Human apolipoprotein E glycosylation and sialylation: from structure to function.

Human apolipoprotein E (ApoE) was first identified as a polymorphic gene in the 1970s; however, the genetic association of ApoE genotypes with late-onset sporadic Alzheimer's disease (sAD) was only discovered 20 years later. Since then, intensive research has been undertaken to understand the molecular effects of ApoE in the development of sAD. Despite three decades' worth of effort and over 10,000 papers published, the greatest mystery in the ApoE field remains: human ApoE isoforms differ by only one or two amino acid residues; what is responsible for their significantly distinct roles in the etiology of sAD, with ApoE4 conferring the greatest genetic risk for sAD whereas ApoE2 providing exceptional neuroprotection against sAD. Emerging research starts to point to a novel and compelling hypothesis that the sialoglycans posttranslationally appended to human ApoE may serve as a critical structural modifier that alters the biology of ApoE, leading to the opposing impacts of ApoE isoforms on sAD and likely in the peripheral systems as well. ApoE has been shown to be posttranslationally glycosylated in a species-, tissue-, and cell-specific manner. Human ApoE, particularly in brain tissue and cerebrospinal fluid (CSF), is highly glycosylated, and the glycan chains are exclusively attached via an O-linkage to serine or threonine residues. Moreover, studies have indicated that human ApoE glycans undergo sialic acid modification or sialylation, a structural alteration found to be more prominent in ApoE derived from the brain and CSF than plasma. However, whether the sialylation modification of human ApoE has a biological role is largely unexplored. Our group recently first reported that the three major isoforms of human ApoE in the brain undergo varying degrees of sialylation, with ApoE2 exhibiting the most abundant sialic acid modification, whereas ApoE4 is the least sialylated. Our findings further indicate that the sialic acid moiety on human ApoE glycans may serve as a critical modulator of the interaction of ApoE with amyloid ß (Aß) and downstream Aß pathogenesis, a prominent pathologic feature in AD. In this review, we seek to provide a comprehensive summary of this exciting and rapidly evolving area of ApoE research, including the current state of knowledge and opportunities for future exploration.

Characterization of the Antibody Response to SARS-CoV-2 Infection in COVID-19 Transplant versus Nontransplant Recipients by Ig-MS.

Solid organ transplant recipients with immunosuppressant regimens to prevent rejection are less able to mount effective immune responses to pathogenic infection. Here, we apply a recently reported mass spectrometry-based serological approach known as Ig-MS to characterize immune responses against infection with SARS-CoV-2 in cohorts of transplant recipients and immunocompetent controls, both at a single early time point following COVID-19 diagnosis as well as over the course of one-month postdiagnosis. We found that the antibody repertoires generated by transplant recipients against SARS-CoV-2 do not differ significantly compared to immunocompetent individuals with regard to repertoire titer, clonality, or glycan composition. Importantly, our study is the first to characterize the evolution of antibody glycan profiles in transplant recipients with COVID-19 disease, presenting evidence that the evolution of glycan composition in these immunocompromised individuals is similar to that in immunocompetent people.

Ultrasound-assisted glycosylation of ovalbumin and dextran conjugate carrier for anthocyanins and their stability evaluation.

Anthocyanins (AC) are vulnerable to degradation when affected by external factors. The present study employed ultrasound-assisted glycosylation of ovalbumin (OVA) and dextran (Dex) to generate conjugate carrier for AC to improve its stability. The results showed that sonication significantly improved the progression of Maillard reaction to OVA. Compared to traditional glycosylation, ultrasound treatment showed a higher degree of grafting, a lower number of free-SH, and smaller particle size and uniform distribution. The SDS-PAGE results indicated covalent interaction. Intrinsic fluorescence (INF), Fourier transform infrared spectroscopy (FTIR), and Circular dichroism (CD) analysis results suggested that ultrasound-assisted glycosylation altered the OVA structure. The scanning electron microscope (SEM) and X-ray diffractometer (XRD) observed that the ultrasound-assisted complex had a more compact and smoother structure and protein unfolding were better. The protein solubility increased significantly after glycosylation. Thermal gravimetric analysis (TGA) and Differential scanning calorimetry (DSC) indicated that the glycosylated conjugates can significantly improve the thermal stability of AC In addition, the AC showed an improved processing and storage stability when conjugated with glycosylated carrier. The glycosylated protein-anthocyanins complex may help provide new ideas and scientific basis for the development of naturally sourced anthocyanins-relevant products in pharmaceutical and food industry applications.

Post-Translational Modifications Control The Signal At The Crossroads Of Plant-Pathogen Interaction.

The co-evolution of plants and pathogens has enabled them to 'outsmart' each other by promoting their own defense responses and suppressing that of the other. While plants are reliant on their sophisticated immune signalling pathways, pathogens make use of effector proteins to achieve the objective. This entails rapid regulation of the underlying molecular mechanisms for prompt induction of the associated signalling events in both plants as well as pathogens. The last decade has witnessed the emergence of post-translational modification (PTM) of proteins as key players in modulating cellular responses. Their ability to expand the functional diversity of the proteome and induce rapid changes at the appropriate time enables them to play crucial roles in the regulation of plant-pathogen interactions. Therefore, this review will delve into the intricate interplay of five major PTMs in plant defense and pathogen countermeasures. The review discusses how plants employ PTMs to fortify their immune networks, and how pathogen effectors utilize/target host modification systems to gain entry into the plant and cause disease. The review also underscores the need for identification of newer PTMs and proposes to use PTM machineries as potential targets for genome editing approaches.

Role of glycosylation-related gene MGAT1 in pancreatic ductal adenocarcinoma.

Background: pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with a very poor prognosis and a complex tumor microenvironment, which plays a key role in tumor progression and treatment resistance. Glycosylation plays an important role in processes such as cell signaling, immune response and protein stability. Materials and methods: single-cell RNA sequencing data and spatial transcriptome data were obtained from GSE197177 and GSE224411, respectively, and RNA-seq data and survival information were obtained from UCSC Xena and TCGA. Multiple transcriptomic data were comprehensively analyzed to explore the role of glycosylation processes in tumor progression, and functional experiments were performed to assess the effects of MGAT1 overexpression on PDAC cell proliferation and migration. Results: In PDAC tumor samples, the glycosylation level of macrophages was significantly higher than that of normal samples. MGAT1 was identified as a key glycosylation-related gene, and its high expression was associated with better patient prognosis. Overexpression of MGAT1 significantly inhibited the proliferation and migration of PDAC cells and affected intercellular interactions in the tumor microenvironment. Conclusion: MGAT1 plays an important role in PDAC by regulating glycosylation levels in macrophages, influencing tumor progression and improving prognosis.MGAT1 is a potential therapeutic target for PDAC and further studies are needed to develop targeted therapeutic strategies against MGAT1 to improve clinical outcomes.

Effects of different HA and NA gene combinations on the growth characteristics of the H3N8 influenza candidate vaccine virus.

Since 2022, three human cases of a novel H3N8 avian influenza virus infection have been reported in three provinces in China. Specific vaccines are important means of preparing for the potential influenza pandemic. Thus, H3N8 viruses [A/Henan/cnic410/2022 (HN410) and A/Changsha/1000/2022(CS1000)] were isolated from the infected patients as prototype viruses to develop candidate vaccine viruses (CVVs) using the reverse genetics (RG) technology. Five reassortant viruses with different HA and NA combinations were constructed based on the two viruses to get a high-yield and safe CVV. The results showed that all viruses had similar antigenicity but different growth characteristics. Reassortant viruses carrying NA from CS1000 exhibited better growth ability and NA enzyme activity than the ones carrying HN410 NA. Furthermore, the NA gene of CS1000 had one more potential N-glycosylation site at position 46 compared with HN410. The substitution of position 46 showed that adding or removing N-glycosylation sites to different reassortant viruses had different effects on growth ability. A reassortant virus carrying HN410 HA and CS1000 NA with high growth ability was selected as a CVV, which met the requirements for a CVV. These data suggest that different surface gene combinations and the presence or absence of potential N-glycosylation sites on position 46 in the NA gene affect the growth characteristics of H3N8 CVVs.

Bisecting N-Acetylglucosamine Correlates with Phospho-Tau181 in Subjective Cognitive Decline but not in Control Cases.

Background: The N-glycan structure bisecting N-acetylglucosamine (bisecting GlcNAc) is present on several N-glycans that are elevated in Alzheimer's disease (AD), and previous studies have shown that bisecting GlcNAc levels correlate with total tau and phospho-tau181 in cerebrospinal fluid at early stages of AD. A recent population-based study showed that bisecting GlcNAc correlates with total tau also in blood and that this correlation could predict conversion to dementia. Objective: In this study, we have further investigated how bisecting GlcNAc relates to total tau and phospho-tau 181 in cerebrospinal fluid samples from controls and cases with early cognitive deficits, stratified by amyloid/tau status and gender. Methods: Relative levels of bisecting GlcNAc in cerebrospinal fluid were measured by an enzyme-linked lectin assay in individuals with subjective cognitive decline, mild cognitive impairment and controls from the Norwegian Dementia Disease Initiation cohort. Results: As in our previous study, the correlation between bisecting GlcNAc and total tau or phospho-tau181 was particularly strong in the subjective cognitive decline group. The correlation was observed in amyloid negative and tau negative as well as amyloid positive and tau positive individuals, both in females and in males. Interestingly, among the amyloid negative and tau negative individuals, the correlation was observed in individuals with subjective cognitive decline but not in the controls. Conclusions: Thus, bisecting GlcNAc could be a biomarker for early cognitive decline.

Post-translational Modifications of α-Synuclein, their Therapeutic Potential and Crosstalk in Health and Neurodegenerative Diseases.

α-Synuclein (α-Syn) aggregation in Lewy bodies and Lewy neurites has emerged as a key pathogenetic feature in Parkinson's disease (PD), Dementia with Lewy Bodies and Multiple System Atrophy. Various factors, including post-translational modifications (PTMs), can influence the propensity of α-Syn to misfold and aggregate. PTMs are biochemical modifications of a protein that occur during or after translation and are typically mediated by enzymes. PTMs modulate several characteristics of proteins including their structure, activity, localization, and stability. α-Syn undergoes various post-translational modifications, including phosphorylation, ubiquitination, SUMOylation, acetylation, glycation, O-GlcNAcylation, nitration, oxidation, polyamination, arginylation, and truncation. Different PTMs of a protein can physically interact with one another or work together to influence a particular physiological or pathological feature in a process known as PTMs crosstalk. The development of detection techniques for the co-occurrence of PTMs in recent years has uncovered previously unappreciated mechanisms of their crosstalk. This has led to the emergence of evidence supporting an association between α-Syn PTMs crosstalk and synucleinopathies. In this review, we provide a comprehensive evaluation of α-Syn PTMs, their impact on misfolding and pathogenicity, the pharmacological means of targeting them, and their potential as biomarkers of disease. We also highlight the importance of the crosstalk between these PTMs in α-Syn function and aggregation. Insight into these PTMS and the complexities of their crosstalk can improve our understanding of the pathogenesis of synucleinopathies and identify novel targets of therapeutic potential. Significance Statement α-Synuclein as a key pathogenic protein in Parkinson's disease and other synucleinopathies, making it a leading therapeutic target for disease modification. Multiple post-translational modifications occur at various sites in α-Synuclein and alter its biophysical and pathological properties, some interacting with one another to add to the complexity of the pathogenicity of this protein. This review details these modifications, their implications in disease and potential therapeutic opportunities.

Systematic mutational analysis reveals an essential role of N275 in IgE stability.

Therapeutic antibodies have predominantly been IgG-based. However, the ongoing clinical trial of MOv18 IgE has highlighted the potential of using IgE antibodies in cancer therapy. While extensive studies targeting IgG glycosylation resulted in a rational basis for the development of enhanced biotherapeutics, IgE glycosylation remains an area with limited analyses. Previous studies on the role of IgE glycosylation present conflicting data with one study emphasizing the importance of N275 and T277 residues for FcεRI binding whereas another asserts the nonsignificance of IgE glycosylation in receptor interaction. While existing literature underscores the significance of glycans at the N275 position for binding to FcεR1 receptor and initiation of anaphylaxis, the role of other IgE glycosylation sites in folding or receptor binding remains elusive. This study systematically investigates the functional significance of N-linked glycosylation sites in the heavy chain of IgE which validates the pivotal role of N275 residue in IgE secretion and stability. Replacement of this asparagine to non-amine group moieties does not affect IgE function in vitro, yet substitution with aspartic acid compromises antibody yield. The deglycosylated IgE variant exhibits superior efficacy, challenging the conventional importance of glycosylation for effector function. In summary, our study unveils an intricate relationship between N-glycosylation sites and the structural-functional dynamics of IgE antibodies. Furthermore, it offers novel insights into the IgE scaffold, paving the way for the development of more effective and stable IgE-based therapeutics.

Biological markers of high risk of thrombotic recurrence in patients with antiphospholipid syndrome: A literature review.

OBJECTIVES: This review aims to identify biological markers associated with the risk of recurrence of thrombotic and/or obstetric events in patients with antiphospholipid syndrome (APS). METHODS: A comprehensive review of literature was conducted to evaluate established and potential novel biological markers associated with thrombosis in APS. To this end, a PubMed literature search was conducted for the last twenty years using the following keywords or their combinations: thrombotic risk, recurrence of thrombosis, risk stratification, severity, predictive value. RESULTS: Previous studies showed that multiple aPL positivity correlates with an increased risk of thrombosis in APS. Moreover, the analysis of N-glycosylation of antiphospholipid antibodies (aPL) revealed that low levels of IgG sialylation, fucosylation or galactosylation increases the pro-inflammatory activity of aPL, predisposing to thrombosis. In addition, quantification of neutrophil extracellular traps (NETs) and antibodies directed against NETs (anti-NETs) in serum demonstrates promising prognostic utility in assessing APS severity. Oxidative stress plays a role in the pathogenicity of APS and paraoxonase 1 (PON1) activity emerges as a promising biomarker of thrombotic risk in APS. Furthermore, identification of novel antigenic targets involved in the pathophysiology of APS, such as lysobisphosphatidic acid (LBPA), had led to the discovery of unconventional aPL, antibodies directed against the LBPA (aLBPA), whose clinical value could make it possible to identify APS patients at high risk of thrombotic recurrence. CONCLUSION: The immunological profile of aPL, N-glycosylation of aPL, quantification of NETs and anti-NETs, analysis of biomarkers of oxidative stress and the discovery of aLBPA offer potential prognostic tools for risk stratification in APS patients.

Mass spectrometry-based structure-specific N-glycoproteomics and biomedical applications.

N-linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, N-glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio. N-glycosylation regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact N-glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact N-glycopeptides and the clinical significance of differentially expressed intact N-glycopeptides. With the help of these modern methods, structure-specific N-glycoproteomics has become an important tool with extensive applications in the biomedical field.

Missense variant in PIGM associated with severe cystic encephalomalacia and portal vein thrombosis: Phenotypic and genotypic expansion of the glycosylphosphatidylinositol biosynthesis defect-1 (GPIBD1).

Glycosylphosphatidylinositols (GPIs) are a type of glycolipid responsible for anchoring many important proteins to the cell membrane surface. Defects in the synthesis of GPIs can lead to a group of multisystem disorders known as the inherited GPI deficiencies (IGDs). Homozygosity for the c.-270C > G variant in the promoter of PIGM has been associated with a IGD subtype known as glycosylphosphatidylinositol biosynthesis defect-1 (GPIBD1). The several cases reported in the literature have been described to have a milder neurologic phenotype in comparison to the other IGDs and have been treated with sodium phenylbutyrate with some degree of success. These patients typically present with portal and hepatic vein thrombosis and mostly develop absence seizures. Here we describe a patient homozygous for a nonsynonymous variant in PIGM who deceased at 9 weeks of life and had multiple physical dysmorphisms (rocker bottom feet, midline cleft palate, thickened and lichenified skin), portal vein thrombosis, CNS structural anomalies (progressive multicystic encephalomalacia and ventriculomegaly), and a neurological phenotype of a diffuse encephalopathy. This is the first known case report of a PIGM-related IGD/CDG due to a coding variant.

Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension.

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Crystal structure of blue laccase BP76, a unique termite suicidal defense weapon.

Aging workers of the termite Neocapritermes taracua can defend their colony by sacrificing themselves by body rupture, mixing the externally stored blue laccase BP76 with hydroquinones to produce a sticky liquid rich in toxic benzoquinones. Here, we describe the crystal structure of BP76 isolated from N. taracua in its native form. The structure reveals several stabilization strategies, including compact folding, glycosylation, and flexible loops with disulfide bridges and tight dimer interface. The remarkable stability of BP76 maintains its catalytic activity in solid state during the lifespan of N. taracua workers, providing old workers with an efficient defensive weapon to protect their colony.

GDNF improves the cognitive ability of PD mice by promoting glycosylation and membrane distribution of DAT.

The core of clinic treatment of Parkinson's disease (PD) is to enhance dopamine (DA) signaling within the brain. The regulation of dopamine transporter (DAT) is integral to this process. This study aims to explore the regulatory mechanism of glial cell line-derived neurotrophic factor (GDNF) on DAT, thereby gaining a profound understanding its potential value in treating PD. In this study, we investigated the effects of GDNF on both cellular and mouse models of PD, including the glycosylation and membrane transport of DAT detected by immunofluorescence and immunoblotting, DA signal measured by neurotransmitter fiber imaging technology, Golgi morphology observed by electron microscopic, as well as cognitive ability assessed by behavior tests. This study revealed that in animal trials, MPTP-induced Parkinson's Disease (PD) mice exhibited a marked decline in cognitive function. Utilizing ELISA and neurotransmitter fiber imaging techniques, we observed a decrease in dopamine levels and a significant reduction in the intensity of dopamine signal release in the Prefrontal Cortex (PFC) of PD mice induced by MPTP. Intriguingly, these alterations were reversed by Glial Cell Line-Derived Neurotrophic Factor (GDNF). In cellular experiments, following MPP + intervention, there was a decrease in Gly-DAT modification in both the cell membrane and cytoplasm, coupled with an increase in Nongly-DAT expression and aggregation of DAT within the cytoplasm. Conversely, GDNF augmented DAT glycosylation and facilitated its membrane transport in damaged dopaminergic neurons, concurrently reversing the effects of GRASP65 depletion and Golgi fragmentation, thereby reducing the accumulation of DAT in the Golgi apparatus. Furthermore, overexpression of GRASP65 enhanced DAT transport in PD cells and mice, while suppression of GRASP65 attenuated the efficacy of GDNF on DAT. Additionally, GDNF potentiated the reutilization of neurotransmitters by the PFC presynaptic membrane, boosting the effective release of dopamine following a single electrical stimulation, ultimately ameliorating the cognitive impairments in PD mice.Therefore, we propose that GDNF enhances the glycosylation and membrane trafficking of DAT by facilitating the re-aggregation of the Golgi apparatus, thereby amplifying the utilization of DA signals. This ultimately leads to the improvement of cognitive abilities in PD mouse models. Our study illuminates, from a novel angle, the beneficial role of GDNF in augmenting DA utilization and cognitive function in PD, providing fresh insights into its therapeutic potential.

Ready chemistry with a rare sugar: Altrobioside synthesis and analysis of conformational characteristics.

We describe the synthesis of the full set of the so far unknown methyl altrobiosides and the initial analysis of the conformational dynamic which occurs in some of the synthesized compounds. d-Altrose chemistry has largely been neglected as it is a rare sugar and has first to be synthesized from glucose or mannose, respectively. Nevertheless, d-altrose is particularly interesting as the energy barrier between the complementary chair conformations is rather low and therefore dynamic mixtures of conformers might occur. We describe the ready synthesis of the selectively protected altrosyl acceptors for the glycosidation from d-mannose and the altrosyl-trichloroacetimidate as useful glycosyl donor to achieve the (1 â†’ 2), (1 â†’ 3), (1 â†’ 4), and (1 â†’ 6)-α-linked altrobiosides. The diastereomeric α- and ß-O-(d-altropyranosyl)-trichloroacetimidates adopt different ring conformations as analyzed by NMR and VCD spectroscopy. Also, the pyranose ring conformations of the obtained altrobiosides apparently differ from a regular 4C1 chair according to NMR analysis and are influenced by the regiochemistry of the interglycosidic linkage.

Towards personalized and rational use of immunoglobulins amid expanding indications and shortages.

The development of intravenous IgG (IVIG) formulations in the 1970s enabled expanded use for treating primary antibody deficiency syndromes and autoimmune conditions. Recent advancements include the use of IVIG in secondary immune deficiencies related to hematologic malignancies and stem cell transplantation, along with the newly emerging prophylactic applications following chimeric antigen receptor T-cell (CAR-T) therapies. Novel therapeutic areas such as bispecific antibodies (BsAbs) for lymphoma and myeloma have increased the use of IgG, given the associated risks of infections. Today, the concept of a rational personalized clinical use of IgG in the context of evolving clinical indications in high-income countries (HIC) is emerging, as unmet challenges in line with managing shortages due to increasing demands globally. The current work aims to review and link the indications for IgG to their characteristics and formulations, their dose, route and frequency of administrations and duration of therapy to meet the needs of individual patients. It will also explore the means to rationalize and monitor IgG use in HIC in the time of shortage, while explaining pragmatic strategies to improve supply and use in low- and middle-income countries (LMIC).

Human organic anion transporting polypeptide 1B3 (OATP1B3) is more heavily N-glycosylated than OATP1B1 in extracellular loops 2 and 5.

Human organic anion transporting polypeptide 1B3 (OATP1B3) and 1B1 are two liver-specific and highly homologous uptake transporters, whose structures consist of 12 transmembrane domains. The present study showed that OATP1B3 is more heavily N-glycosylated than OATP1B1 in extracellular loop 2 (EL2) and EL5. OATP1B3 has six N-glycosylation sites, namely N134, N145, N151, N445, N503, and N516, which is twice of that of OATP1B1. Single removal of individual N-glycans seems to have minimal influence on the surface expression and function of OATP1B3. However, simultaneous removal of all N-glycans will lead to OATP1B3's large retention in the endoplasmic reticulum and cellular degradation and thus significantly disrupts its surface expression. While N-glycosylation plays a crucial role in the surface expression of OATP1B3, it also has some effect on the transport function of OATP1B3 per se, which is not due to a decrease of substrate binding affinity but due to a reduced transporter's turnover number. Taken together, N-glycosylation is essential for normal surface expression and function of OATP1B3. Its disruption by some liver diseases such as NASH might alter the pharmacokinetic/pharmacodynamic properties of OATP1B3's substrate drugs.

Clinical glycoprotein mass spectrometry: The future of disease detection and monitoring.

Protein glycosylation is the co- and/or post-translational modification of proteins with oligosaccharides (glycans). This process is not template based and can introduce a heterogeneous set of glycan modifications onto substrate proteins. Glycan structures preserve biomolecular information from the cell, with glycoproteins from different cell types and tissues displaying distinct patterns of glycosylation. Several decades of research have revealed that glycan structures also differ between normal physiology and disease. This suggests that the information stored in glycoproteins and glycans can be utilized for disease diagnosis and monitoring. Methods that enable sensitive and site-specific measurement of protein glycosylation in clinical settings, such as nano-flow liquid chromatography tandem mass spectrometry, are therefore essential. The purpose of this perspective is to discuss recent advances in mass spectrometry and the potential of these advances to facilitate the detection and monitoring of disease-specific glycoprotein glycoforms. Glycoproteomics, the system-wide characterization of glycoprotein identity inclusive of site-specific characterization of carbohydrate modifications on proteins, and glycomics, the characterization of glycan structures, will be discussed in this context. Quantitative measurement of glycopeptide markers via parallel reaction monitoring is highlighted. The development of promising glycopeptide markers for autoimmune disease, liver disease, and liver cancer is discussed. Synthetic glycopeptide standards, ambient ionization mass spectrometry, and consideration of glyco-biomarkers in two- and three-dimensional space within tissue will be critical to the advancement of this field. The authors envision a future in which glycoprotein mass spectrometry workflows will be integrated into clinical settings, to aid in the rapid diagnosis and monitoring of disease.

Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.