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 envel ope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerev isiae and Kluyveromyces lactis, using a yeast signal peptide sequence to di rect the viral glycoprotein to the endoplasmic reticulum (ER) pathway of se cretion. Characterization of secreted E2 showed that the protein is endogly cosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycos ylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosyl ated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreduci ng conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with a n average molecular mass of 770-1000 kDa in both yeasts. However, variation s were observed in the binding of the glycoprotein secreted by the two yeas ts to a mannose-specific lectin, and also in its reactivity with anti-E2-sp ecific antibodies. This denotes differences between the two yeasts in foldi ng and/or modification of the E2 glycoprotein. (C) Elsevier, Paris.