression of Human Influenza A/WSN/33 HA protein in S. cerevisiae [ 47 ]. However this reported protein is truncated, hyperglycosylated and is also cell-associated and have not demonstrated its in vivo applications.…”

Alternative Experimental Models for Studying Influenza Proteins, Host–Virus Interactions and Anti-Influenza Drugs

“…The yeast system was used to study structure-function relationships within the HA protein by expressing different deletion mutants. HA500 (lacking the C-terminal transmembrane anchor and cytoplasmic domain) and HA325 (containing only HA1 region and lacking the entire HA2 region) were glycosylated and stable compared to the other mutants [127]. However, HA500 was retained intracellularly, while HA325 was found to be secreted to the extracellular medium and was hyperglycosylated.…”

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

“…Failure of yeast to produce active human virus surface glycoproteins indicates principal difference between yeast and mammalian cell secretion pathways. There are some reports where possible limitations of yeast expression systems to produce viral surface proteins are described including rather low expression level [ 12 , 13 ], formation of insoluble multimers [ 9 , 10 ] and inactivity due to hyperglycosylation [ 14 ]. However, earlier studies were focused only on the analysis of recombinant products, whereas the molecular processes that influence synthesis of these complex transmembrane proteins in yeast cells were not examined.…”