title: Mass spectrometry approach and ELISA reveal the effect of codon optimization on N-linked glycosylation of HIV-1 gp120.
abstract: The genes encoding many viral proteins such as HIV-1 envelope glycoprotein gp120 have a tendency for codons that are poorly used by the human genome. Why these codons are frequently present in the HIV genome is not known. The presence of these codons limits expression of HIV-1 gp120 for biochemical studies. The poor codons are replaced by synonymous codons that are frequently present in the highly expressed human genes to overexpress this protein. Whether this codon optimization affects functional properties of gp120 such as its N-linked glycosylation is unknown. We applied a bottom-up mass-spectrometry-based workflow for the direct measurement of deglycosylated and unglycosylated peptides with putative N-linked glycosylation sites, that is, NxS/T motifs. Using this mass-spectrometry approach in combination with ELISA, it is found that codon optimization significantly reduces the frequency with which the dolichol pyrophosphate-linked oligosaccharide is added by the catalytic subunits of oligosaccharide transferase complex to the glycosylation sites. This reduction affects binding of glycan-dependent broadly neutralizing antibodies. These data are essential for biochemical studies of gp120 and successful development of a vaccine against HIV-1. Furthermore, they demonstrate a mass-spectrometry approach for studying the site-specific N-linked glycosylation efficiency of glycoproteins.
authors.affiliation: Department of Infectious Diseases and ‡Mass Spectrometry and Proteomics, The Scripps Research Institute , Scripps Florida, 130 Scripps Way No. 2A2, Jupiter, Florida 33458, United States .
authors.forename: Kourosh
authors.initials: K
authors.lastname: Honarmand Ebrahimi
authors.forename: Graham M
authors.initials: GM
authors.lastname: West
authors.forename: Ricardo
authors.initials: R
authors.lastname: Flefil
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003062:Codon; D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D009844:Oligosaccharides; D010455:Peptides; C514793:gp120 protein, Human immunodeficiency virus 1
keywords: HIV-1; codon optimization; envelope glycoprotein; glycosylation; gp-120