The influence of N-glycosylation and C-terminal sequence on secretion of HBV large surface antigen from S. cerevisiae.
In Saccharomyces cerevisiae, we synthesized and secreted L-HBVsAg (named as pre-S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre-S degree degree::S (Asn15Gln and Asn123Gln), pre-S degree degree::S degree (Asn15Gln, Asn123Gln, and Asn320Gln), and pre-S degree degree::S degree degree (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre-S::S was N-glycosylated, i.e., hyper-mannosylated. In the secretion of pre-S degree degree::S and pre-S degree degree::S degree, besides the hyper-mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre-S degree degree::S and pre-S degree degree::S degree. Only a part of the secreted pre-S degree degree::S or pre-S degree degree::S degree molecules was N-glycosylated, and the site for the partial N-glycosylation seems to be Asn233 in S-antigen region. Compared to the N-glycosylated pre-S degree degree::S and pre-S degree degree::S degree, pre-S degree degree::S degree degree (non-N-glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti-S antigen monoclonal Ab. Interestingly, unlike pre-S degree degree::S degree degree with authentic C-terminus, the recombinant pre-S degree degree::S degree degree with C-terminal myc or poly-histidine tag (pre-S degree degree::S degree degree::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C-terminal sequence and glycosylation in S-antigen region seem to be of crucial importance in determining the secretion efficiency of L-HBVsAg in S. cerevisiae.
Park
Jin-Seung
JS
Department of Chemical and Biological Engineering, Korea University, Sungbuk-Ku, Seoul.
Seo
Hyuk-Seong
HS
Yum
Jung-Sun
JS
Moon
Hong Mo
HM
Lee
Jeewon
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D006031:Glycosylation; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics*
C096362:L protein, hepatitis B virus; D014759:Viral Envelope Proteins