Related Experiment Videos
Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells
1Biochemisches Institut, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Abstract:
A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus-inducing virus plus the remainder of the genome of an amphoropic murine leukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2-3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and fractionated by high-performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing. The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4- and 2,6-branched triantennary and tetraantennary complex type N-glycans with 0-4 sialic acid residues and only small amounts of high-mannose type species with 5-9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(alpha 1-3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4-branched triantennary oligosaccharides with 1-3 sialic acid residues, in addition to trace amounts of high-mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N-glycans of the retroviral envelope glycoprotein display cell-specific variations including differences in oligosaccharide branching, sialylation and substitution by additional Gal(alpha 1-3) residues.
Insights
This study analyzed retroviral envelope glycoproteins synthesized in different cell types. Results show cell-specific variations in N-glycan structures, impacting viral properties.
Area of Science:
- Virology
- Glycobiology
- Molecular Biology
Background:
- Retroviral envelope glycoproteins play a crucial role in viral entry and host interactions.
- Understanding the glycosylation patterns of these proteins is essential for characterizing viral behavior.
Purpose of the Study:
- To investigate and compare the N-glycan structures of retroviral envelope glycoproteins synthesized in mouse embryo fibroblasts and mink lung cells.
- To identify cell-specific variations in oligosaccharide branching, sialylation, and substitution.
Main Methods:
- Propagation of a recombinant retrovirus in distinct cell lines (mouse embryo fibroblasts, mink lung cells).
- Metabolic labeling of viral particles with [2-3H]mannose.
- Isolation and purification of viral envelope glycoprotein using immunoaffinity chromatography and SDS-PAGE.
- Liberation and fractionation of oligosaccharides using enzymatic treatments (endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F) and HPLC.
- Characterization of glycans via chromatography, methylation analysis, and enzymic microsequencing.
Main Results:
- Viral glycoproteins from mouse embryo fibroblasts predominantly featured complex-type N-glycans (di-, tri-, and tetra-antennary) with partial fucosylation and 0-4 sialic acid residues, alongside minor high-mannose species.
- A notable finding was the presence of additional Gal(alpha 1-3) substituents on some complex-type glycans from mouse cells.
- Glycoproteins from mink lung cells showed predominantly partially fucosylated di- and tri-antennary oligosaccharides with 1-3 sialic acid residues and trace high-mannose types.
Conclusions:
- The N-glycans of retroviral envelope glycoproteins exhibit significant cell-specific variations.
- These variations include differences in oligosaccharide branching, degree of sialylation, and the presence of specific substitutions like Gal(alpha 1-3).
- These structural differences likely influence the biological properties and interactions of the retrovirus.