Glycosylated Form Research Articles

Effective and Selective Extraction of Quercetin from Onion (Allium cepa L.) Skin Waste Using Water Dilutions of Acid-Based Deep Eutectic Solvents.

Deep Eutectic Solvents (DESs) are experiencing growing interest as substitutes of polluting organic solvents for their low or absent toxicity and volatility. Moreover, they can be formed with natural bioavailable and biodegradable molecules; they are synthesized in absence of hazardous solvents. DESs are, inter alia, successfully used for the extraction/preconcentration of biofunctional molecules from complex vegetal matrices. Onion skin is a highly abundant waste material which represents a reservoir of molecules endowed with valuable biological properties such as quercetin and its glycosylated forms. An efficient extraction of these molecules from dry onion skin from “Dorata di Parma” cultivar was obtained with water dilution of acid-based DESs. Glycolic acid (with betaine 2/1 molar ratio and L-Proline 3/1 molar ratio as counterparts) and of p-toluensulphonic acid (with benzyltrimethylammonium methanesulfonate 1/1 molar ratio)-based DESs exhibited more than 3-fold higher extraction efficiency than methanol (14.79 µg/mL, 18.56 µg/mL, 14.83 µg/mL vs. 5.84 µg/mL, respectively). The extracted quercetin was also recovered efficaciously (81% of recovery) from the original extraction mixture. The proposed extraction protocol revealed to be green, efficacious and selective for the extraction of quercetin from onion skin and it could be useful for the development of other extraction procedures from other biological matrixes.

Human Brain and Blood N-Glycome Profiling in Alzheimer's Disease and Alzheimer's Disease-Related Dementias.

Glycosylation, the process of adding glycans (i.e., sugars) to proteins, is the most abundant post-translational modification. N-glycosylation is the most common form of glycosylation, and the N-glycan moieties play key roles in regulating protein functions and many other biological processes. Thus, identification and quantification of N-glycome (complete repertoire of all N-glycans in a sample) may provide new sources of biomarkers and shed light on health and disease. To date, little is known about the role of altered N-glycome in Alzheimer’s Disease and Alzheimer’s Disease-related Dementias (AD/ADRD). The current study included 45 older adults who had no cognitive impairment (NCI) at baseline, followed and examined annually, and underwent brain autopsy after death. During about 12-year follow-up, 15 developed mild cognitive impairment (MCI), 15 developed AD, and 15 remained NCI. Relative abundances of N-glycans in serum at 2 time points (baseline and proximate to death, ∼12.3 years apart) and postmortem brain tissue (dorsolateral prefrontal cortex) were quantified using MALDI-TOF-MS. Regression models were used to test the associations of N-glycans with AD/ADRD phenotypes. We detected 71 serum and 141 brain N-glycans, of which 46 were in common. Most serum N-glycans had mean fold changes less than one between baseline and proximate to death. The cross-tissue N-glycan correlations were weak. Baseline serum N-glycans were more strongly associated with AD/ADRD compared to change in serum N-glycans over time and brain N-glycans. The N-glycan associations were observed in both AD and non-AD neuropathologies. To our knowledge, this is the first comprehensive glycomic analysis in both blood and brain in relation to AD pathology. Our results suggest that altered N-glycans may serve as mechanistic biomarkers for early diagnosis and progression of AD/ADRD.

Distribution of seven ApoC-III glycoforms in plasma, VLDL, IDL, LDL and HDL of healthy subjects

Glycosylation of ApoC-III modulates its function in TG metabolism, with some variants being associated with a more atherogenic lipid profile. These associations have been studied in whole plasma but rarely in individual lipoprotein fractions. In this study, we aimed to measure the relative content of ApoC-III glycoforms in each lipoprotein fraction as a potential biomarker for TG metabolism and cardiovascular risk. Lipoprotein fractions were separated by differential ultracentrifugation of plasma samples from healthy subjects. Relative concentrations of seven ApoC-III variants were measured by MSIA. ApoC-III1, ApoC-III0b and ApoC-III2 were the most abundant glycoforms. There was high interindividual variability in the distribution of glycoforms across the study population but a uniform proportion in all lipoprotein fractions of each given subject. Two ApoC-III variants, ApoC-III0b and ApoC-III1d, negatively correlated with plasma and VLDL triglycerides irrespectively of VLDL size and were associated with increased LDL size when transported in LDL particles. ApoC-III0b also showed a negative correlation with lipoprotein-insulin resistance score. We have been able to measure seven ApoC-III glycoforms in each lipoprotein fraction, setting the basis for future studies exploring their role on cardiovascular risk. Some glycoforms suggest a less proatherogenic role on TG and lipoprotein metabolism. SignificanceApo CIII has an important role on plasma TG metabolism through different mechanisms and it is also involved in type 1 and type 2 Diabetes Mellitus. Different glycosylated forms of Apo CIII exist and they show different roles. For this reason, this protein has gained interest in the las years and the relationship between ApoC-III glycoforms and lipids, lipoproteins and metabolic disorders has been increasingly studied in the last years.Apo CIII glycoforms have been previously analysed in plasma, and the function of the main four glycoforms has been assessed in a variety of cohorts; but in the present study, ApoC-III glycoforms are measured in each lipoprotein fraction, which may be of clinical interest.

Caffeate may play an important role in the somatic embryogenesis of oil palm (Elaeis guineensis Jacq.)

Somatic embryogenesis (SE) is a key step in plant regeneration and propagation, but the mechanisms underlying SE in oil palm still stay unclear. Integrated analysis of metabolome and transcriptome data showed that 13 metabolites are specifically accumulated in somatic embryo, of which caffeate was 16.6-fold (p = 0.0002) higher than in callus. Moreover, caffeate was significantly correlated with phytohormone signaling genes including ethylene receptor (ETR2-2), auxin efflux carrier component 3a (PIN3A), auxin response factor (ARF15-2), and several SE transcription factors (TFs) including WUSCHEL-related homeobox (WOX), VIVIPAROUS1(VP1), BABY BOOM2 (BBM2). Furthermore, staining and microscopic observation showed that phenolic acids highly accumulated in the center of newly formed callus clusters and lignin is predominant in somatic embryo than in callus, indicated that caffeate (the major phenolic acids in glycosylated form and the precursor of lignin biosynthesis) may play an important role in SE. Our results provide a better understanding of the SE mechanism in oil palm.

Metabolic Labeling of Primary Neurons Using Carbohydrate Click Chemistry.

Glycans play an important role in many neuronal processes, such as neurotransmitter release and reuptake, cell-cell communication and adhesion, modulation of ion channel activity, and immune function. Carbohydrate click chemistry is a powerful technique for studying glycan function and dynamics in vitro, in vivo, and ex vivo. Here, we use commercially available synthetic tetraacetylated azido sugars, copper and copper-free click chemistry to metabolically label and analyze primary rat cortical neurons. In addition, we use high resolution confocal and STED microscopy to image and analyze different forms of glycosylation in ultrahigh resolution. We observe different patterns of GlcNAz, GalNAz, and ManNAz distribution at different stages of neuronal development. We also observe highly sialylated structureson the neuronal plasma membrane, which warrant further investigation.

A Stable Gold Nanoparticle-Based Vaccine for the Targeted Delivery of Tumor-Associated Glycopeptide Antigens.

We have developed a novel antigen delivery system based on polysaccharide-coated gold nanoparticles (AuNPs) targeted to antigen-presenting cells (APCs) expressing Dectin-1. AuNPs were synthesized de novo using yeast-derived β-1,3-glucans (B13G) as the reductant and passivating agent in a microwave-catalyzed procedure, yielding highly uniform and serum-stable particles. These were further functionalized with both a peptide and a specific glycosylated form from the tandem repeat sequence of mucin 4 (MUC4), a glycoprotein overexpressed in pancreatic tumors. The glycosylated sequence contained the Thomsen–Friedenreich disaccharide, a pan-carcinoma, tumor-associated carbohydrate antigen (TACA), which has been a traditional target for antitumor vaccine design. These motifs were prepared with a cathepsin B protease cleavage site (Gly-Phe-Leu-Gly), loaded on the B13G-coated particles, and these constructs were examined for Dectin-1 binding, APC processing, and presentation in a model in vitro system and for immune responses in mice. We showed that these particles elicit strong in vivo immune responses through the production of both high-titer antibodies and priming of antigen-recognizing T-cells. Further examination showed that a favorable antitumor balance of expressed cytokines was generated, with limited expression of immunosuppressive Il-10. This system is modular in that any range of antigens can be conjugated to our particles and efficiently delivered to APCs expressing Dectin-1.

JS01.3.A Oncogenic chaperoning of Hsp90 in glioma with FGFR3-TACC3

Abstract BACKGROUND Fusion genes are chromosomal aberrations in malignancies that can be used as prognostic markers as well as therapeutic targets. The FGFR3-TACC3 (F3-T3) was initially discovered as an oncogenic molecule in glioblastoma and bladder cancer and subsequently found in many other cancer types. Based on clinical evidence, F3-T3 was found in glioblastoma patients before and after TMZ and radiotherapy treatment, suggesting that targeting F3-T3 is a valid strategy for glioblastoma treatment. MATERIAL AND METHODS We profiled the proteins that interacted with F3-T3 fusion protein in U-251 MG cells with F3-T3 through 2-D liquid chromatography-tandem mass spectrometry. To validate the result of proteomic analysis, we performed reverse immunoprecipitation by pulling down Hsp90 or Cdc37 in U-251 MG cells stably expressing F3-T3. To inhibit the association between F3-T3 and the Hsp90-Cdc37 complex, we treated U-251 MG and LN-229 cells stably expressing F3-T3 with Hsp90 inhibitors or siRNA of Cdc37. We applied the CCK8 assay to evaluate the sensitivity of glioblastoma cells stably expressing F3-T3, wild-type FGFR3, kinase-dead F3-T3 (K508R), and empty vectors to TMZ. Immunoblot and immunofluorescence staining were used to detect DNA damage marker pH2AX. The drug combination effect index was analyzed using software CalcuSyn. U-251 MG cells stably expressing F3-T3 infected with luciferase virus were intracranially injected in nude mice. The experimental group was administered with temozolomide (5mg/kg/day) by oral gavage, Hsp90 inhibitor Onalespib (30mg/kg/day) by tail vein injection or the combination of the two for indicated days. RESULTS We identified the proteins that showed increased binding ratios to F3-T3 over full-length FGFR3, the molecular chaperone proteins encoded by the genes HSP90AB1, HSP90AA1, and CDC37 emerged as 5th, 6th, and 7th on the top ten list, showing an approximately 4-fold increase in normalized spectral counts. Using Hsp90 inhibitors or Cdc37 siRNA disrupted the formation of the F3-T3/Hsp90/Cdc37 complex. Disruption of Hsp90-Cdc37 chaperoning caused a ubiquitination-mediated degradation of the glycosylated form of F3-T3 and abrogated the maturation of nascent F3-T3, resulting in suppression of F3-T3 signaling pathways. Additionally, our results provide evidence that the F3-T3 signaling pathway confers drug resistance to TMZ induced DNA damage. However, the resistance of TMZ was disrupted in glioblastoma cells harboring kinase-dead F3-T3 (K508R). We also demonstrated Hsp90 inhibitor significantly sensitized glioblastoma cells harboring the F3-T3 fusion gene to TMZ treatment and improved survival of xenograft model bearing F3-T3 tumor in vivo. CONCLUSION F3-T3 is a strong Hsp90 client that shows strong addiction to the Hsp90-Cdc37 chaperone system. Combination therapy with Hsp90 inhibitor overcomes the TMZ resistance conferred by F3-T3.

Dissecting the Role of N-Glycan at N413 in Toll-like Receptor 3 via Molecular Dynamics Simulations.

Toll-like receptor 3 (TLR3) is an endosomal receptor involved in initiating immune responses upon viral infection by directly recognizing double-stranded RNA (dsRNA). As one of the most heavily glycosylated TLR family members, the role of glycan at N413 of TLR3 in ligand recognition has been in debate for decades. Herein, to investigate the role of glycans in TLR3, specifically at amino acid residue N413, molecular dynamic simulations were performed. The loop region of LRR12 (residues 323-355), which protrudes from the dsRNA binding TLR3 lateral surface was found to be vital for interacting with dsRNA via the formation of hydrogen bonds. The glycan at N413 not only prevented dsRNA from being exposed to the bulk water during the binding process but further stabilized dsRNA in the TLR3 binding site. When N413 was in the glycosylated form, the binding free energy of TLR3 interacting with dsRNA was significantly lower than that of TLR3 in the N413 unglycosylated form. Additionally, as the glycan at N413 functioned to alter the dynamics of the dsRNA binding process, its flexibility was meanwhile influenced by dsRNA. In all, these results demonstrate that the size, length, and branch of glycan at N413 affect the thermodynamics and dynamics of TLR3 recognition with dsRNA. This study further extends our understanding of the biological role of glycans in the innate immune recognition of dsRNA by TLR3 and provides a new perspective for modulating TLR3 function.

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins

BackgroundPlasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M−protein). Some patients have more than one identifiable M−protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M−proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M−protein, (2) an M−protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M−protein with light chain glycosylation, or (4) two distinct biclonal M−proteins. MethodsPatient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M−proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. ResultsEighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. ConclusionsWe demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M−proteins is useful for monitoring of patients with PCDs.

Selectively Deoxyfluorinated N-Acetyllactosamine Analogues as 19 F NMR Probes to Study Carbohydrate-Galectin Interactions.

Galectins are widely expressed galactose-binding lectins implied, for example, in immune regulation, metastatic spreading, and pathogen recognition. N-Acetyllactosamine (Galβ1-4GlcNAc, LacNAc) and its oligomeric or glycosylated forms are natural ligands of galectins. To probe substrate specificity and binding mode of galectins, we synthesized a complete series of six mono-deoxyfluorinated analogues of LacNAc, in which each hydroxyl has been selectively replaced by fluorine while the anomeric position has been protected as methyl β-glycoside. Initial evaluation of their binding to human galectin-1 and -3 by ELISA and 19 F NMR T2 -filter revealed that deoxyfluorination at C3, C4' and C6' completely abolished binding to galectin-1 but very weak binding to galectin-3 was still detectable. Moreover, deoxyfluorination of C2' caused an approximately 8-fold increase in the binding affinity towards galectin-1, whereas binding to galectin-3 was essentially not affected. Lipophilicity measurement revealed that deoxyfluorination at the Gal moiety affects log P very differently compared to deoxyfluorination at the GlcNAc moiety.

Facile Generation of Strained Peptidyl Thiolactones from Hydrazides and Its Application in Assembling MUC‐1 VNTR Oligomers†

Main observation and conclusionAn efficient method for the activation of C‐terminal 4‐mecaptoproline‐ or penicillamine‐containing peptide hydrazides in ligation reactions is reported herein. The corresponding peptide hydrazides can be readily prepared using solid‐phase peptide synthesis, and subsequently activated by acetylacetone (acac) without exogenous thiol additives. Strained peptidyl thiolactones could be the possible reactive intermediates that drastically accelerate the reaction rates at the sterically demanding proline and valine sites. This developed protocol allows for sequential peptide ligations in a one‐pot manner, and expedites the assembly of mucin 1 (MUC‐1) variable number tandem repeat (VNTR) trimers in various glycosylated forms.

O-Linked-N-Acetylglucosaminylation of the RNA-Binding Protein EWS N-Terminal Low Complexity Region Reduces Phase Separation and Enhances Condensate Dynamics.

Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. O-linked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O-GlcNAcylation reduces the phase separation propensity of the EWS N-terminal low complexity region (LCRN) under different conditions, including in the presence of the arginine- and glycine-rich RNA-binding domains (RBD). O-GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCRN condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCRN+RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCRN do not. In HeLa cells, EWS is less O-GlcNAcylated following OGT knockdown, which correlates with its increased accumulation in a filter retardation assay. Relative to the human proteome, O-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O-GlcNAcylation in regulation of biomolecular condensates.

Clitorienolactones and Isoflavonoids of Clitorea ternatea Roots Alleviate Stress-Like Symptoms in a Reserpine-Induced Zebrafish Model.

Clitorea ternatea has been used in Ayurvedic medicine as a brain stimulant to treat mental illnesses and mental functional disorders. In this study, the metabolite profiles of crude C. ternatea root extract (CTRE), ethyl acetate (EA), and 50% aqueous methanol (50% MeOH) fractions were investigated using ultrahigh-performance liquid chromatography–diode array detector–tandem mass spectrometry (UHPLC–DAD–MS/MS), while their effect on the stress-like behavior of zebrafish, pharmacologically induced with reserpine, was investigated. A total of 32 compounds were putatively identified, among which, a series of norneolignans, clitorienolactones, and various flavonoids (flavone, flavonol, isoflavone, and isoflavanone) was found to comprise the major constituents, particularly in the EA and 50% MeOH fractions. The clitorienolactones, presently unique to the species, were present in both the free and glycosylated forms in the roots. Both the EA and 50% MeOH fractions displayed moderate effects on the stress-induced zebrafish model, significantly decreasing freezing duration and elevating the total distance travelled and average velocity, 72 h post-treatment. The results of the present study provide further evidence that the basis for the use of C. ternatea roots in traditional medicine to alleviate brain-related conditions, such as stress and depression, is attributable to the presence of clitorienolactones and the isoflavonoidal constituents.

Understanding the Clinical Impact of MUC5AC Expression on Pancreatic Ductal Adenocarcinoma.

Simple SummaryManagement of pancreatic cancer is challenging as there are limited treatment options, and most cases are diagnosed at advanced stages. In addition, there are no dependable tests available to predict bad outcomes or treatment responses in current clinical practice. Here, we shed light on the available evidence on mucin, MUC5AC in predicting the outcome of pancreatic cancers. We also discuss variants of MUC5AC believed to have a role in the malignant transformation of pancreatic tissues. Mucin-5AC (MUC5AC) is a heavily glycosylated gel-forming secreted mucin with a reliable prognostic value when detected in multiple malignancies. It is highly prevalent (70%) in PDA and is nonexistent in normal pancreatic tissues. Retrospective studies on PDA tumor tissue (detected by immunohistochemistry or IHC)) have investigated the prognostic value of MUC5AC expression but were equivocal. Some studies associated it with poor outcomes (survival or pathological features such as lymph node disease, vascular/neural invasion in resected tumors), while others have concluded that it is a good prognostic marker. The examination of expression level threshold (5%, 10%, or 25%) and the detected region (apical vs. cytoplasmic) were variable among the studies. The maturation stage and glycoform of MUC5AC detected also differed with the Monoclonal antibody (Mab) employed for IHC. CLH2 detects less mature/less glycosylated versions while 45M1 or 21-1 detect mature/more glycosylated forms. Interestingly, aberrantly glycosylated variants of MUC5AC were detected using lectin assays (Wheat Germ Agglutinin-MUC5AC), and Mabs such as NPC-1C and PAM4 have are more specific to malignant pancreatic tissues. NPC-1C and PAM4 antibody reactive epitopes on MUC5AC are immunogenic and could represent specific changes on the native MUC5AC glycoprotein linked to carcinogenesis. It was never studied to predict treatment response.

C1GALT1 in health and disease.

O-linked glycosylation (O-glycosylation) and N-linked glycosylation (N-glycosylation) are the two most important forms of protein glycosylation, which is an important post-translational modification. The regulation of protein function involves numerous mechanisms, among which protein glycosylation is one of the most important. Core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) serves an important role in the regulation of O-glycosylation and is an essential enzyme for synthesizing the core 1 structure of mucin-type O-glycans. Furthermore, C1GALT1 serves a vital role in a number of biological functions, such as angiogenesis, platelet production and kidney development. Impaired C1GALT1 expression activity has been associated with different types of human diseases, including inflammatory or immune-mediated diseases, and cancer. O-glycosylation exists in normal tissues, as well as in tumor tissues. Previous studies have revealed that changes in the level of glycosyltransferase in different types of cancer may be used as potential therapeutic targets. Currently, numerous studies have reported the dual role of C1GALT1 in tumors (carcinogenesis and cancer suppression). The present review reports the role of C1GALT1 in normal development and human diseases. Since the mechanism and regulation of C1GALT1 and O-glycosylation remain elusive, further studies are required to elucidate their effects on development and disease.

Synthetic Biology towards Improved Flavonoid Pharmacokinetics.

Flavonoids are a structurally diverse class of natural products that have been found to have a range of beneficial activities in humans. However, the clinical utilisation of these molecules has been limited due to their low solubility, chemical stability, bioavailability and extensive intestinal metabolism in vivo. Recently, the view has been formed that site-specific modification of flavonoids by methylation and/or glycosylation, processes that occur in plants endogenously, can be used to improve and adapt their biophysical and pharmacokinetic properties. The traditional source of flavonoids and their modified forms is from plants and is limited due to the low amounts present in biomass, intrinsic to the nature of secondary metabolite biosynthesis. Access to greater amounts of flavonoids, and understanding of the impact of modifications, requires a rethink in terms of production, more specifically towards the adoption of plant biosynthetic pathways into ex planta synthesis approaches. Advances in synthetic biology and metabolic engineering, aided by protein engineering and machine learning methods, offer attractive and exciting avenues for ex planta flavonoid synthesis. This review seeks to explore the applications of synthetic biology towards the ex planta biosynthesis of flavonoids, and how the natural plant methylation and glycosylation pathways can be harnessed to produce modified flavonoids with more favourable biophysical and pharmacokinetic properties for clinical use. It is envisaged that the development of viable alternative production systems for the synthesis of flavonoids and their methylated and glycosylated forms will help facilitate their greater clinical application.

Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte

RationaleAbout 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance.ObjectiveTo evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes.Methods and resultsWe evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization.ConclusionsThe upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

Macroprolactin Molecular Heterogeneity and Variable Immunoassay Reactivity

Circulating prolactin (PRL) exhibits molecular heterogeneity the clinical significance of which is not known. Macroprolactin (mPRL) is widely reported, however, its heterogeneity is unknown. In addition to determining its molecular nature, the relative impact of the various forms on routinely available immunoassays needs to be examined. This study, applied various proteomics techniques to define the nature of mPRL and examined the effect of those forms on its measurement. Methods: Samples suspected of false hyperprolactinemia were subjected to; 1) precipitation by polyethylene glycol, 2) gel permeation chromatography, 3) protein-G affinity chromatography to identify presence of PRL-antibody complex and, 4) samples negative for antibody by step (3) were applied onto a lectin concanavalin A column to examine the presence of glycosylated PRL forms. Analysis was according to manufacturer’s instructions. All study samples and their chromatography fractions were measured using highly sensitive ELISA (DuoSet, R&D Systems, MN). Samples identified above as containing different mPRL forms were analyzed using four immunoassay analyzers in routine clinical use, namely; Advia Centaur® and Atellica® (Siemens, PA), Alinity-ci (Abbott Laboratories, IL), and Cobas 6000® (Roche Diagnostics, IN). Results: A total of 13 samples were entered into the study. PRL levels ranged from 21.4 to 1,469 ng/mL (median 48.8). Samples positive for mPRL (n=8) (with <40% recovery by PEG) exhibited predominant (52.3 to 95.0%) PRL activity in the (H) range (≥150kDa), with significant but relatively lower amount (3.6 to 34.1%) in the (M) range (≥30<150kDa) and (1.4 to 34.5%) at the (L) range (<30kDa). Samples negative for mPRL exhibited little PRL activity (1.2 to 5.1%) in (H) range, predominantly (60.0 to 79.4%) in the (M) range and moderate presence (15.4 to 38.9 %) in the (L) range. Two samples indeterminate for mPRL contained prolactin forms at all molecular weight levels, (H) (7.9, 27.1%), (M) (67.0, 40.7%), and (L) (5.9, 51.4%). Samples with mPRL exhibited significant binding to protein G affinity column indicating presence of PRL-antibody complex. Samples with (M) range mPRL forms exhibited significant lectin affinity binding. Samples with (H) mPRL showed markedly high PRL by two of the analyzers (Atellica®, Abbott Laboratories) and Cobas 6000®, (Roche Diagnostics). The deviation was more marked when using the Atellica® compared to Cobas 6000®. Samples with (M) mPRL showed marked deviation using the Roche Cobas 6000® compared to the others. In conclusion: Macroprolactin is heterogenous with antibody-PRL complex and aggregated glycosylated forms. Those forms exhibit variable immunoassay reactivity in different routinely used assays. Knowledge of circulating form as well as of their immunoreactivity is important when suspecting false hyperprolactinemia due to macroprolactin.

Hepatic stellate cell as a Mac-2-binding protein-producing cell in patients with liver fibrosis.

Mac-2 binding protein (M2BP) glycosylated isomer (M2BPGi) is a serum marker of liver fibrosis; M2BPGi is a glycosylated form of M2BP. Hepatocytes and hepatic stellate cells (HSCs) have been studied to determine the source of M2BP. This study proposes to identify the origin of M2BP in fibrotic liver. Using liver fibrosis tissue specimens from 15 patients with liver cancer, M2BP mRNA and M2BP were detected by in situ hybridization and immunohistochemistry, respectively. The expression levels of M2BP mRNA were evaluated with scores of 3, 2, and 1. Fluorescent in situ hybridization was carried out to evaluate the distribution of M2BP mRNA and the activated-HSC marker αSMA mRNA; multicolor fluorescent immunohistochemistry was used for protein localization of M2BP, αSMA, and CD68. The Kruskal-Wallis test analyzed the relationship between M2BP mRNA expression and existing serum fibrosis markers. M2BP mRNA was expressed in spindle-shaped cells along the fibrous septa and in the perisinusoidal area of the fibrotic liver. The HSC markers αSMA mRNA and M2BP mRNA were colocalized in the spindle-shaped cells; on the protein level, M2BP was expressed in Kupffer cells. M2BP mRNA expression was positively correlated with serum M2BPGi levels. Aspartate transaminase-to-platelet ratio index, Fibrosis-4, hyaluronic acid, and the 15-minute indocyanine green retention rate were significantly correlated with M2BP mRNA expression. M2BP mRNA transcription in fibrotic liver was primarily observed in HSCs but not at the M2BP level, which suggests that HSCs might produce and introduce M2BP to Kupffer cells and serum.

Binding of hydroxychloroquine and chloroquine dimers to palmitoyl-protein thioesterase 1 (PPT1) and its glycosylated forms: a computational approach

The lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1) removes thioester-linked fatty acid groups from membrane-bound proteins to facilitate their proteolysis. A lack of PPT1 (due to gene mutations) causes the progressive death of cortical neurons and is responsible for infantile neural ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder in children. Conversely, PPT1 is often over-expressed in cancer, and considered as a valid target to control tumor growth. Potent and selective inhibitors of PPT1 have been designed, in particular 4-amino-7-chloro-quinoline derivatives such as hydroxychloroquine (HCQ) and the dimeric analogues Lys05 and DC661. We have modeled the interaction of these three compounds with the enzyme, taking advantage of the PPT1 crystallographic structure. The molecules can fit into the palmitate site of the protein, with the dimeric compounds forming more stable complexes than the monomer. But the molecular modeling suggests that the most favorable binding sites are located outside the active site. Two sites centered on residues Met112 and Gln144 were identified, offering suitable cavities for drug binding. According to the calculated empirical energies of interaction (ΔE), the dimer DC661 forms the most stable complex at site Met112 of palmitate-bound PPT1. N-glycosylated forms of PPT1 were elaborated. Paucimannosidic glycans (M2FA and M3F) and a bulkier tetra-antennary complex glycan were introduced at asparagine residues N197, N212 and N232. These N-glycans do not impede drug binding, thus suggesting that all glycoforms of PPT1 can be targeted with these compounds. Communicated by Ramaswamy H. Sarma