Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry

Abstract

The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades.The systematic mapping and characterization of protein glycosylation provide a wealth of molecular information that is crucial for understanding a wide variety of biochemical and cellular processes. However, comprehensive analysis of protein glycosylation has proven to be difficult due to the wide dynamic range of glycopeptide concentrations and immense structural diversity of glycans. Glycan modifications on proteins undergo a series of glycan processing steps from the endoplasmic reticulum (ER) to the Golgi apparatus with a diverse array of glycan processing enzymes that compete for available substrate, resulting in multiple glycosylation patterns for each glycosylation site for a given protein and variation in glycosylation site occupancy (35,52,53). Moreover, protein glycosylation varies significantly across different cell types, cell states, tissues, and organisms (3,12,16,34,35,53,56,62). Despite these challenges, recent advances in proteomics have accelerated the pace of the development of efficient methods and technologies that can be tailored for the analysis of protein glycosylation (6,51,66). To date, the analysis of protein glycosylation by mass spectrometry (MS) is underpinned by an array of sample preparation methods that include affinity/enrichment schemes (6, 18, 26), modern chromatographic methods (31,50,54), and remarkable improvements in mass spectrometry instrumentation (47,48,51). When used effectively, these methods provi...

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

“…Consistent with the notion that uninterpretable glycan density results from sample heterogeneity rather than from refinement problems, we found unambiguous density at positions that should be homogenous, e.g. at N332 gp120 , a high-mannose-only site (Behrens et al, 2016), whereas sites predicted to be more heterogeneous, such as N156 gp120 (Behrens et al, 2016;Go et al, 2011), exhibited some uninterpretable heterogeneous electron density. Although the relatively low resolution of our crystal structures and heterogeneous glycosylation compounded inherent difficulties in making unambiguous glycan assignments, we built coordinates into very extensive densities (e.g.…”

“…In other cases, the interpretation of our electrondensity maps was partially based on supporting experimental data: e.g. for the N301 gp120 glycan a core fucose was not ordered in our maps, but the N301 gp120 glycan was modeled as complextype in our structures based on mass-spectrometric data (Go et al, 2011). As expected given the large degree of glycan heterogeneity in HIV-1 Env (Doores, 2015), the glycan density was sometimes ambiguous.…”

“…Glycans were assigned as complextype if there was density for a core fucose and/or based on mass-spectrometry assignments (Behrens et al, 2016;Go et al, 2011). A core fucose was sometimes visible in one structure but not in the other.…”

Role of donor genital tract HIV-1 diversity in the transmission bottleneck

“…We and others have reported that founder viruses generally have fewer glycosylation sites and more compact Envs (9,(31)(32)(33), and our earlier studies also demonstrated that newly transmitted viruses were not escape variants from PL-neutralizing antibodies in the donor (9). Recent studies have suggested that subtype A and C founder viruses bind efficiently to the gut homing receptor α4β7 expressed on a subset of highly susceptible CD4 T lymphocytes and that this property can be reduced with evolution of the virus after transmission (34).…”

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

“…Pal et al showed that gp41 from viruses prepared in MOLT-3 cells was Endo H resistant, but when prepared in the presence of an ER mannosidase inhibitor, 1-deoxymannojirimycin, gp41 became sensitive to Endo H digestion (59). More recent work by us and by Go and colleagues has shown that the gp41 glycans on recombinant gp140 trimers (13,60,61), including the SOSIP trimer (14), are predominantly complex type and that these structures are generally more dependent on producer cells. In this study, we were able to record the chromatograms of labeled glycans from PGT151-isolated pseudovirion-derived material and cell surface-associated Env to provide a more detailed description of glycan structures present on native virion-derived gp41.…”