Glycosaminoglycan Chains Research Articles

Azide-Functionalized Naphthoxyloside as a Tool for Glycosaminoglycan Investigations.

We present a xylosylated naphthoxyloside carrying a terminal azide functionality that can be used for conjugation using click chemistry. We show that this naphthoxyloside serves as a substrate for β4GalT7 and induces the formation of soluble glycosaminoglycan (GAG) chains with physiologically relevant lengths and sulfation patterns. Finally, we demonstrate its usefulness by conjugation to the Alexa Fluor 647 and TAMRA fluorophores and coupling to a surface plasmon resonance chip for interaction studies with the hepatocyte growth factor known to interact with the GAG heparan sulfate.

Inherited Proteoglycan Biosynthesis Defects—Current Laboratory Tools and Bikunin as a Promising Blood Biomarker

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases.

Analytical challenges of glycosaminoglycans at biological interfaces

The analysis of glycosaminoglycans (GAGs) is a challenging task due to their high structural heterogeneity, which results in diverse GAG chains with similar chemical properties. Simultaneously, it is of high importance to understand their role and behavior in biological systems. It has been known for decades now that GAGs can interact with lipid molecules and thus contribute to the onset of atherosclerosis, but their interactions at and with biological interfaces, such as the cell membrane, are yet to be revealed. Here, analytical approaches that could yield important knowledge on the GAG-cell membrane interactions as well as the synthetic and analytical advances that make their study possible are discussed. Due to recent developments in laser technology, we particularly focus on nonlinear spectroscopic methods, especially vibrational sum-frequency generation spectroscopy, which has the potential to unravel the structural complexity of heterogeneous biological interfaces in contact with GAGs, in situ and in real time.Graphical abstract

Myopathy Associated With Dermatan Sulfate-Deficient Decorin and Myostatin in Musculocontractural Ehlers-Danlos Syndrome: A Mouse Model Investigation.

Carbohydrate sulfotransferase 14 (CHST14) encodes dermatan 4-O-sulfotransferase 1, a critical enzyme for dermatan sulfate (DS) biosynthesis. Musculocontractural Ehlers-Danlos syndrome (mcEDS) is associated with biallelic pathogenic variants of CHST14 and is characterized by malformations and manifestations related to progressive connective tissue fragility. We identified myopathy phenotypes in Chst14-deficient mice using an mcEDS model. Decorin is a proteoglycan harboring a single glycosaminoglycan chain containing mainly DS, which are replaced with chondroitin sulfate (CS) in mcEDS patients with CHST14 deficiency. We studied the function of decorin in the skeletal muscle of Chst14-deficient mice because decorin is important for collagen-fibril assembly and has a myokine role in promoting muscle growth. Although decorin was present in the muscle perimysium of wild-type (Chst14+/+) mice, decorin was distributed in the muscle perimysium as well as in the endomysium of Chst14–/– mice. Chst14–/– mice had small muscle fibers within the spread interstitium; however, histopathological findings indicated milder myopathy in Chst14–/– mice. Myostatin, a negative regulator of protein synthesis in the muscle, was upregulated in Chst14–/– mice. In the muscle of Chst14–/– mice, decorin was downregulated compared to that in Chst14+/+ mice. Chst14–/– mice showed altered cytokine/chemokine balance and increased fibrosis, suggesting low myogenic activity in DS-deficient muscle. Therefore, DS deficiency in mcEDS causes pathological localization and functional abnormalities of decorin, which causes disturbances in skeletal muscle myogenesis.

Assays for Evaluation of Substrates for and Inhibitors of β-1,4-Galactosyltransferase 7.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by β4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl β-D-xylopyranoside. We have cloned and expressed β4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of β4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of β4GalT7.

Imaging Glycosaminoglycan Modification Patterns In Vivo.

Glycosaminoglycans (GAGs) such as heparan sulfates (HS) or chondroitin sulfates (CS) are long unbranched polymers of a disaccharide comprised of hexuronic acid and hexosamine. Attached to a protein backbone via a characteristic tetrasaccharide, the GAG chains are non-uniformly modified by sulfations, epimerizations, and deacetylations. The resultant glycan chains contain highly modified domains, separated by sections of sparse or no modifications. These GAG domains are central to the role of glycans in binding to proteins and mediating protein-protein interactions. Since HS and CS domains are not genetically encoded, they cannot be visualized and studied with conventional methods in vivo. We describe a transgenic approach using single chain variable fragment (scFv) antibodies that bind HS or CS. By transgenically expressing fluorescently tagged scFv antibodies, we can directly visualize both HS and CS domains in live Caenorhabditis elegans revealing unprecedented cellular specificity and evolutionary conservation (Attreed et al., Nat Methods 9(5): 477-479, 2012; Attreed et al., Glycobiology 26(8): 862-870, 2016) (unpublished). The approach allows concomitant co-labeling of multiple GAG domains, the study of GAG dynamics, and could lend itself to a genetic analysis of GAG domain biosynthesis or function.

Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis and Sorting in Filter-Grown and Polarized Epithelial Cells.

Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown on filters for more than three decades. Growing such cells on permeable support allows for nutrient uptake via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter. While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with 35S-labeled sulfate enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG) chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and transport.

Identification of Cell Autonomous and Non-Cell Autonomous Functions of Heparan Sulfate Glycosaminoglycan Chains by Creating Chimeric Mouse Embryos.

Cell surface-tethered heparan sulfate glycosaminoglycan chains primarily function in a cell autonomous manner, while extracellular matrix-associated heparan sulfate glycosaminoglycan chains function in a non-cell autonomous manner. In addition, the cleaved forms of cell surface-tethered heparan sulfate chains enzymatically released by proteases and heparanases, called shedding, can contribute to non-cell autonomous mechanisms. The movement of heparan sulfate chains to surrounding cells mediated by transcytosis or filopodia also involves another non-cell autonomous mechanism. To determine cell autonomous or non-cell autonomous roles of heparan sulfate glycosaminoglycan chains during early embryogenesis, direct conclusions can be drawn by analyzing chimeric embryos which are composed of wild-type and heparan sulfate glycosaminoglycan chain-deficient cells. Here, we describe methods of production of these chimeric embryos and analysis of their cellular phenotypes with immunohistochemistry at a single-cell level.

Xyloside Derivatives as Molecular Tools to Selectively Inhibit Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects, making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner. Reversible and selective inhibition of GAG biosynthesis would allow us to examine the importance of endogenous GAGs to specific cellular, tissue, or organ systems. In this chapter, we describe the chemical synthesis and biological evaluation of xyloside derivatives as selective inhibitors of heparan sulfate and chondroitin/dermatan sulfate proteoglycan biosynthesis.

Natural Presentation of Glycosaminoglycans in Synthetic Matrices for 3D Angiogenesis Models.

Glycosaminoglycans (GAGs) are long, linear polysaccharides that occur in the extracellular matrix of higher organisms and are either covalently attached to protein cores, as proteoglycans or in free form. Dependent on their chemical composition and structure, GAGs orchestrate a wide range of essential functions in tissue homeostasis. Accordingly, GAG-based biomaterials play a major role in tissue engineering. Current biomaterials exploit crosslinks between chemically modified GAG chains. Due to modifications along the GAG chains, they are limited in their GAG-protein interactions and accessibility to dissect the biochemical and biophysical properties that govern GAG functions. Herein, a natural presentation of GAGs is achieved by a terminal immobilization of GAGs to a polyethylene glycol (PEG) hydrogel. A physicochemical characterization showed that different end-thiolated GAGs can be incorporated within physiological concentration ranges, while the mechanical properties of the hydrogel are exclusively tunable by the PEG polymer concentration. The functional utility of this approach was illustrated in a 3D cell culture application. Immobilization of end-thiolated hyaluronan enhanced the formation of capillary-like sprouts originating from embedded endothelial cell spheroids. Taken together, the presented PEG/GAG hydrogels create a native microenvironment with fine-tunable mechanobiochemical properties and are an effective tool for studying and employing the bioactivity of GAGs.

Modification of gingival proteoglycans by reactive oxygen species: potential mechanism of proteoglycan degradation during periodontal diseases

Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.

Conformational Properties of Glycosaminoglycan Disaccharides: A Molecular Dynamics Study.

The structure and conformation of glycosaminoglycans (GAGs) are of central importance to understand the mechanisms behind their functions in biological systems. Due to the inherent chemical and structural heterogeneity of GAGs, focusing on longer, naturally existing GAG chains hinders drawing conclusions on the influence of the chemical functionalization on the basic conformational degree of freedom, that is, the dynamic shape of glycosidic linkage present in the particular disaccharide repeating unit. In the present study, we have considered the complete set of 106 GAG-related disaccharides, being potential building blocks for longer GAG chains (including hyaluronan, chondroitin, keratan, dermatan, and heparan). Both the unfunctionalized units and all possible combinations of either partially or fully sulfated derivatives contribute to this number. The unbiased and enhanced sampling molecular dynamics simulations provide a link to understand the influence of sulfation on the conformational properties of GAG glycosidic linkages. Residue-residue hydrogen bonding is not significant for either the glycosidic linkage conformation or its flexibility. It was found that in the majority of cases, the dominating conformation of the linkage is weakly affected by sulfation and the main role is played by the steric and stereoelectronic effects. However, there exist numerous cases where sulfation increases the contribution of alternative conformations to a nonnegligible extent and, in some rare cases (restricted to disaccharides building heparan), leads to the reorientation of the glycosidic linkage. The identified sulfation sites, being the most important in this context, are C6 and C3 at the GlcNAc residue. Finally, the full set of free energy maps relying on the glycosidic dihedral angle values for diverse GAG disaccharides are provided; they may be used for further studies, focused on longer GAG chains.

Role of Glycosylation in Vascular Calcification.

Glycosylation is an important step in post-translational protein modification. Altered glycosylation results in an abnormality that causes diseases such as malignancy and cardiovascular diseases. Recent emerging evidence highlights the importance of glycosylation in vascular calcification. Two major types of glycosylation, N-glycosylation and O-glycosylation, are involved in vascular calcification. Other glycosylation mechanisms, which polymerize the glycosaminoglycan (GAG) chain onto protein, resulting in proteoglycan (PG), also have an impact on vascular calcification. This paper discusses the role of glycosylation in vascular calcification.

Inhibition of glucose use improves structural recovery of injured Achilles tendon in mice.

Injured tendons do not regain their native structure except at fetal or very young ages. Healing tendons often show mucoid degeneration involving accumulation of sulfated glycosaminoglycans (GAGs), but its etiology and molecular base have not been studied substantially. We hypothesized that quality and quantity of gene expression involving the synthesis of proteoglycans having sulfated GAGs are altered in injured tendons and that a reduction in synthesis of sulfated GAGs improves structural and functional recovery of injured tendons. C57BL6/j mice were subjected to Achilles tendon tenotomy surgery. The injured tendons accumulated sulfate proteoglycans as early as 1-week postsurgery and continued so by 4-week postsurgery. Transcriptome analysis revealed upregulation of a wide range of proteoglycan genes that have sulfated GAGs in the injured tendons 1 and 3 weeks postsurgery. Genes critical for enzymatic reaction of initiation and elongation of chondroitin sulfate GAG chains were also upregulated. After the surgery, mice were treated with the 2-deoxy-d-glucose (2DG) that inhibits conversion of glucose to glucose-6-phosphate, an initial step of glucose metabolism as an energy source and precursors of monosaccharides of GAGs. The 2DG treatment reduced accumulation of sulfated proteoglycans, improved collagen fiber alignment, and reduced the cross-sectional area of the injured tendons. The modulus of the 2DG-treated groups was higher than that in the vehicle group, but not of statistical significance. Our findings suggest that mucoid degeneration in injured tendons may result from the upregulated expression of genes involved the synthesis of sulfate proteoglycans and can be inhibited by reduction of glucose utilization.

Phenotypic Characterization of Immortalized Chondrocytes from a Desbuquois Dysplasia Type 1 Mouse Model: A Tool for Studying Defects in Glycosaminoglycan Biosynthesis

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

YY-11, a camel milk-derived peptide, inhibits TGF-β-mediated atherogenic signaling in human vascular smooth muscle cells.

Atherosclerosis, the major underlying pathology of cardiovascular disease, commences with the binding and trapping of lipids on modified proteoglycans, with hyperelongated glycosaminoglycan chains. Transforming growth factor (TGF)-β stimulates glycosaminoglycan elongation in vascular smooth muscle cells. We have recently shown that this TGF-β signaling pathway involves reactive oxygen species (ROS). YY-11 is a dodecapeptide derived from camel milk and it has antioxidant activity. We have investigated the role of YY-11 in blocking ROS signaling and downstream atherogenic responses. YY-11 inhibited TGF-β stimulated ROS production and inhibited the expression of genes for glycosaminoglycan chain elongation as a component of an invitro model of atherosclerosis. This study provides a biochemical mechanism for the role of camel milk as a potential nutritional product to contribute to the worldwide amelioration of cardiovascular disease. PRACTICAL APPLICATIONS: The identification of readily accessible foods with antioxidant properties would provide a convenient and cost-effective approach community wide reducing oxidative stress induced pathologies such as atherosclerosis. We demonstrate that camel milk-derived peptide is an antioxidant that can inhibit growth factor-mediated proteoglycan modification in vitro. As proteoglycan modification is being recognized as one of the earliest atherogenic responses, these data support the notion of camel milk as a suitable nutritional product to contribute to the prevention of early stage of atherosclerosis development.

Proteoglycan synthesis in conserved oligomeric Golgi subunit deficient HEK293T cells is affected differently, depending on the lacking subunit.

The Conserved Oligomeric Golgi (COG) complex is an eight subunit protein complex associated with Golgi membranes. Genetic defects affecting individual COG subunits cause congenital disorders of glycosylation (CDGs), due to mislocalization of Golgi proteins involved in glycosylation mechanisms. While the resulting defects in N-and O-glycosylation have been extensively studied, no corresponding study of proteoglycan (PG) synthesis has been undertaken. We here show that glycosaminoglycan (GAG) modification of PGs is significantly reduced, regardless which COG subunit that is missing in HEK293T cells. Least reduction was observed for cells lacking COG1 and COG8 subunits, that bridge the A and B lobes of the complex. Lack of these subunits did not reduce GAG chain lengths of secreted PGs, which was reduced in cells lacking any other subunit (COG2-7). COG3 knock out (KO) cells had particularly reduced ability to polymerize GAG chains. For cell-associated GAGs, the mutant cell lines, except COG4 and COG7 KO, displayed longer GAG chains than wild-type cells, indicating that COG subunits play a role in cellular turnover of PGs. In light of the important roles PGs play in animal development, the effects KO of individual COG subunits have on GAG synthesis could explain the variable severity of COG associated CDGs.

Sulfation of Glycosaminoglycans Modulates the Cell Cycle of Embryonic Mouse Spinal Cord Neural Stem Cells.

In the developing spinal cord neural stem and progenitor cells (NSPCs) secrete and are surrounded by extracellular matrix (ECM) molecules that influence their lineage decisions. The chondroitin sulfate proteoglycan (CSPG) DSD-1-PG is an isoform of receptor protein tyrosine phosphatase-beta/zeta (RPTPβ/ζ), a trans-membrane receptor expressed by NSPCs. The chondroitin sulfate glycosaminoglycan chains are sulfated at distinct positions by sulfotransferases, thereby generating the distinct DSD-1-epitope that is recognized by the monoclonal antibody (mAb) 473HD. We detected the epitope, the critical enzymes and RPTPβ/ζ in the developing spinal cord. To obtain insight into potential biological functions, we exposed spinal cord NSPCs to sodium chlorate. The reagent suppresses the sulfation of glycosaminoglycans, thereby erasing any sulfation code expressed by the glycosaminoglycan polymers. When NSPCs were treated with chlorate and cultivated in the presence of FGF2, their proliferation rate was clearly reduced, while NSPCs exposed to EGF were less affected. Time-lapse video microscopy and subsequent single-cell tracking revealed that pedigrees of NSPCs cultivated with FGF2 were strongly disrupted when sulfation was suppressed. Furthermore, the NSPCs displayed a protracted cell cycle length. We conclude that the inhibition of sulfation with sodium chlorate interferes with the FGF2-dependent cell cycle progression in spinal cord NSPCs.

Investigation of the Experimental Parameters of Ultraviolet Photodissociation for the Structural Characterization of Chondroitin Sulfate Glycosaminoglycan Isomers.

Glycosaminoglycans (GAGs) are linear polysaccharides that participate in a broad range of biological functions. Their incomplete biosynthesis pathway leads to nonuniform chains and complex mixtures. For this reason, the characterization of GAGs has been a difficult hurdle for the analytical community. Recently, ultraviolet photodissociation (UVPD) has emerged as a useful tool for determining sites of modification within a GAG chain. Here, we investigate the ability for UVPD to distinguish chondroitin sulfate epimers and the effects of UVPD experimental parameters on fragmentation efficiency. Chondroitin sulfate A (CS-A) and chondroitin sulfate B (CS-B), commonly referred to as dermatan sulfate (DS), differ only in C-5 uronic acid stereochemistry. This uronic acid difference can influence GAG-protein binding and therefore can alter the specific biological function of a GAG chain. Prior tandem mass spectrometry methods investigated for the elucidation of GAG structures also have difficulty differentiating 4-O from 6-O sulfation in chondroitin sulfate GAGs. Preliminary data using UVPD to characterize GAGs showed a promising ability to characterize 4-O sulfation in CS-A GAGs. Here, we look in depth at the capability of UVPD to distinguish chondroitin sulfate C-5 diastereomers and the role of key experimental parameters in making this distinction. Results using a 193 nm excimer laser and a 213 nm solid-state laser are compared for this study. The effect of precursor ionization state, the number of laser pulses (193 or 213 nm UVPD), and the use of the low-pressure versus high-pressure trap are investigated.

Specific heparan sulfate modifications stabilize the synaptic organizer MADD-4/Punctin at Caenorhabditis elegans neuromuscular junctions.

Heparan sulfate (HS) proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by HS modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding HS modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions (NMJs). Using single chain antibodies that recognize different HS modification patterns, we show in vivo that these two HS epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan ortholog, at NMJs. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique HS modification patterns at different NMJs. Moreover, while most enzymes are individually dispensable for proper organization of NMJs, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.