Enabling Lipidomic Biomarker Studies for Protected Populations by Combining Noninvasive Fingerprint Sampling with MS Analysis and Machine Learning.

Triacylglycerols and wax esters are two lipid classes that have been linked to diseases, including autism, Alzheimer's disease, dementia, cardiovascular disease, dry eye disease, and diabetes, and thus are molecules worthy of biomarker exploration studies. Since triacylglycerols and wax esters make up the majority of skin-surface lipid secretions, a viable sampling method for these potential biomarkers would be that of groomed latent fingerprints. Currently, however, blood-based sampling protocols predominate in the field. The invasiveness of a blood draw limits its utility to protected populations, including children and the elderly. Herein we describe a noninvasive means for sample collection (from fingerprints) paired with fast MS data-acquisition (MassIVE data set MSV000092742) and efficient data analysis via machine learning. Using both supervised and unsupervised classification, we demonstrate the usefulness of this method in determining whether a variable of interest imparts measurable change within the lipidomic data set. As a proof-of-concept, we show that the method is capable of distinguishing between the fingerprints of different individuals as well as between anatomical sebum collection regions. This noninvasive, high-throughput approach enables future lipidomic biomarker researchers to more easily include underrepresented, protected populations, such as children and the elderly, thus moving the field closer to definitive disease diagnoses that apply to all.

Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein.

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E, and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer.

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.

Mass Spectrometry-Based Disulfide Mapping of Lysyl Oxidase-like 2.

Lysyl oxidase-like 2 (LOXL2) catalyzes the oxidative deamination of peptidyl lysines and hydroxylysines to promote extracellular matrix remodeling. Aberrant activity of LOXL2 has been associated with organ fibrosis and tumor metastasis. The lysine tyrosylquinone (LTQ) cofactor is derived from Lys653 and Tyr689 in the amine oxidase domain via post-translational modification. Based on the similarity in hydrodynamic radius and radius of gyration, we recently proposed that the overall structures of the mature LOXL2 (containing LTQ) and the precursor LOXL2 (no LTQ) are very similar. In this study, we conducted a mass spectrometry-based disulfide mapping analysis of recombinant LOXL2 in three forms: a full-length LOXL2 (fl-LOXL2) containing a nearly stoichiometric amount of LTQ, Δ1-2SRCR-LOXL2 (SRCR1 and SRCR2 are truncated) in the precursor form, and Δ1-3SRCR-LOXL2 (SRCR1, SRCR2, SRCR3 are truncated) in a mixture of the precursor and the mature forms. We detected a set of five disulfide bonds that is conserved in both the precursor and the mature recombinant LOXL2s. In addition, we detected a set of four alternative disulfide bonds in low abundance that is not associated with the mature LOXL2. These results suggest that the major set of five disulfide bonds is retained post-LTQ formation.

Antigenicity and Immunogenicity of HIV-1 Envelope Trimers Complexed to a Small-Molecule Viral Entry Inhibitor.

Small-molecule viral entry inhibitors, such as BMS-626529 (BMS-529), allosterically block CD4 binding to HIV-1 envelope (Env) and inhibit CD4-induced structural changes in Env trimers. Here, we show that the binding of BMS-529 to clade C soluble chimeric gp140 SOSIP (ch.SOSIP) and membrane-bound trimers with intact transmembrane domain (gp150) prevented trimer conformational transitions and enhanced their immunogenicity. When complexed to BMS-529, ch.SOSIP trimers retained their binding to broadly neutralizing antibodies (bNAbs) and to their unmutated common ancestor (UCA) antibodies, while exposure of CD4-induced (CD4i) non-bNAb epitopes was inhibited. BMS-529-complexed gp150 trimers in detergent micelles, which were isolated from CHO cells, bound to bNAbs, including UCA and intermediates of the CD4 binding site (bs) CH103 bNAb lineage, and showed limited exposure of CD4i epitopes and a glycosylation pattern with a preponderance of high-mannose glycans. In rabbits, BMS-529-complexed V3 glycan-targeting ch.SOSIP immunogen induced in the majority of immunized animals higher neutralization titers against both autologous and select high mannose-bearing heterologous tier 2 pseudoviruses than those immunized with the noncomplexed ch.SOSIP. In rhesus macaques, BMS-529 complexed to CD4 bs-targeting ch.SOSIP immunogen induced stronger neutralization against tier 2 pseudoviruses bearing high-mannose glycans than noncomplexed ch.SOSIP trimer immunogen. When immunized with gp150 complexed to BMS-529, rhesus macaques showed neutralization against tier 2 pseudoviruses with targeted glycan deletion and high-mannose glycan enrichment. These results demonstrated that stabilization of Env trimer conformation with BMS-529 improved the immunogenicity of select chimeric SOSIP trimers and elicited tier 2 neutralizing antibodies of higher potency than noncomplexed trimers.IMPORTANCE Soluble forms of HIV-1 envelope trimers exhibit conformational heterogeneity and undergo CD4-induced (CD4i) exposure of epitopes of non-neutralizing antibodies that can potentially hinder induction of broad neutralizing antibody responses. These limitations have been mitigated through recent structure-guided approaches and include trimer-stabilizing mutations that resist trimer conformational transition and exposure of CD4i epitopes. The use of small-molecule viral inhibitors that allosterically block CD4 binding represents an alternative strategy for stabilizing Env trimer in the pre-CD4-triggered state of both soluble and membrane-bound trimers. In this study, we report that the viral entry inhibitor BMS-626529 restricts trimer conformational transition and improves the immunogenicity of select Env trimer immunogens.

High level stable expression of recombinant HIV gp120 in glutamine synthetase gene deficient HEK293T cells.

Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.

The opportunity cost of automated glycopeptide analysis: case study profiling the SARS-CoV-2 S glycoprotein.

Glycosylation analysis of viral glycoproteins contributes significantly to vaccine design and development. Among other benefits, glycosylation analysis allows vaccine developers to assess the impact of construct design or producer cell line choices for vaccine production, and it is a key measure by which glycoproteins that are produced for use in vaccination can be compared to their native viral forms. Because many viral glycoproteins are multiply glycosylated, glycopeptide analysis is a preferrable approach for mapping the glycans, yet the analysis of glycopeptide data can be cumbersome and requires the expertise of an experienced analyst. In recent years, a commercial software product, Byonic, has been implemented in several instances to facilitate glycopeptide analysis on viral glycoproteins and other glycoproteomics data sets, and the purpose of the study herein is to determine the strengths and limitations of using this software, particularly in cases relevant to vaccine development. The glycopeptides from a recombinantly expressed trimeric S glycoprotein of the SARS-CoV-2 virus were first analyzed using an expert-based analysis strategy; subsequently, analysis of the same data set was completed using Byonic. Careful assessment of instances where the two methods produced different results revealed that the glycopeptide assignments from Byonic contained more false positives than true positives, even when the data were assessed using a 1% false discovery rate. The work herein provides a roadmap for removing the spurious assignments that Byonic generates, and it provides an assessment of the opportunity cost for relying on automated assignments for glycopeptide data sets from viral glycoproteins.

Stabilization of the V2 loop improves the presentation of V2 loop-associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers.

A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development.

The Aristotle Classifier: Using the Whole Glycomic Profile To Indicate a Disease State.

"The totality is not, as it were, a mere heap, but the whole is something besides the parts."-Aristotle. We built a classifier that uses the totality of the glycomic profile, not restricted to a few glycoforms, to differentiate samples from two different sources. This approach, which relies on using thousands of features, is a radical departure from current strategies, where most of the glycomic profile is ignored in favor of selecting a few features, or even a single feature, meant to capture the differences in sample types. The classifier can be used to differentiate the source of the material; applicable sources may be different species of animals, different protein production methods, or, most importantly, different biological states (disease vs healthy). The classifier can be used on glycomic data in any form, including derivatized monosaccharides, intact glycans, or glycopeptides. It takes advantage of the fact that changing the source material can cause a change in the glycomic profile in many subtle ways: some glycoforms can be upregulated, some downregulated, some may appear unchanged, yet their proportion-with respect to other forms present-can be altered to a detectable degree. By classifying samples using the entirety of their glycan abundances, along with the glycans' relative proportions to each other, the "Aristotle Classifier" is more effective at capturing the underlying trends than standard classification procedures used in glycomics, including PCA (principal components analysis). It also outperforms workflows where a single, representative glycomic-based biomarker is used to classify samples. We describe the Aristotle Classifier and provide several examples of its utility for biomarker studies and other classification problems using glycomic data from several sources.

Recombinant Human Lysyl Oxidase-like 2 Secreted from Human Embryonic Kidney Cells Displays Complex and Acidic Glycans at All Three N-Linked Glycosylation Sites.

Human lysyl oxidase-like 2 (hLOXL2), a glycoprotein implicated in tumor progression and organ fibrosis, is a molecular target for anticancer and antifibrosis treatment. This glycoprotein contains three predicted N-linked glycosylation sites; one is near the protein's active site, and at least one more is known to facilitate the protein's secretion. Because the glycosylation impacts the protein's biology, we sought to characterize the native, mammalian glycosylation profile and to determine how closely this profile is recapitulated when the protein is expressed in insect cells. All three glycosylation sites on the protein, expressed in human embryonic kidney (HEK) cells, were characterized individually using a mass spectrometry-based glycopeptide analysis workflow. These data were compared to the glycosylation profile of the same protein expressed in insect cells. We found that the producer cell type imparts a substantial influence on the glycosylation of this important protein. The more-relevant version, expressed in HEK cells, contains large, acidic glycoforms; these glycans are not generated in insect cells. The glycosylation differences likely have structural and functional consequences, and these data should be considered when generating protein for functional studies or for high-throughput screening campaigns.

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers.

HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV-1 envelope glycoprotein vaccine candidate.

We describe the properties of BG505 SOSIP.664 HIV-1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native-like trimers that are the basis for many structure-guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV-1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum-free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size-exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10 . The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native-like as judged by negative-stain electron microscopy, and were stable over a multi-month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native-like Env glycoprotein trimers of various designs and genotypes.

Two New Tools for Glycopeptide Analysis Researchers: A Glycopeptide Decoy Generator and a Large Data Set of Assigned CID Spectra of Glycopeptides.

The glycopeptide analysis field is tightly constrained by a lack of effective tools that translate mass spectrometry data into meaningful chemical information, and perhaps the most challenging aspect of building effective glycopeptide analysis software is designing an accurate scoring algorithm for MS/MS data. We provide the glycoproteomics community with two tools to address this challenge. The first tool, a curated set of 100 expert-assigned CID spectra of glycopeptides, contains a diverse set of spectra from a variety of glycan types; the second tool, Glycopeptide Decoy Generator, is a new software application that generates glycopeptide decoys de novo. We developed these tools so that emerging methods of assigning glycopeptides' CID spectra could be rigorously tested. Software developers or those interested in developing skills in expert (manual) analysis can use these tools to facilitate their work. We demonstrate the tools' utility in assessing the quality of one particular glycopeptide software package, GlycoPep Grader, which assigns glycopeptides to CID spectra. We first acquired the set of 100 expert assigned CID spectra; then, we used the Decoy Generator (described herein) to generate 20 decoys per target glycopeptide. The assigned spectra and decoys were used to test the accuracy of GlycoPep Grader's scoring algorithm; new strengths and weaknesses were identified in the algorithm using this approach. Both newly developed tools are freely available. The software can be downloaded at http://glycopro.chem.ku.edu/GPJ.jar.

GlycoPep MassList: software to generate massive inclusion lists for glycopeptide analyses.

Protein glycosylation drives many biological processes and serves as markers for disease; therefore, the development of tools to study glycosylation is an essential and growing area of research. Mass spectrometry can be used to identify both the glycans of interest and the glycosylation sites to which those glycans are attached, when proteins are proteolytically digested and their glycopeptides are analyzed by a combination of high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) methods. One major challenge in these experiments is collecting the requisite MS/MS data. The digested glycopeptides are often present in complex mixtures and in low abundance, and the most commonly used approach to collect MS/MS data on these species is data-dependent acquisition (DDA), where only the most intense precursor ions trigger MS/MS. DDA results in limited glycopeptide coverage. Semi-targeted data acquisition is an alternative experimental approach that can alleviate this difficulty. However, due to the massive heterogeneity of glycopeptides, it is not obvious how to expediently generate inclusion lists for these types of analyses. To solve this problem, we developed the software tool GlycoPep MassList, which can be used to generate inclusion lists for liquid chromatography tandem-mass spectrometry (LC-MS/MS) experiments. The utility of the software was tested by conducting comparisons between semi-targeted and untargeted data-dependent analysis experiments on a variety of proteins, including IgG, a protein whose glycosylation must be characterized during its production as a biotherapeutic. When the GlycoPep MassList software was used to generate inclusion lists for LC-MS/MS experiments, more unique glycopeptides were selected for fragmentation. Generally, ∼30 % more unique glycopeptides can be analyzed per protein, in the simplest cases, with low background. In cases where background ions from proteins or other interferents are high, usage of an inclusion list is even more advantageous. The software is freely publically accessible. Graphical abstract Software increases the number of glycopeptides that get selected for MS/MS analysis.

Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins.

UNLABELLED: We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds. IMPORTANCE: It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140.

UNLABELLED: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140. IMPORTANCE: A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers.

UNLABELLED: We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCE: Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Glycosylation and disulfide bond analysis of transiently and stably expressed clade C HIV-1 gp140 trimers in 293T cells identifies disulfide heterogeneity present in both proteins and differences in O-linked glycosylation.

The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV vaccine is the presence of the complex set of post-translational modifications (PTMs) on Env, namely, glycosylation and disulfide bonds, that affect protein folding, epitope accessibility, and immunogenecity. Although these PTMs vary with expression systems, variations in Env's PTMs due to changes in the expression method are not yet well established. In this study, we compared the disulfide bond network and glycosylation profiles of clade C recombinant HIV-1 Env trimers, C97ZA012 gp140, expressed by stable and transient transfections using an integrated mass mapping workflow that combines collision induced dissociation (CID) and electron transfer dissociation (ETD). Site-specific analysis of the N- and O-glycosylation profiles revealed that C97ZA012 gp140 produced by both transfection methods displayed a high degree of similarity in N-glycosylation profiles and site occupancy except for one site. By contrast, different O-glycosylation profiles were detected. Analysis of the disulfide bond networks of the Env revealed that both transfection methods yielded C97ZA012 gp140 adopting the expected disulfide bond pattern identified for the monomeric gp120 and gp41 as well as alternative disulfide bond patterns in the C1, V1/V2, and C2 regions. The finding that disulfide bonding is consistently heterogeneous in these proteins is perhaps the most significant outcome of these studies; this disulfide heterogeneity has been reported for multiple other recombinant gp140s, and it is likely present in most recombinantly expressed Env immunogens.

Post-translational modifications of recombinant human lysyl oxidase-like 2 (rhLOXL2) secreted from Drosophila S2 cells.

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2-6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.