Abstract
The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking. Here, we integrate metabolic labeling and click chemistry technology with recombinant gp120 expression to demonstrate that gp120 glycans could be specifically labeled and directly detected. Selective labeling of gp120 by N-azidoacetylmannosamine (ManNAz) and N-azidoacetylgalactosamine (GalNAz) incorporation into the gp120 glycan shield was characterized by MS of tryptic glycopeptides. By using metabolically labeled gp120, we investigated the impact of gp120 glycosylation on its interaction with host cells and demonstrated that oligomannose enrichment and sialic acid deficiency drastically enhanced gp120 uptake by bone marrow-derived dendritic cells. Collectively, our data reveal an effective labeling and detection method for gp120, serving as a tool for functional characterization of the gp120 glycans and potentially other glycosylated proteins.
Highlights
The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1
One prominent example is the interaction of the HIV-1 envelope spike with the host protein CD4, a key step for viral entry, and infectivity of CD4ϩ T cells [3,4,5]. gp120 has remarkably high levels of surface glycosylation with glycans contributing to nearly half of its molecular weight and gp120 recognition by broadly neutralizing antibodies2 [6]
These results demonstrated that metabolically labeled gp120 with azido analogs provides a valuable tool to trace and study gp120 glycan function. gp120 glycosylation profile is important in mediating gp120 – host interaction, as oligomannose structures promote, whereas terminal sialic acids inhibit, the gp120 internalization by antigen-presenting cells (APCs)
Summary
To metabolically label gp120 glycans, the azido-modified metabolic glycoprotein-labeling reagents GlcNAz, ManNAz, and GalNAz were added together into the FreeStyleTM 293-F cells transfected with gp120 expression plasmid. Increasing amounts of gp120 proteins were loaded on SDSpolyacrylamide gel and reacted with Alexa Fluor 488 DIBO alkyne to detect by fluorescence scanning (Fig. 2D, left) or stained with Coomassie Blue as a protein-loading control (Fig. 2D, right) These results demonstrate that azido sugars ManNAz and GalNAz, but not GlcNAz, can be effectively incorporated into gp120 glycans during protein expression. Gp120 proteins before or after PNGase F treatment were reacted with fluorophore-labeled DIBO alkyne or biotinylated DIBO alkyne and detected by fluorescence scanning or Western blotting Both the fluorescent and biotin signals were absent after PNGase F treatment in both ManNAz- and GalNAz-incorporated gp120 proteins (Fig. 4 (A and B), top and bottom), demonstrating the specific labeling of gp120 by those azido sugars. ManNAz was metabolically incorporated into terminal sialic acid of gp120 glycans, which can be efficiently removed by sialidase treatment
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