Glycosaminoglycans Research Articles

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background. IDS-/- NSG mice lacked detectable IDS activity in plasma and all analyzed tissues and exhibited elevated levels of GAGs in those same tissues and in the urine. Histopathology revealed vacuolized cells in both the periphery and CNS of NSG-MPS II mice. This model recapitulates skeletal disease manifestations, such as increased zygomatic arch diameter and decreased femur length. Neurocognitive deficits in spatial memory and learning were also observed in the NSG-MPS II model. We anticipate that this new immunodeficient model will be appropriate for preclinical studies involving xenotransplantation of human cell products intended for the treatment of MPS II.

Study protocol of a multicentre double-blind RCT, comparing a traditional RCT with an aggregated N-of-1 trial: GAG therapy Efficacy Trial Solution for Bladder pain syndrome/Interstitial cystitis (GETSBI study)

IntroductionObtaining level 1 evidence on efficacy of glycosaminoglycan (GAG) therapy is difficult, due to low incidence of bladder pain syndrome/interstitial cystitis (BPS/IC) and heterogeneous symptoms experienced by patients with BPS/IC....

Designing Smaller, Synthetic, Functional Mimetics of Sulfated Glycosaminoglycans as Allosteric Modulators of Coagulation Factors.

Natural glycosaminoglycans (GAGs) are arguably the most diverse collection of natural products. Unfortunately, this bounty of structures remains untapped. Decades of research has realized only one GAG-like synthetic, small-molecule drug, fondaparinux. This represents an abysmal output because GAGs present a frontier that few medicinal chemists, and even fewer pharmaceutical companies, dare to undertake. GAGs are heterogeneous, polymeric, polydisperse, highly water soluble, synthetically challenging, too rapidly cleared, and difficult to analyze. Additionally, GAG binding to proteins is not very selective and GAG-binding sites are shallow. This Perspective attempts to transform this negative view into a much more promising one by highlighting recent advances in GAG mimetics. The Perspective focuses on the principles used in the design/discovery of drug-like, synthetic, sulfated small molecules as allosteric modulators of coagulation factors, such as antithrombin, thrombin, and factor XIa. These principles will also aid the design/discovery of sulfated agents against cancer, inflammation, and microbial infection.

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.

In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.

Rational engineering of glycosaminoglycan-based Dickkopf-1 scavengers to improve bone regeneration

The WNT signaling pathway is a central regulator of bone development and regeneration. Functional alterations of WNT ligands and inhibitors are associated with a variety of bone diseases that affect bone fragility and result in a high medical and socioeconomic burden. Hence, this cellular pathway has emerged as a novel target for bone-protective therapies, e.g. in osteoporosis. Here, we investigated glycosaminoglycan (GAG) recognition by Dickkopf-1 (DKK1), a potent endogenous WNT inhibitor, and the underlying functional implications in order to develop WNT signaling regulators. In a multidisciplinary approach we applied in silico structure-based de novo design strategies and molecular dynamics simulations combined with synthetic chemistry and surface plasmon resonance spectroscopy to Rationally Engineer oligomeric Glycosaminoglycan derivatives (REGAG) with improved neutralizing properties for DKK1. In vitro and in vivo assays show that the GAG modification to obtain REGAG translated into increased WNT pathway activity and improved bone regeneration in a mouse calvaria defect model with critical size bone lesions. Importantly, the developed REGAG outperformed polymeric high-sulfated hyaluronan (sHA3) in enhancing bone healing up to 50% due to their improved DKK1 binding properties. Thus, rationally engineered GAG variants may represent an innovative strategy to develop novel therapeutic approaches for regenerative medicine.

Solvent Model Benchmark for Molecular Dynamics of Glycosaminoglycans.

In computational studies of glycosaminoglycans (GAGs), a group of anionic, periodic linear polysaccharides, so far there has been very little discussion about the role of solvent models in the molecular dynamics simulations of these molecules. Predominantly, the TIP3P water model is commonly used as one of the most popular explicit water models in general. However, there are numerous alternative explicit and implicit water models that are neglected in the computational research of GAGs. Since solvent-mediated interactions are particularly important for GAG dynamic and structural properties, it would be of great interest for the GAG community to establish the solvent model that is suited the best in terms of the quality of theoretically obtained GAG parameters and, at the same time, would be reasonably demanding in terms of computational resources required. In this study, heparin (HP) was simulated using five implicit and six explicit solvent models with the aim to find out how different solvent models influence HP's molecular descriptors in the molecular dynamics simulations. Here, we initiate the search for the most appropriate solvent representation for GAG systems and we hope to encourage other groups to contribute to this highly relevant subject.

Cationic Carrier Mediated Delivery of Anionic Contrast Agents in Low Doses Enable Enhanced Computed Tomography Imaging of Cartilage for Early Osteoarthritis Diagnosis.

Cartilage tissue exhibits early degenerative changes with onset of osteoarthritis (OA). Early diagnosis is critical as there is only a narrow time window during which therapeutic intervention can reverse disease progression. Computed tomography (CT) has been considered for cartilage imaging as a tool for early OA diagnosis by introducing radio-opaque contrast agents like ioxaglate (IOX) into the joint. IOX, however, is anionic and thus repelled by negatively charged cartilage glycosaminoglycans (GAGs) that hinders its intra-tissue penetration and partitioning, resulting in poor CT attenuation. This is further complicated by its short intra-tissue residence time owing to rapid clearance from joints, which necessitates high doses causing toxicity concerns. Here we engineer optimally charged cationic contrast agents based on cartilage negative fixed charge density by conjugating cartilage targeting a cationic peptide carrier (CPC) and multi-arm avidin nanoconstruct (mAv) to IOX, such that they can penetrate through the full thickness of cartilage within 6 h using electrostatic interactions and elicit similar CT signal with about 40× lower dose compared to anionic IOX. Their partitioning and distribution correlate strongly with spatial GAG distribution within healthy and early- to late-stage arthritic bovine cartilage tissues at 50-100× lower doses than other cationic contrast agents used in the current literature. The use of contrast agents at low concentrations also allowed for delineation of cartilage from subchondral bone as well as other soft tissues in rat tibial joints. These contrast agents are safe to use at current doses, making CT a viable imaging modality for early detection of OA and staging of its severity.

Marine Gelatin-Methacryloyl-Based Hydrogels as Cell Templates for Cartilage Tissue Engineering.

Marine-origin gelatin has been increasingly used as a safe alternative to bovine and porcine ones due to their structural similarity, avoiding the health-related problems and sociocultural concerns associated with using mammalian-origin materials. Another benefit of marine-origin gelatin is that it can be produced from fish processing-products enabling high production at low cost. Recent studies have demonstrated the excellent capacity of gelatin-methacryloyl (GelMA)-based hydrogels in a wide range of biomedical applications due to their suitable biological properties and tunable physical characteristics, such as tissue engineering applications, including the engineering of cartilage. In this study, fish gelatin was obtained from Greenland halibut skins by an acidic extraction method and further functionalized by methacrylation using methacrylic anhydride, developing a photosensitive gelatin-methacryloyl (GelMA) with a degree of functionalization of 58%. The produced marine GelMA allowed the fabrication of photo-crosslinked hydrogels by incorporating a photoinitiator and UV light exposure. To improve the biological performance, GelMA was combined with two glycosaminoglycans (GAGs): hyaluronic acid (HA) and chondroitin sulfate (CS). GAGs methacrylation reaction was necessary, rendering methacrylated HA (HAMA) and methacrylated CS (CSMA). Three different concentrations of GelMA were combined with CSMA and HAMA at different ratios to produce biomechanically stable hydrogels with tunable physicochemical features. The 20% (w/v) GelMA-based hydrogels produced in this work were tested as a matrix for chondrocyte culture for cartilage tissue engineering with formulations containing both HAMA and CSMA showing improved cell viability. The obtained results suggest these hybrid hydrogels be used as promising biomaterials for cartilage tissue engineering applications.

Hyaluronan and Reactive Oxygen Species Signaling-Novel Cues from the Matrix?

Hyaluronan (HA) is a naturally occurring non-sulfated glycosaminoglycan (GAG) localized to the cell surface and the tissue extracellular matrix (ECM). It is composed of disaccharides containing glucuronic acid and N-acetylglucosamine, is synthesized by the HA synthase (HAS) enzymes and is degraded by hyaluronidase (HYAL) or reactive oxygen and nitrogen species (ROS/RNS) actions. HA is deposited as a high molecular weight (HMW) polymer and degraded to low molecular weight (LMW) fragments and oligosaccharides. HA affects biological functions by interacting with HA-binding proteins (hyaladherins). HMW HA is anti-inflammatory, immunosuppressive, and antiangiogenic, whereas LMW HA has pro-inflammatory, pro-angiogenetic, and oncogenic effects. ROS/RNS naturally degrade HMW HA, albeit at enhanced levels during tissue injury and inflammatory processes. Thus, the degradation of endothelial glycocalyx HA by increased ROS challenges vascular integrity and can initiate several disease progressions. Conversely, HA exerts a vital role in wound healing through ROS-mediated HA modifications, which affect the innate immune system. The normal turnover of HA protects against matrix rigidification. Insufficient turnover leads to increased tissue rigidity, leading to tissue dysfunction. Both endogenous and exogenous HMW HA have a scavenging capacity against ROS. The interactions of ROS/RNS with HA are more complex than presently perceived and present an important research topic.

Gene therapy for cross-correction of somatic organs and the CNS in mucopolysaccharidosis II in rodents and non-human primates

Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.

Fucosylated heparan sulfate from the midgut gland of Patinopecten yessoensis

The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1H NMR detected significant signals near 1.2–1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC–MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl-L-fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl-L-fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities.

Simultaneous production of N-acetylheparosan and recombinant chondroitin using gene-engineered Escherichia coli K5

N-Acetylheparosan and chondroitin are increasingly needed as alternative sources of animal-derived sulfated glycosaminoglycans (GAGs) and as inert substances in medical devices and pharmaceuticals. The N-acetylheparosan productivity of E. coli K5 has achieved levels of industrial applications, whereas E.coli K4 produces a relatively lower amount of fructosylated chondroitin. In this study, the K5 strain was gene-engineered to co-express K4-derived, chondroitin-synthetic genes, namely kfoA and kfoC. The productivities of total GAG and chondroitin in batch culture were 1.2 g/L and 1.0 g/L respectively, which were comparable to the productivity of N-acetylheparosan in the wild K5 strain (0.6–1.2 g/L). The total GAG of the recombinant K5 was partially purified by DEAE-cellulose chromatography and was subjected to degradation tests with specific GAG-degrading enzymes combined with HPLC and 1H NMR analyses. The results indicated that the recombinant K5 simultaneously produced both 100-kDa chondroitin and 45-kDa N-acetylheparosan at a weight ratio of approximately 4:1. The content of chondroitin in total GAG partially purified was 73.2%. The molecular weight of recombinant chondroitin (100 kDa) was 5–10 times higher than that of commercially available chondroitin sulfate. These results indicated that the recombinant K5 strain acquired the chondroitin-producing ability without altering the total GAG productivity of the host.

Urinary Glycosaminoglycans Are Associated with Recurrent UTI and Urobiome Ecology in Postmenopausal Women

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units of uronic acid and amino sugars. The luminal surface of the bladder epithelium is coated with a GAG layer. These urothelial GAGs are thought to provide a protective barrier and serve as a potential interaction site with the urinary microbiome (urobiome). Previous studies have profiled urinary GAG composition in mixed cohorts, but the urinary GAG composition in postmenopausal women remains undefined. To investigate the relationship between GAGs and recurrent urinary tract infection (rUTI), we profiled urinary GAGs in a controlled cohort of postmenopausal women. We found that chondroitin sulfate (CS) is the major urinary GAG in postmenopausal women and that urinary CS was elevated in women with active rUTI. We also associated urinary GAGs with urobiome composition and identified bacterial species that significantly associated with urinary GAG concentration. Corynebacterium amycolatum, Porphyromonas somerae, and Staphylococcus pasteuri were positively associated with heparin sulfate or hyaluronic acid, and bacterial species associated with vaginal dysbiosis were negatively correlated with urinary CS. Altogether, this work defines changes in urinary GAG composition associated with rUTI and identifies new associations between urinary GAGs and the urobiome that may play a role in rUTI pathobiology.

Interleukin-1β and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro

To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via real-time polymerase chain reaction (rtPCR). Culture media matrix metalloproteinase (MMP) -3 and -13 concentrations were quantified via ELISA. Total glycosaminoglycan (GAG) content in the cell pellets and culture media was also assessed. IL-1β and IL-1β combined with MPA significantly downregulated ECM gene expression to a greater extent in NBF cells compared with DDFT cells. IL-1β and IL-1β combined with MPA significantly upregulated MMP-3 culture media concentrations in DDFT cells only, and MMP-13 culture media concentrations to a greater extent in NBF cells compared with DDFT cells. NBF cells were more susceptible to IL-1β and MPA-mediated ECM gene expression downregulation in vitro. These results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolve the inflammation as well as take into consideration NBF cell biosynthesis in horses with navicular disease, for which currently no information exists.

Mucopolysaccharidoses Differential Diagnosis by Mass Spectrometry-Based Analysis of Urine Free Glycosaminoglycans-A Diagnostic Prediction Model.

Impaired glycosaminoglycans (GAGs) catabolism may lead to a cluster of rare metabolic and genetic disorders called mucopolysaccharidoses (MPSs). Each subtype is caused by the deficiency of one of the lysosomal hydrolases normally degrading GAGs. Affected tissues accumulate undegraded GAGs in cell lysosomes and in the extracellular matrix, thus leading to the MPS complex clinical phenotype. Although each MPS may present with recognizable signs and symptoms, these may often overlap between subtypes, rendering the diagnosis difficult and delayed. Here, we performed an exploratory analysis to develop a model that predicts MPS subtypes based on UHPLC-MS/MS measurement of a urine free GAG profile (or GAGome). We analyzed the GAGome of 78 subjects (38 MPS, 37 healthy and 3 with other MPS symptom-overlapping disorders) using a standardized kit in a central-blinded laboratory. We observed several MPS subtype-specific GAGome changes. We developed a multivariable penalized Lasso logistic regression model that attained 91.2% balanced accuracy to distinguish MPS type II vs. III vs. any other subtype vs. not MPS, with sensitivity and specificity ranging from 73.3% to 91.7% and from 98.4% to 100%, depending on the predicted subtype. In conclusion, the urine GAGome was revealed to be useful in accurately discriminating the different MPS subtypes with a single UHPLC-MS/MS run and could serve as a reliable diagnostic test for a more rapid MPS biochemical diagnosis.

Type III mucopolysaccharidosis: literature review and clinical observation

Introduction. Mucopolysaccharidosis type III (Sanfilippo syndrome) is a rare multi-stem disease caused by the accumulation of glycosaminoglycans (GAG) in the cells of various organs, leading to a violation of their function, specific phentopic signs and progressive neurocognitive disorders. Neurologic manifestations are leading in the clinical picture of the disease, as by the time of manifestation, and by severity. In most patients, in the first and even second years of life, children have normal development or indistinctly pronounced deviations. In the absence of substitute enzyme therapy for this type of MPS, a severe delay in intellectual and speech development develops rapidly, recurrent respiratory episodes in the form of pneumonia and bronchial obstruction are observed, the child is disabled early. Polymorphism and non-specificity of clinical manifestations, lack of alertness of doctors to orphan diseases are a common cause of late diagnosis of MPS. The article presents an overview of data on the prevalence, genetic and phenotypic variants of type III mucopolysaccharidosis, features of patient management and presentation of clinical observation of a child with this pathology.Clinical observation. We present our own prolonged clinical observation of a type III MPS case in a patient who has been under our supervision for 12 years. The diagnosis was established and confirmed at the age of three years. The disease was manifested by neuropsychiatric regression and systemic somatic manifestations. Motor deficits, cognitive impairments with the development of dementia and recurrent aspiration syndrome progressed in the dynamics of the child.Conclusions. The classical clinical picture of type III MPS is distinguished by the aggressive development of cognitive and motor disorders at 2–3 years of life, characteristic phenotypic and somatic manifestations of the disease. This case demonstrates the multiplicity of problems and the need for interaction between doctors of various specialties.

Endoscopic and Image Analysis of the Airway in Patients with Mucopolysaccharidosis Type IVA.

Mucopolysaccharidosis (MPS) is a hereditary disorder arising from lysosomal enzymes deficiency, with glycosaminoglycans (GAGs) storage in connective tissues and bones, which may compromise the airway. This retrospective study evaluated patients with MPS type IVA with airway obstruction detected via endoscopy and imaging modalities and the effects of surgical interventions based on symptoms. The data of 15 MPS type IVA patients (10 males, 5 females, mean age 17.8 years) were reviewed in detail. Fiberoptic bronchoscopy (FB) was used to distinguish adenotonsillar hypertrophy, prolapsed soft palate, secondary laryngomalacia, vocal cord granulation, cricoid thickness, tracheal stenosis, shape of tracheal lumen, nodular deposition, tracheal kinking, tracheomalacia with rigid tracheal wall, and bronchial collapse. Computed tomography (CT) helped to measure the deformed sternal angle, the cross-sectional area of the trachea, and its narrowest/widest ratio (NW ratio), while angiography with 3D reconstruction delineated tracheal torsion, kinking, or framework damage and external vascular compression of the trachea. The NW ratio correlated negatively with age (p < 0.01), showing that airway obstruction progressed gradually. Various types of airway surgery were performed to correct the respiratory dysfunction. MPS type IVA challenges the management of multifactorial airway obstruction. Preoperative airway evaluation with both FB and CT is strongly suggested to assess both intraluminal and extraluminal factors causing airway obstruction.

The Interplay of Glycosaminoglycans and Cysteine Cathepsins in Mucopolysaccharidosis.

Mucopolysaccharidosis (MPS) consists of a group of inherited lysosomal storage disorders that are caused by a defect of certain enzymes that participate in the metabolism of glycosaminoglycans (GAGs). The abnormal accumulation of GAGs leads to progressive dysfunctions in various tissues and organs during childhood, contributing to premature death. As the current therapies are limited and inefficient, exploring the molecular mechanisms of the pathology is thus required to address the unmet needs of MPS patients to improve their quality of life. Lysosomal cysteine cathepsins are a family of proteases that play key roles in numerous physiological processes. Dysregulation of cysteine cathepsins expression and activity can be frequently observed in many human diseases, including MPS. This review summarizes the basic knowledge on MPS disorders and their current management and focuses on GAGs and cysteine cathepsins expression in MPS, as well their interplay, which may lead to the development of MPS-associated disorders.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Quantification of Glycosaminoglycans in Urine by Isotope‐Dilution Liquid Chromatography‐Electrospray Ionization Tandem Mass Spectrometry

Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of the methanolysis time Support Protocol 5: Measurement of the concentration of methanolic HCl.