Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

Cao, Liwei; Diedrich, Jolene K.; Kulp, Daniel W.; Pauthner, Matthias; He, Lin; Park, Sung-Kyu Robin; Sok, Devin; Su, Ching Yao; Delahunty, Claire; Menis, Sergey; Andrabi, Raiees; Guenaga, Javier; Georgeson, Erik; Kubitz, Michael; Adachi, Yumiko; Burton, Dennis R.; Schief, William R.; Yates, John R.; Paulson, James C.

doi:10.1038/ncomms14954

Cited by 189 publications

(311 citation statements)

References 90 publications

(199 reference statements)

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“…Recent studies using chromatography coupled mass spectrometry analysis of soluble Env proteins, suggested that the N156 glycan position is occupied largely by a high mannose glycan (Behrens et al, 2016; Panico et al, 2016). Another study (Cao et al, 2017) indicated that N156 is occupied either by high mannose or complex glycans for several soluble SOSIP.664 trimers, including the CRF250 trimer used in this study. Further studies are required to fully understand the compositions of the glycans in the V2 apex region on different isolates and expressed from different cell types.…”

Section: Resultsmentioning

confidence: 83%

Glycans Function as Anchors for Antibodies and Help Drive HIV Broadly Neutralizing Antibody Development

Andrabi

Liang

et al. 2017

Immunity

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Summary Apex broadly neutralizing HIV antibodies (bnAbs) recognize glycans and protein surface close to the 3-fold axis of the envelope (Env) trimer and are among the most potent and broad Abs described. The evolution of apex bnAbs from one donor (CAP256) has been studied in detail and many Abs at different stages of maturation have been described. Using diverse engineering tools, we investigated the involvement of glycan recognition in the development of the CAP256.VRC26 Ab lineage. We found that sialic acid-bearing glycans were recognized by germline-encoded and somatically mutated residues on the Ab heavy chain. This recognition provided an “anchor” for the Abs as the core protein epitope varies, prevented complete neutralization escape, and eventually led to broadening of the response. These findings illustrate how glycan-specific maturation enables a human Ab to cope with pathogen escape mechanisms and will aid in optimization of immunization strategies to induce V2 apex bnAb responses.

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Section: Resultsmentioning

confidence: 83%

Glycans Function as Anchors for Antibodies and Help Drive HIV Broadly Neutralizing Antibody Development

Andrabi

Liang

et al. 2017

Immunity

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“…This study showed a very high occupancy level of recombinant gp120 N-glycosylation sites, however, the effect of metabolic engineering on protein occupancy during biosynthesis is unknown. A methodology to assess occupancy on a site-specific level has been developed and applied to various Env strains [79]. Here, protease-generated (glyco)peptides are first treated with Endo H and then digested with PNGase F in O 18 -water.…”

Section: Assessment Of Glycan Site Occupancymentioning

confidence: 99%

“…Cao et al , reported that almost all sites are more than 90 % occupied on soluble Env trimers of 6 different strains [79]. …”

Section: Assessment Of Glycan Site Occupancymentioning

confidence: 99%

“…It has been hypothesized that the induction of bNAbs could be achieved by the use of immunogens primers that help expand B-cell linages corresponding to the desired bNAbs [82–86]. Glycosylation analytics have shown that deletion of multiple glycans has minimal effect of neighboring glycans and potentially multiple holes can be incorporated into a single immunogen without adversely affecting the remaining glycan-dependent epitopes [79]. Overall, glycan, glycopeptide and intact glycoprotein analytics have emerged as an indispensable tool in the evaluation of immunogens against HIV-1 and in the understanding of the epitopes of broadly neutralizing antibodies.…”

Section: Expert Commentarymentioning

confidence: 99%

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Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

Behrens

Struwe

Crispin

2017

Expert Review of Proteomics

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Introduction Much of the efforts to develop a vaccine against the human immunodeficiency virus (HIV) have focused on the design of recombinant mimics of the viral attachment glycoprotein (Env). The leading immunogens exhibit native-like antigenic properties and are being investigated for their ability to induce broadly neutralizing antibodies (bNAbs). Understanding the relative abundance of glycans at particular glycosylation sites on these immunogens is important as most bNAbs have evolved to recognize or evade the dense coat of glycans that masks much of the protein surface. Understanding the glycan structures on candidate immunogens enables triaging between native-like conformations and immunogens lacking key structural features as steric constraints limit glycan processing. The sensitivity of the processing state of a particular glycan to its structural environment has led to the need for quantitative glycan profiling and site-specific analysis to probe the structural integrity of immunogens. Areas covered We review analytical methodologies for HIV immunogen evaluation and discuss how these studies have led to a greater understanding of the structural constraints that control the glycosylation state of the HIV attachment and fusion spike. Expert commentary Total composition and site-specific glycosylation profiling are emerging as standard methods in the evaluation of Env-based immunogen candidates.

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“…Glycomics is the systematic study of glycan structures, including monosaccharide compositions, glycan sequence, branching pattern and linkage specifications, which are essential to the functions of glycoconjugates [11–14]. Nevertheless, glycomics analysis is inherently challenging due to their structure complexity and heterogeneity, low natural abundance and the lack of amplification methods [5,14,15]. …”

Section: Introductionmentioning

confidence: 99%

Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans

Zhang

Wang

Jin

et al. 2019

Analytica Chimica Acta

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Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n = 3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16 μg ovalbumin could be detected. The assay demonstrated good stability (RSD≤2.95%, within 10 days), reliable reproducibility (RSD = 2.96%, n = 7) and a desirable linear dynamic range from 78 nmol/mL to 10 μmol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.

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Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

Cited by 189 publications

References 90 publications

Glycans Function as Anchors for Antibodies and Help Drive HIV Broadly Neutralizing Antibody Development

Glycans Function as Anchors for Antibodies and Help Drive HIV Broadly Neutralizing Antibody Development

Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans

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