Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems

Glycosaminoglycans (GAGs) are natural, linear and negatively charged heteropolysaccharides which are incident in every mammalian tissue. They consist of repeating disaccharide units, which are composed of either sulfated or non-sulfated monosaccharides. Depending on tissue types, GAGs exhibit structural heterogeneity such as the position and degree of sulfation or within their disaccharide units composition being heparin, heparan sulfate, chondroitine sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. They are covalently linked to a core protein (proteoglycans) or as free chains (hyaluronan). GAGs affect cell properties and functions either by direct interaction with cell receptors or by sequestration of growth factors. These evidences of divert biological roles of GAGs make their characterization at cell and tissue levels of importance. Thus, non-invasive techniques are interesting to investigate, to qualitatively and quantitatively characterize GAGs in vitro in order to use them as diagnostic biomarkers and/or as therapeutic targets in several human diseases including cancer. Infrared and Raman microspectroscopies and imaging are sensitive enough to differentiate and classify GAG types and subtypes in spite of their close molecular structures. Spectroscopic markers characteristic of reference GAG molecules were identified. Beyond these investigations of the standard GAG spectral signature, infrared and Raman spectral signatures of GAG were searched in complex biological systems like cells. The aim of the present review is to describe the implementation of these complementary vibrational spectroscopy techniques, and to discuss their potentials, advantages and disadvantages for GAG analysis. In addition, this review presents new data as we show for the first time GAG infrared and Raman spectral signatures from conditioned media and live cells, respectively.     

A chip‐based amide‐HILIC LC/MS platform for glycosaminoglycan glycomics profiling

A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable, and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation, and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple MS with an on‐line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide–silica hydrophilic interaction chromatography (HILIC) in a chip‐based format for LC/MS of heparin, heparan sulfate (HS) GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built‐in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate (Ac) groups and acidic sulfate groups. In summary, chip based amide‐HILIC LC/MS is an enabling technology for GAG glycomics profiling.     

Orbitrap mass spectrometry characterization of hybrid chondroitin/dermatan sulfate hexasaccharide domains expressed in brain

In the central nervous system, chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) modulate neurotrophic effects and glial cell maturation during brain development. Previous reports revealed that GAG composition could be responsible for CS/DS activities in brain. In this work, for the structural characterization of DS- and CS-rich domains in hybrid GAG chains extracted from neural tissue, we have developed an advanced approach based on high-resolution mass spectrometry (MS) using nanoelectrospray ionization Orbitrap in the negative ion mode. Our high-resolution MS and multistage MS approach was developed and applied to hexasaccharides obtained from 4- and 14-week-old mouse brains by GAG digestion with chondroitin B and in parallel with AC I lyase. The expression of DS- and CS-rich domains in the two tissues was assessed comparatively. The analyses indicated an age-related structural variability of the CS/DS motifs. The older brain was found to contain more structures and a higher sulfation of DS-rich regions, whereas the younger brain was found to be characterized by a higher sulfation of CS-rich regions. By multistage MS using collision-induced dissociation, we also demonstrated the incidence in mouse brain of an atypical [4,5-Δ-GlcAGalNAc(IdoAGalNAc)₂], presenting a bisulfated CS disaccharide formed by 3-O-sulfate-4,5-Δ-GlcA and 6-O-sulfate-GalNAc moieties.     

An automated mass spectrometry-based screening method for analysis of sulfated glycosaminoglycans

Glycosaminoglycans (GAGs) are linear polysaccharides, consisting of repeated disaccharide units, attached to core proteins in all multicellular organisms. Chondroitin sulfate (CS) and dermatan sulfate (DS) constitute a subgroup of sulfated GAGs for which the degree of sulfation varies between species and tissues. One major goal in GAG characterization is to correlate structure to function. A common approach is to exhaustively degrade the GAG chains and thereafter determine the amount of component disaccharide units. In large-scale studies, there is a need for high-throughput screening methods since existing methods are either very time- or samples consuming. Here, we present a new strategy applying MALDI-TOF MS in positive ion mode for semi-qualitative and quantitative analysis of CS/DS derived disaccharide units. Only a few picomoles of sample are required per analysis and 10 samples can be analyzed in 25 min, which makes this approach an attractive alternative to many established assay methods. The total CS/DS concentration in 19 samples derived from Caenorhabditis elegans and mammalian tissues and cells was determined. The obtained results were well in accordance with concentrations determined by a standard liquid chromatography-based method, demonstrating the applicability of the method for samples from various biological matrices containing CS/DS of different sulfation degrees.     

Galectin 1 inhibits incorporation of vitronectin and chondroitin sulfate B into the extracellular matrix of human vascular smooth muscle cells

Galectin-1, a beta-galactoside-binding dimeric lectin, interacts with the extracellular matrix (ECM) of smooth muscle cells (SMCs) and with particular ECM proteins. Enrichment of the ECM with galectin-1 affects adhesion and proliferation of cultured SMCs. Here we investigated whether galectin-1 (1) interacts with glycosaminoglycan (GAG) chains, (2) cross-links between ligands and facilitates the incorporation of GAGs, vitronectin and plasma fibronectin in the ECM of vascular SMCs. A recombinant galectin-1 fusion protein GalH, used in this study, formed dimers and interacted with ECM proteins. GAG chains inhibited these interactions. Among the studied GAG chains, only chondroitin sulfate B interacted with GalH in beta-galactoside-dependent manner. GalH did not bridge between ECM proteins on solid phase and [125I]-labelled ECM proteins or GAGs in solution. The ECM incorporated less vitronectin in the presence of soluble GalH. GalH-enriched ECM incorporated less vitronectin and chondroitin sulfate B. The ECM partially depleted of endogenous galectins incorporated more chondroitin sulfate B compared to untreated ECM. These results suggest that galectin-1 is likely to be involved in the ECM assembly affecting incorporation of some ECM components important for SMC behaviour.     

Glycosaminoglycan-dependent and -independent inhibition of neurite outgrowth by agrin

Agrin is a proteoglycan that can inhibit neurite outgrowth from multiple neuronal types when present as a substrate. Agrin's neurite inhibitory activity is confined to the N-terminal segment of the protein (agrin N150), which contains heparan sulfate (HS) and chondroitin sulfate (CS) side chains. We have examined the activities of various purified recombinant agrin fragments and their glycosaminoglycan (GAG) side chains in neurite outgrowth inhibition. Inhibitory activity was tested using dissociated chick ciliary ganglion neurons or dorsal root ganglion explants growing on laminin or N-cadherin. Initial experiments demonstrated that agrin N150 lacking GAG chains inhibited neurite outgrowth. Both halves of N150, each containing HS and/or CS side chains, could also inhibit neurite growth. Experiments using agrin fragments in which the GAG acceptor residues were mutated, or using agrin fragments purified from cells deficient in GAG synthesis, demonstrated that inhibition by the N-terminal portion of N150 requires GAGs, but that inhibition from the C-terminal part of N150 does not. Thus, the core protein or other types of glycosylation are important for inhibition from the more C-terminal region. Our results suggest that there are two distinct mechanisms for neurite outgrowth inhibition by agrin, one that is GAG-dependent and one that is GAG-independent.     

A New C-Xyloside Induces Modifications of GAG Expression,Structure and Functional Properties

Proteoglycans (PGs) are critically involved in major cellular processes. Most PG activities are due to the large interactive properties of their glycosaminoglycan (GAG) polysaccharide chains, whose expression and fine structural features are tightly controlled by a complex and highly regulated biosynthesis machinery. Xylosides are known to bypass PG-associated GAG biosynthesis and prime the assembly of free polysaccharide chains. These are, therefore, attractive molecules to interfere with GAG expression and function. Recently, we have developed a new xyloside derivative, C-Xyloside, that shares classical GAG-inducing xyloside activities while exhibiting improved metabolic stability. We have previously shown that C-Xyloside had beneficial effects on skin homoeostasis/regeneration using a number of models, but its precise effects on GAG expression and fine structure remained to be addressed. In this study, we have therefore investigated this in details, using a reconstructed dermal tissue as model. Our results first confirmed that C-Xyloside strongly enhanced synthesis of GAG chains, but also induced significant changes in their structure. C-Xyloside primed GAGs were exclusively chondroitin/dermatan sulfate (CS/DS) that featured reduced chain size, increased O-sulfation, and changes in iduronate content and distribution. Surprisingly, C-Xyloside also affected PG-borne GAGs, the main difference being observed in CS/DS 4-O/6-O-sulfation ratio. Such changes were found to affect the biological properties of CS/DS, as revealed by the significant reduction in binding to Hepatocyte Growth Factor observed upon C-Xyloside treatment. Overall, this study provides new insights into the effect of C-Xyloside on GAG structure and activities, which opens up perspectives and applications of such compound in skin repair/regeneration. It also provides a new illustration about the use of xylosides as tools for modifying GAG fine structure/function relationships.     

Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters

Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.     

Interactions of hepatocyte growth factor/scatter factor with various glycosaminoglycans reveal an important interplay between the presence of iduronate and sulfate density

Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N- and 6-O- positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.     

Comparative glycomics of connective tissue glycosaminoglycans

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. GAGs were extracted from joint tissues (cartilage, tendon, ligment, muscle, and synovium) and partially depolymerized using chondroitinase enzymes. The oligosaccharide products were differentially stable isotope labeled by reductive amination using 2-anthranilic acid-d(0) or -d(4) and subjected to amide-hydrophilic interaction chromatography (HILIC) online LC-MS/MS. The analysis presented herein enables simultaneous profiling of the expression of nonreducing end, linker region, and Delta-unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results provide important new information on the changes to the expression of CS GAG chains during disease and development.     

Glycosaminoglycan Chains Affect Exocytic and Endocytic Protein Traffic

Protein glycosylation such as N- and O-linked glycans as well as glycosaminoglycans (GAGs) have been shown to contribute to polarized sorting in epithelial cells. Here, we analyzed the effect of GAGs more generally on protein traffic also in non-polarized cells. Using short sequence tags of 10–17 amino acids encoding known GAG attachment sites, we have converted the asialoglycoprotein receptor H1, which constitutively cycles between the plasma membrane and endosomes, into a proteoglycan. Expressed in HeLa cells, the receptor was almost completely modified with a chondroitin sulfate chain and could be efficiently labeled by [³⁵S]sulfation. GAG attachment altered the steady-state distribution of the receptor by inhibiting endocytosis, while recycling was not affected. The reduced internalization is not the result of immobilization by interaction with the extracellular matrix, because fluorescence recovery after photobleaching did not detect an increased immobile fraction nor even a significant change in mobility. GAG chains furthermore accelerated Golgi-to-cell surface transport of H1. The same acceleration of export was also observed for a GAG-tagged version of the secretory protein α1-protease inhibitor, suggesting that this effect acts generally on proteoglycans, possibly by directing them into distinct carriers. Our results show novel roles of GAGs in protein sorting also in non-polarized cells.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

Anticoagulant motifs of marine sulfated glycans

Sulfated polysaccharides, like the glycosaminoglycan (GAG) heparin, are known to exhibit anticoagulant properties when certain structural features are present. The structural requirement for this action is well-established for heparin, in which a pentasaccharide motif plays a key role for keeping the high-affinity interaction to antithrombin. Over the last years of this glycomic era, several novel anticoagulant sulfated glycans have been described. Those from marine sources have been awakening special attention mainly because of their impressive anticoagulant effects together with structural uniqueness. The commonest of these glycans are the sulfated fucans (SFs), the sulfated galactans (SGs), and the marine invertebrate GAGs like the fucosylated chondroitin sulfate and ascidian dermatan sulfate. Since these marine sulfated glycans do not bear within their polymeric chains the specific pentasaccharide motif of heparin, other structural features must be necessary to trigger the anticoagulant effect. The objective of this report is to present the anticoagulant motifs of the marine SFs, SGs and GAGs.     

Glycosaminoglycans: Sorting determinants in intracellular protein traffic

Intracellular transport of proteins to their appropriate destinations is crucial for the maintenance of cellular integrity and function. Sorting information is contained either directly in the amino acid sequence or in a protein's post-translational modifications. Glycosaminoglycans (GAGs) are characteristic modifications of proteoglycans. GAGs are long unbranched polysaccharide chains with unique structural and functional properties also contributing to protein sorting in various ways. By deletion or insertion of GAG attachment sites it has been shown that GAGs affect polarized sorting in epithelial cells, targeting to and storage in secretory granules, and endocytosis. Most recently, the role of GAGs as signals for rapid trans-Golgi-to-cell surface transport, dominant over the cytosolic sorting motifs in the core protein, was demonstrated. Here, we provide an overview on existing data on the roles of GAGs on protein and proteoglycan trafficking.     

A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression

Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-β-D-naphthol and xyloside-β-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.     

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.     

Sequence Analysis and Domain Motifs in the Porcine Skin Decorin Glycosaminoglycan Chain

Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences.     

Glycosaminoglycan–Protein Interactions: The First Draft of the Glycosaminoglycan Interactome

The six mammalian glycosaminoglycans (GAGs), chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronan, and keratan sulfate, are linear polysaccharides. Except for hyaluronan, they are sulfated to various extent, and covalently attached to proteins to form proteoglycans. GAGs interact with growth factors, morphogens, chemokines, extracellular matrix proteins and their bioactive fragments, receptors, lipoproteins, and pathogens. These interactions mediate their functions, from embryonic development to extracellular matrix assembly and regulation of cell signaling in various physiological and pathological contexts such as angiogenesis, cancer, neurodegenerative diseases, and infections. We give an overview of GAG–protein interactions (i.e., specificity and chemical features of GAG- and protein-binding sequences), and review the available GAG–protein interaction networks. We also provide the first comprehensive draft of the GAG interactome composed of 832 biomolecules (827 proteins and five GAGs) and 932 protein–GAG interactions. This network is a scaffold, which in the future should integrate structures of GAG–protein complexes, quantitative data of the abundance of GAGs in tissues to build tissue-specific interactomes, and GAG interactions with metal ions such as calcium, which plays a major role in the assembly of the extracellular matrix and its interactions with cells. This contextualized interactome will be useful to identify druggable GAG–protein interactions for therapeutic purpose: