2 research outputs found
Mass Spectrometry Approach and ELISA Reveal the Effect of Codon Optimization on N‑Linked Glycosylation of HIV‑1 gp120
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