Comparison of secretion of a hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis.
A C-terminally truncated form of the hepatitis C virus (HCV) putative envelope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, using a yeast signal peptide sequence to direct the viral glycoprotein to the endoplasmic reticulum (ER) pathway of secretion. Characterization of secreted E2 showed that the protein is endoglycosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycosylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosylated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreducing conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with an average molecular mass of 770-1000 kDa in both yeasts. However, variations were observed in the binding of the glycoprotein secreted by the two yeasts to a mannose-specific lectin, and also in its reactivity with anti-E2-specific antibodies. This denotes differences between the two yeasts in folding and/or modification of the E2 glycoprotein.
Mustilli
A C
AC
Chiron Vaccines, I.R.I.S., Siena, Italy.
Izzo
E
E
Houghton
M
M
Galeotti
C L
CL
D004220:Disulfides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014415:Tunicamycin / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014415:Tunicamycin; C091015:glycoprotein E2, Hepatitis C virus; D006026:Glycoside Hydrolases