The retinoid X receptor has a critical role in synthetic rexinoid-induced increase in cellular all-trans-retinoic acid.

Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.

Current Understanding of Complement Proteins as Therapeutic Targets for the Treatment of Immunoglobulin A Nephropathy.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and a frequent cause of kidney failure. Currently, the diagnosis necessitates a kidney biopsy, with routine immunofluorescence microscopy revealing IgA as the dominant or co-dominant immunoglobulin in the glomerular immuno-deposits, often with IgG and sometimes IgM or both. Complement protein C3 is observed in most cases. IgAN leads to kidney failure in 20-40% of patients within 20 years of diagnosis and reduces average life expectancy by about 10 years. There is increasing clinical, biochemical, and genetic evidence that the complement system plays a paramount role in the pathogenesis of IgAN. The presence of C3 in the kidney immuno-deposits differentiates the diagnosis of IgAN from subclinical glomerular mesangial IgA deposition. Markers of complement activation via the lectin and alternative pathways in kidney-biopsy specimens are associated with disease activity and are predictive of poor outcome. Levels of select complement proteins in the circulation have also been assessed in patients with IgAN and found to be of prognostic value. Ongoing genetic studies have identified at least 30 loci associated with IgAN. Genes within some of these loci encode complement-system regulating proteins that can interact with immune complexes. The growing appreciation for the central role of complement components in IgAN pathogenesis highlighted these pathways as potential treatment targets and sparked great interest in pharmacological agents targeting the complement cascade for the treatment of IgAN, as evidenced by the plethora of ongoing clinical trials.

Soluble prefusion-closed HIV-envelope trimers with glycan-covered bases.

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N-linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers -even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response.

Next-generation retinoid X receptor agonists increase ATRA signaling in organotypic epithelium cultures and have distinct effects on receptor dynamics.

Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 µM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.

Conformationally Defined Rexinoids for the Prevention of Inflammation and Nonmelanoma Skin Cancers.

Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.

Racial heterogeneity of IgA1 hinge-region O-glycoforms in patients with IgA nephropathy.

Galactose (Gal)-deficient IgA1 (Gd-IgA1) is involved in IgA nephropathy (IgAN) pathogenesis. To reflect racial differences in clinical characteristics, we assessed disease- and race-specific heterogeneity in the O-glycosylation of the IgA1 hinge region (HR). We determined serum Gd-IgA1 levels in Caucasians (healthy controls [HCs], n = 31; IgAN patients, n = 63) and Asians (HCs, n = 20; IgAN patients, n = 60) and analyzed profiles of serum IgA1 HR O-glycoforms. Elevated serum Gd-IgA1 levels and reduced number of Gal residues per HR were observed in Caucasians. Reduced number of N-acetylgalactosamine (GalNAc) residues per HR and elevated relative abundance of IgA1 with three HR O-glycans were common features in IgAN patients; these features were associated with elevated blood pressure and reduced renal function. We speculate that the mechanisms underlying the reduced GalNAc content in IgA1 HR may be relevant to IgAN pathogenesis.

Immunoglobulin A Glycosylation and Its Role in Disease.

Human IgA is comprised of two subclasses, IgA1 and IgA2. Monomeric IgA (mIgA), polymeric IgA (pIgA), and secretory IgA (SIgA) are the main molecular forms of IgA. The production of IgA rivals all other immunoglobulin isotypes. The large quantities of IgA reflect the fundamental roles it plays in immune defense, protecting vulnerable mucosal surfaces against invading pathogens. SIgA dominates mucosal surfaces, whereas IgA in circulation is predominately monomeric. All forms of IgA are glycosylated, and the glycans significantly influence its various roles, including antigen binding and the antibody effector functions, mediated by the Fab and Fc portions, respectively. In contrast to its protective role, the aberrant glycosylation of IgA1 has been implicated in the pathogenesis of autoimmune diseases, such as IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN). Furthermore, detailed characterization of IgA glycosylation, including its diverse range of heterogeneity, is of emerging interest. We provide an overview of the glycosylation observed for each subclass and molecular form of IgA as well as the range of heterogeneity for each site of glycosylation. In many ways, the role of IgA glycosylation is in its early stages of being elucidated. This chapter provides an overview of the current knowledge and research directions.

Aberrantly Glycosylated IgA1 in IgA Nephropathy: What We Know and What We Don't Know.

IgA nephropathy (IgAN), the most common primary glomerular disease worldwide, is characterized by glomerular deposition of IgA1-containing immune complexes. The IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine usually with ß1,3-linked galactose and variable sialylation. Circulating levels of IgA1 with abnormally O-glycosylated HR, termed galactose-deficient IgA1 (Gd-IgA1), are increased in patients with IgAN. Current evidence suggests that IgAN is induced by multiple sequential pathogenic steps, and production of aberrantly glycosylated IgA1 is considered the initial step. Thus, the mechanisms of biosynthesis of aberrantly glycosylated IgA1 and the involvement of aberrant glycoforms of IgA1 in disease development have been studied. Furthermore, Gd-IgA1 represents an attractive biomarker for IgAN, and its clinical significance is still being evaluated. To elucidate the pathogenesis of IgAN, it is important to deconvolute the biosynthetic origins of Gd-IgA1 and characterize the pathogenic IgA1 HR O-glycoform(s), including the glycan structures and their sites of attachment. These efforts will likely lead to development of new biomarkers. Here, we review the IgA1 HR O-glycosylation in general and the role of aberrantly glycosylated IgA1 in the pathogenesis of IgAN in particular.

Stability of the Retinoid X Receptor-α Homodimer in the Presence and Absence of Rexinoid and Coactivator Peptide.

Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-α ligand binding domain (RXRα LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXRα LBD homodimer displayed a single thermal unfolding transition with a Tm of 58.7 °C and an unfolding enthalpy (ΔH) of 673 kJ/mol (12.5 J/g), much lower than average value (35 J/g) of small globular proteins. Using a heat capacity change (ΔCp) of 15 kJ/(mol K) determined by measurements at different pH values, the free energy of unfolding (ΔG) of the native state was 33 kJ/mol at 37 °C. Rexinoid binding to the apo-homodimer increased Tm by 5 to 9 °C and increased the ΔG of the native homodimer by 12 to 20 kJ/mol at 37 °C, consistent with the nanomolar dissociation constant (Kd) of the rexinoids. GRIP-1 binding to holo-homodimers containing rexinoid resulted in additional increases in ΔG of 14 kJ/mol, a value that was the same for all three rexinoids. Binding of rexinoid and GRIP-1 resulted in a combined 50% increase in unfolding enthalpy, consistent with reduced structural fluidity and more compact folding observed in other published structural studies. The complexes of UAB110 and UAB111 are each more stable than the UAB30 complex by 8 kJ/mol due to enhanced hydrophobic interactions in the binding pocket because of their larger end groups. This increase in thermodynamic stability positively correlates with their improved RXR activation potency. Thermodynamic measurements are thus valuable in predicting agonist potency.

Quantitative assessment of successive carbohydrate additions to the clustered O-glycosylation sites of IgA1 by glycosyltransferases.

Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or immunoglobulin A1 (IgA1). Here, we describe a series of nano-liquid chromatography (LC)-mass spectrometry (MS) analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgA1. By utilizing nano-LC-MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgA1 O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrasnfersase (GalNAc-T EC 2.4.1.41) and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and α2,3- and α2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgA1, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgA1 O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.

Glycan Positioning Impacts HIV-1 Env Glycan-Shield Density, Function, and Recognition by Antibodies.

HIV-1 envelope (Env) N-glycosylation impact virus-cell entry and immune evasion. How each glycan interacts to shape the Env-protein-sugar complex and affects Env function is not well understood. Here, analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation sites affected the Env structure at distant locations and had a ripple effect on Env-wide glycan processing, virus infectivity, antibody recognition, and virus neutralization. Specifically, the N262 glycan, although not in the CD4-binding site, modulated Env binding to the CD4 receptor, affected Env recognition by several glycan-dependent neutralizing antibodies, and altered site-specific glycosylation heterogeneity, with, for example, N448 displaying limited glycan processing. Molecular-dynamic simulations visualized differences in glycan density and how specific oligosaccharide positions can move to compensate for a glycan loss. This study demonstrates how changes in individual glycans can alter molecular dynamics, processing, and function of the Env-glycan shield.

Mass spectrometry for the identification and analysis of highly complex glycosylation of therapeutic or pathogenic proteins.

INTRODUCTION: Protein glycosylation influences characteristics such as folding, stability, protein interactions, and solubility. Therefore, glycan moieties of therapeutic proteins and proteins that are likely associated with disease pathogenesis should be analyzed in-depth, including glycan heterogeneity and modification sites. Recent advances in analytical methods and instrumentation have enabled comprehensive characterization of highly complex glycosylated proteins. AREA COVERED: The following aspects should be considered when analyzing glycosylated proteins: sample preparation, chromatographic separation, mass spectrometry (MS) and fragmentation methods, and bioinformatics, such as software solutions for data analyses. Notably, analysis of glycoproteins with heavily sialylated glycans or multiple glycosylation sites requires special considerations. Here, we discuss recent methodological advances in MS that provide detailed characterization of heterogeneous glycoproteins. EXPERT OPINION: As characterization of complex glycosylated proteins is still analytically challenging, the function or pathophysiological significance of these proteins is not fully understood. To reproducibly produce desired forms of therapeutic glycoproteins or to fully elucidate disease-specific patterns of protein glycosylation, a highly reproducible and robust analytical platform(s) should be established. In addition to advances in MS instrumentation, optimization of analytical and bioinformatics methods and utilization of glycoprotein/glycopeptide standards is desirable. Ultimately, we envision that an automated high-throughput MS analysis will provide additional power to clinical studies and precision medicine.

Analysis of O-glycoforms of the IgA1 hinge region by sequential deglycosylation.

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with ß1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T236, followed by S230, T233, T228, and S232. The new workflow for quantitative profiling of IgA1 HR O-glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O-glycoforms in IgAN.

Glycosylation of viral surface proteins probed by mass spectrometry.

Glycosylation is a common and biologically significant post-translational modification that is found on numerous virus surface proteins (VSPs). Many of these glycans affect virulence through modulating virus receptor binding, masking antigenic sites, or by stimulating the host immune response. Mass spectrometry (MS) has arisen as a pivotal technique for the characterization of VSP glycosylation. This review will cover how MS-based analyses, such as released glycan profiles, glycan site localization, site-occupancy, and site-specific heterogeneity, are being utilized to map VSP glycosylation. Furthermore, this review will provide information on how MS glycoprofiling data are being used in conjunction with molecular and structural experiments to provide a better understanding of the role of specific glycans in VSP function.

Glycosylation in health and disease.

The glycome describes the complete repertoire of glycoconjugates composed of carbohydrate chains, or glycans, that are covalently linked to lipid or protein molecules. Glycoconjugates are formed through a process called glycosylation and can differ in their glycan sequences, the connections between them and their length. Glycoconjugate synthesis is a dynamic process that depends on the local milieu of enzymes, sugar precursors and organelle structures as well as the cell types involved and cellular signals. Studies of rare genetic disorders that affect glycosylation first highlighted the biological importance of the glycome, and technological advances have improved our understanding of its heterogeneity and complexity. Researchers can now routinely assess how the secreted and cell-surface glycomes reflect overall cellular status in health and disease. In fact, changes in glycosylation can modulate inflammatory responses, enable viral immune escape, promote cancer cell metastasis or regulate apoptosis; the composition of the glycome also affects kidney function in health and disease. New insights into the structure and function of the glycome can now be applied to therapy development and could improve our ability to fine-tune immunological responses and inflammation, optimize the performance of therapeutic antibodies and boost immune responses to cancer. These examples illustrate the potential of the emerging field of 'glycomedicine'.

Pyrazolopyrimidinones, a novel class of copper-dependent bactericidal antibiotics against multi-drug resistant S. aureus.

The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections poses a therapeutic challenge as even last resort drugs become increasingly ineffective. As the demand for antibiotics with novel modes of action is growing, new approaches are needed to probe a greater spectrum of antimicrobial activities for their potential efficacy against drug-resistant pathogens. The use of copper (Cu) by the innate immune system to mount an antimicrobial response against bacterial invaders has created an opportunity to explore a role for Cu in antimicrobial therapy. Here we describe pyrazolopyrimidinones (PZP) as novel copper-dependent inhibitors (CDI) of S. aureus. 5-Benzyl-3-(4-chlorophenyl)-2-methyl-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one (PZP-915) showed potent bactericidal properties at sub-micromolar concentrations and activity against clinical MRSA isolates and biofilms cultures. This cupricidal activity is founded on the molecule's ability to coordinate Cu and induce accumulation of Cu ions inside S. aureus cells. We demonstrate that exposure to 915 + Cu led to an almost instantaneous collapse of the membrane potential which was accompanied by a complete depletion of cellular ATP, loss of cell-associated K+, a substantial gain of cell associated Na+, and an inability to control the influx of protons in slightly acidic medium, while the integrity of the cell membrane remained intact. These findings highlight PZP-915 as a novel membrane-directed metalloantibiotic against S. aureus that is likely to target a multiplicity of membrane associated protein functions rather than imposing physical damage to the membrane structure.