Cross-Reactive Polyclonal Antibodies Raised Against GalNAc-Conjugated siRNA Recognize Mostly the GalNAc Moiety.

Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

Research progress on asialoglycoprotein receptor-targeted radiotracers designed for hepatic nuclear medicine imaging.

Asialoglycoprotein receptor (ASGPR) specifically recognizes glycans terminated with ß-d-galactose or N-acetylgalactosamine. Its exclusive expression in mammalian hepatocytes renders it an ideal hepatic-targeted biomarker. To date, ASGPR-targeted ligands have been actively developed for drug delivery and hepatic imaging. This review provides a comprehensive summary of the progress achieved to-date in the field of developing ASGPR-targeted nuclear medicine imaging (NMI) radiotracers, highlighting the recent advancements over the last decade in terms of structure, radionuclides and labeling strategies. The biodistribution patterns, imaging characteristics, challenges and future prospective are discussed.

Deficiency of polypeptide N-acetylgalactosamine transferase 9 contributes to a risk for Parkinson's disease via mitochondrial dysfunctions.

Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to serine/threonine in a protein. To unravel the association of GALNT9 with Parkinson's disease (PD), a progressive neurodegenerative disorder, GALNT9 levels were evaluated in the patients with PD and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and statistically analyzed based on the GEO datasets of GSE114918 and GSE216281. Glycoproteins with exposing GalNAc were purified using lectin affinity chromatography and identified by LC-MS/MS. The influence of GALNT9 on cells was evaluated via introducing a GALNT9-specific siRNA into SH-SY5Y cells. Consequently, GALNT9 deficiency was found to occur under PD conditions. GALNT9 silencing contributed to a causative factor in PD pathogenesis via reducing the levels of intracellular dopamine, tyrosine hydroxylase and soluble α-synuclein, and promoting α-synuclein aggregates. MS identification revealed 14 glycoproteins. 5 glycoproteins, including ACO2, ATP5B, CKB, CKMT1A, ALDOC, were associated with energy metabolism. GALNT9 silencing resulted in mitochondrial dysfunctions via increasing ROS accumulation, mitochondrial membrane depolarization, mPTPs opening, Ca2+ releasing and activation of the CytC-related apoptotic pathway. The dysfunctional mitochondria then triggered mitophagy, possibly intermediated by adenine nucleotide translocase 1. Our study suggests that GALNT9 is potentially developed into an auxiliary diagnostic index and therapeutic target of PD.

RNA interference in the era of nucleic acid therapeutics.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.

Application of improved GalNAc conjugation in development of cost-effective siRNA therapies targeting cardiovascular diseases.

N-Acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) therapies have received approval for treating both orphan and prevalent diseases. To improve in vivo efficacy and streamline the chemical synthesis process for efficient and cost-effective manufacturing, we conducted this study to identify better designs of GalNAc-siRNA conjugates for therapeutic development. Here, we present data on redesigned GalNAc-based ligands conjugated with siRNAs against angiopoietin-like 3 (ANGPTL3) and lipoprotein (a) (Lp(a)), two target molecules with the potential to address large unmet medical needs in atherosclerotic cardiovascular diseases. By attaching a novel pyran-derived scaffold to serial monovalent GalNAc units before solid-phase oligonucleotide synthesis, we achieved increased GalNAc-siRNA production efficiency with fewer synthesis steps compared to the standard triantennary GalNAc construct L96. The improved GalNAc-siRNA conjugates demonstrated equivalent or superior in vivo efficacy compared to triantennary GalNAc-conjugated siRNAs.

Safety and Tolerability of GalNAc3-Conjugated Antisense Drugs Compared to the Same-Sequence 2'-O-Methoxyethyl-Modified Antisense Drugs: Results from an Integrated Assessment of Phase 1 Clinical Trial Data.

The triantennary N-acetylgalactosamine (GalNAc3) cluster has demonstrated the utility of receptor-mediated uptake of ligand-conjugated antisense drugs targeting RNA expressed by hepatocytes. GalNAc3-conjugated 2'-O-methoxyethyl (2'MOE) modified antisense oligonucleotides (ASOs) have demonstrated a higher potency than the unconjugated form to support lower doses for an equivalent pharmacological effect. We utilized the Ionis integrated safety database to compare four GalNAc3-conjugated and four same-sequence unconjugated 2'MOE ASOs. This assessment evaluated data from eight randomized placebo-controlled dose-ranging phase 1 studies involving 195 healthy volunteers (79 GalNAc3 ASO, 24 placebo; 71 ASO, 21 placebo). No safety signals were identified by the incidence of abnormal threshold values in clinical laboratory tests for either ASO group. However, there was a significant increase in mean alanine transaminase levels compared with placebo in the upper dose range of the unconjugated 2'MOE ASO group. The mean percentage of subcutaneous injections leading to local cutaneous reaction was 30-fold lower in the GalNAc3-conjugated ASO group compared with the unconjugated ASO group (0.9% vs. 28.6%), with no incidence of flu-like reactions (0.0% vs. 0.7%). Three subjects (4.2%) in the unconjugated ASO group discontinued dosing. An improvement in the overall safety and tolerability profile of GalNAc3-conjugated 2'MOE ASOs is evident in this comparison of short-term clinical data in healthy volunteers.

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Gold nanoparticles decorated with monosaccharides and sulfated ligands as potential modulators of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS).

N-Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers (i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the ß-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, ß-D-galactosides/sulfated ligands do not show better inhibition than the ß-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with ß-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.

N-acetylgalactosamine delivery systems for RNA therapeutics: a patent perspective.

INTRODUCTION: SiRNA molecules with a feature of good gene-silencing are critical for drug discovery and development based on RNA interference. GalNAc-RNA therapeutics is a rapid growing area in RNA therapeutics. AREAS COVERED: This article provides patent landscape and modification feature of GalNAc-RNA therapeutics. The US-granted patents from January 2004 to April 2023 were retrieved and analyzed. EXPERT OPINION: Globally, our study is the first one to holistically depict a map of modifications and therapeutic applications for GalNAc-RNA therapeutics by patent data analysis. The results showed there were 8 major modifications and 5 new emerged modifications for GalNAc-RNA therapeutic agents. Especially, the study provides recent new emerged modifications in sugar, base, and internucleotide linkage of GalNAc-RNA therapeutic agents, e.g. morpholino-type ring, 5-methylcytosine, and phosphorodithioates. In addition, our study systematically demonstrated major therapeutic applications for GalNAc-RNA therapeutics, including liver or gallbladder disorders, anticancer, antihyperlipidemics, and disorders of the nervous system etc.

Targeting Angiotensinogen With N-Acetylgalactosamine-Conjugated Small Interfering RNA to Reduce Blood Pressure.

Blood pressure management involves antihypertensive therapies blocking the renin-angiotensin system (RAS). Yet, it might be inadequate due to poor patient adherence or the so-called RAS escape phenomenon, elicited by the compensatory renin elevation upon RAS blockade. Recently, evidence points toward targeting hepatic AGT (angiotensinogen) as a novel approach to block the RAS pathway that could circumvent the RAS escape phenomenon. Removing AGT, from which all angiotensins originate, should prevent further angiotensin generation, even when renin rises. Furthermore, by making use of a trivalent N-acetylgalactosamine ligand-conjugated small interfering RNA that specifically targets the degradation of hepatocyte-produced mRNAs in a highly potent and specific manner, it may be possible in the future to manage hypertension with therapy that is administered 1 to 2× per year, thereby supporting medication adherence. This review summarizes all current findings on AGT small interfering RNA in preclinical models, making a comparison versus classical RAS blockade with either ACE (angiotensin-converting enzyme) inhibitors or AT1 (angiotensin II type 1) receptor antagonists and AGT suppression with antisense oligonucleotides. It ends with discussing the first-in-human study with AGT small interfering RNA.

Plasma Pharmacokinetics of N-Acetylgalactosamine-Conjugated Small-Interfering Ribonucleic Acids (GalNAc-Conjugated siRNAs).

Small-interfering ribonucleic acids (siRNAs) with N-acetylgalactosamine (GalNAc) conjugation for improved liver uptake represent an emerging class of drugs that modulate liver-expressed therapeutic targets. The pharmacokinetics of GalNAc-siRNAs are characterized by a rapid distribution from plasma to tissue (hours) and a long terminal plasma half-life, analyzed in the form of the antisense strand, driven by redistribution from tissue (weeks). Understanding how clinical pharmacokinetics relate to the dose and type of siRNA chemical stabilizing method used is critical, e.g., to design studies, to investigate safety windows, and to predict the pharmacokinetics of new preclinical assets. To this end, we collected and analyzed pharmacokinetic data from the literature regarding nine GalNAc-siRNAs. Based on this analysis, we showed that the clinical plasma pharmacokinetics of GalNAc-siRNAs are approximately dose proportional and similar between chemical stabilizing methods. This holds for both the area under the concentration-time curve (AUC) and the maximum plasma concentration (Cmax). Corresponding rat and monkey pharmacokinetic data for a subset of the nine GalNAc-siRNAs show dose-proportional Cmax, supra-dose-proportional AUC, and similar pharmacokinetics between chemical stabilizing methods​. Together, the animal and human pharmacokinetic data indicate that plasma clearance divided by bioavailability follows allometric principles and scales between species with an exponent of 0.75. Finally, the clinical plasma concentration-time profiles can be empirically described by standard one-compartment kinetics with first-order absorption up to 24 h after subcutaneous dosing, and by three-compartment kinetics with first-order absorption in general. To describe the system more mechanistically, we report a corrected and unambiguously defined version of a previously published physiologically based pharmacokinetic model.

Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses.

Inducing antigen-specific tolerance during an established immune response typically requires non-specific immunosuppressive signalling molecules. Hence, standard treatments for autoimmunity trigger global immunosuppression. Here we show that established antigen-specific responses in effector T cells and memory T cells can be suppressed by a polymer glycosylated with N-acetylgalactosamine (pGal) and conjugated to the antigen via a self-immolative linker that allows for the dissociation of the antigen on endocytosis and its presentation in the immunoregulatory environment. We show that pGal-antigen therapy induces antigen-specific tolerance in a mouse model of experimental autoimmune encephalomyelitis (with programmed cell-death-1 and the co-inhibitory ligand CD276 driving the tolerogenic responses), as well as the suppression of antigen-specific responses to vaccination against a DNA-based simian immunodeficiency virus in non-human primates. Our findings show that pGal-antigen therapy invokes mechanisms of immune tolerance to resolve antigen-specific inflammatory T-cell responses and suggest that the therapy may be applicable across autoimmune diseases.

Molecular characterisation of Entamoeba histolytica UDP-glucose 4-epimerase, an enzyme able to provide building blocks for cyst wall formation.

In the human host, the protozoan parasite Entamoeba histolytica is adapted to a non-invasive lifestyle in the colon as well as to an invasive lifestyle in the mesenterial blood vessels and the liver. This means to cope with bacteria and human cells as well as various metabolic challenges. Galactose and N-acetylgalactosamine (GalNAc) are sugars of great importance for the amoebae, they attach to the host mucus and enterocytes via their well-studied Gal/GalNAc specific lectin, they carry galactose residues in their surface glycans, and they cleave GalNAc from host mucins. The enzyme UDP-glucose 4-epimerase (GalE) works as a bridge between the galactose and glucose worlds, it can help to generate glucose for glycolysis from phagocytosis products containing galactose as well as providing UDP-galactose necessary for the biosynthesis of galactose-containing surface components. E. histolytica contains a single galE gene. We recombinantly expressed the enzyme in Escherichia coli and used a spectrophotometric assay to determine its temperature and pH dependency (37°C, pH 8.5), its kinetics for UDP-glucose (Km = 31.82 µM, Vmax = 4.31 U/mg) and substrate spectrum. As observed via RP-HPLC, the enzyme acts on UDP-Glc/Gal as well as UDP-GlcNAc/GalNAc. Previously, Trypanosoma brucei GalE and the bloodstream form of the parasite were shown to be susceptible to the three compounds ebselen, a selenoorganic drug with antioxidant properties, diethylstilbestrol, a mimic of oestrogen with anti-inflammatory properties, and ethacrynic acid, a loop diuretic used to treat oedema. In this study, the three compounds had cytotoxic activity against E. histolytica, but only ebselen inhibited the recombinant GalE with an IC50 of 1.79 µM (UDP-Gal) and 1.2 µM (UDP-GalNAc), suggesting that the two other compounds are active against other targets in the parasite. The importance of the ability of GalE to interconvert UDP-GalNAc and UDP-GlcNAc may be that the trophozoites can generate precursors for their own cyst wall from the sugar subunits cleaved from host mucins. This finding advances our understanding of the biochemical interactions of E. histolytica in its colonic environment.

Assessment of changes in Streptococcus pyogenes levels using N-acetylgalactosamine-6-sulfatase marker and pharyngeal airway space with appliance therapy in mouth breathers - An ELISA-based study.

Background: The frequency of adenotonsillar hypertrophy in mouth-breathing children when compared to the average found in the general population is considered to be higher. Mouth breathing is considered as one of the causative factors for tonsillitis in children. Through continuous irritation on tonsillar wall, tonsils swell up and inflammation develops. Purpose: The purpose of the study is to evaluate Streptococcus pyogenes count using colony-forming units (CFUs) and N-acetylgalactosamine-6-sulfatase side chain marker on ELISA (enzyme linked immunosorbent assay) in mouth breathers and to establish its correlation with pharyngeal airway space pre- and post-oral screen appliance therapy. Materials and Methods: A total number of 24 (n) mouth breathers aged between 5 and 12 years were included in the study and given oral screen appliance therapy. The subjects were evaluated for the various parameters before the delivery of a habit-breaking appliance and then reevaluated for the same parameters (presence of S. pyogenes and its counts, size of tonsils, and pharyngeal airway space dimensions) after 6 months of appliance usage. Results: A statistically significant difference was seen in levels of S. pyogenes using ELISA and CFUs. Furthermore, statistically significant difference was observed in Friedman tonsil scoring and pharyngeal airway space and pre- and post-oral screen appliance therapy. Conclusion: Oral screen appliance therapy reduced the frequency of occurrence of tonsillitis in mouth breathers by decreasing the counts of S. pyogenes bacteria. Upper and lower pharyngeal airway space dimensions were increased after 6 months of appliance therapy in mouth breathers.

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

In Vivo Metabolite Profiles of an N-Acetylgalactosamine-Conjugated Antisense Oligonucleotide AZD8233 Using Liquid Chromatography High-Resolution Mass Spectrometry: A Cross-Species Comparison in Animals and Humans.

AZD8233, a liver-targeting antisense oligonucleotide (ASO), inhibits subtilisin/kexin type 9 protein synthesis. It is a phosphorothioated 3-10-3 gapmer with a central DNA sequence flanked by constrained 2'-O-ethyl 2',4'-bridged nucleic acid (cEt-BNA) wings and conjugated to a triantennary N-acetylgalactosamine (GalNAc) ligand at the 5'-end. Herein we report the biotransformation of AZD8233, as given by liver, kidney, plasma and urine samples, after repeated subcutaneous administration to humans, mice, rats, rabbits, and monkeys. Metabolite profiles were characterized using liquid chromatography high-resolution mass spectrometry. Metabolite formation was consistent across species, mainly comprising hydrolysis of GalNAc sugars, phosphodiester-linker hydrolysis releasing the full-length ASO, and endonuclease-mediated hydrolysis within the central DNA gap followed by exonuclease-mediated 5'- or 3'-degradation. All metabolites contained the 5'- or 3'-cEt-BNA terminus. Most shortmer metabolites had the free terminal alcohol at 5'- and 3'-positions of ribose, although six were found retaining the terminal 5'-phosphorothioate group. GalNAc conjugated shortmer metabolites were also observed in urine. Synthesized metabolite standards were applied for (semi)quantitative metabolite assessment. Intact AZD8233 was the major component in plasma, whereas the unconjugated full-length ASO was predominant in tissues. In plasma, most metabolites were shortmers retaining the 3'-cEt-BNA terminus, whereas metabolites containing the 5'- or 3'-cEt-BNA terminus were detected in both tissues and urine. All metabolites in human plasma were also detected in all nonclinical species, and all human urine metabolites were detected in monkey urine. In general, metabolite profiles in animal species were qualitatively similar and quantitatively exceeded the exposures of the circulating metabolites in humans at the doses studied. SIGNIFICANCE STATEMENT: This study presents metabolite identification and profiling of AZD8233, an N-acetylgalactosamine-conjugated antisense oligonucleotide (ASO), across species. A biotransformation strategy for ASOs was established by utilizing biologic samples collected from toxicology and/or clinical studies and liquid chromatography high-resolution mass spectrometry analysis without conducting bespoke radiolabeled absorption, distribution, metabolism, and excretion studies. The generated biotransformation package was considered adequate by health authorities to progress AZD8233 into a phase 3 program, proving its applicability to future metabolism studies of ASOs in drug development.

Precolumn derivatization LC-MS/MS method to simultaneous quantitation of 10 monosaccharides in rat plasma.

Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100-5.00 µg/mL for rhamnose, 0.0500-25.0 µg/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00-500 µg/mL for galactose, 10.0-5000 µg/mL for mannose, and 50.0-25,000 µg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.