Abstract
The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.
Highlights
From the Biotechnology Research Laboratories and the §Biology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka 532, Japan
After the fused-chicken-lysozyme signal peptide (C-SIG) peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular ticles (22-nm sphericaland filamentous particles),along with 42-nm Hepatitis B virus (HBV) virions
It is indicated that theS and M proteins expressed fore, the pre-S1peptide may involve weak or reduced in yeast cells are initially translocated across the endoplasmic reticulum (ER) memsignal peptide activity forrecognition by the secretory brane, theproteinsassemble themapparatus and/or for the transportof the pre-Speptide selves into subviral particles inthe yeast secretory apparatus
Summary
Zymolyase lOOT was purchased from Seikagaku Kogyo Co.(Tokyo, Japan). Other enzymes were from Takara Shuzo Co. (Kyoto, Japan). Purified L particles (50 fig)were dialyzed against 0.1 M sodium citrate, pH 4.5, and 0.1 mM ZnSO., incubated a t 37 "C with either endo-p-N-acetylglucosaminidas(e2pg)or a-mannosidase (5 pg) for 2 or 4 h, respectively.The reacted materials were immunoprecipitated with the anti-Sprotein monoclonal antibody (HBs 2-06) as described previously [18]. The purified L particles (50 pg) weredialyzed against 0.85% NaCl, 10 mM sodium phosphate, pH 7.2, with or without 0.5% SDS, incubated with either L-1-tosylethylchloromethylketone-treatedtrypsin (1pg) or Staphylococcusaureus V8 proteinase (50 pg) at 37 "C for 2 or 4 h, respectively.The reacted materials were immunoprecipitated with HBs 2-06 as described previously [18]. Ultra thin sections were prepared using an Ultrotome I11 (Pharmacia-LKB),doubly stained with uranyl acetate and lead citrate, and observed under the JEM1200 EX transmission electron microscope
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