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.     

Analysis of the Glycosaminoglycan Chains of Proteoglycans

Glycosaminoglycans (GAGs) are heterogeneous, negatively charged, macromolecules that are found in animal tissues. Based on the form of component sugar, GAGs have been categorized into four different families: heparin/heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate, and hyaluronan. GAGs engage in biological pathway regulation through their interaction with protein ligands. Detailed structural information on GAG chains is required to further understanding of GAG–ligand interactions. However, polysaccharide sequencing has lagged behind protein and DNA sequencing due to the non-template-driven biosynthesis of glycans. In this review, we summarize recent progress in the analysis of GAG chains, specifically focusing on techniques related to mass spectroscopy (MS), including separation techniques coupled to MS, tandem MS, and bioinformatics software for MS spectrum interpretation. Progress in the use of other structural analysis tools, such as nuclear magnetic resonance (NMR) and hyphenated techniques, is included to provide a comprehensive perspective.     

Glycosaminoglycans of the porcine central nervous system

Glycosaminoglycans (GAGs) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth. In this paper, we report an initial glycomics study of GAGs from the porcine central nervous system. GAGs of the porcine central nervous system, brain and spinal cord were isolated and purified by defatting, proteolysis, anion-exchange chromatography, and methanol precipitation. The isolated GAG content in brain was 5 times higher than in spinal cord (0.35 mg/g of dry sample, compared to 0.07 mg/g of dry sample). In both tissues, chondroitin sulfate (CS) and heparan sulfate (HS) were the major and the minor GAG, respectively. The average molecular masses of CS from brain and spinal cord were 35.5 and 47.1 kDa, respectively, and those for HS from brain and spinal cord were 56.9 and 34 kDa, respectively. The disaccharide analysis showed that the compositions of CS from brain and spinal cords are similar, with uronic acid (1→3) 4-O-sulfo-N-acetylgalactosamine residue corresponding to the major disaccharide unit (CS type A) along with five minor disaccharide units. The major disaccharides of both brain and spinal cord HS were uronic acid (1→4) N-acetylglucosamine and uronic acid (1→4) 6-O-sulfo-N-sulfoglucosamine, but their composition of minor disaccharides differed. Analysis by (1)H and two-dimensional NMR spectroscopy confirmed these disaccharide analyses and provided the glucuronic/iduronic acid ratio. Finally, both purified CS and HS were biotinylated and immobilized on BIAcore SA biochips. Interactions between these GAGs and fibroblast growth factors (FGF1 and FGF2) and sonic hedgehog (Shh) were investigated by surface plasmon resonance.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Glycosaminoglycanomics: where we are

Glycosaminoglycans regulate numerous physiopathological processes such as development, angiogenesis, innate immunity, cancer and neurodegenerative diseases. Cell surface GAGs are involved in cell-cell and cell-matrix interactions, cell adhesion and signaling, and host-pathogen interactions. GAGs contribute to the assembly of the extracellular matrix and heparan sulfate chains are able to sequester growth factors in the ECM. Their biological activities are regulated by their interactions with proteins. The structural heterogeneity of GAGs, mostly due to chemical modifications occurring during and after their synthesis, makes the development of analytical techniques for their profiling in cells, tissues, and biological fluids, and of computational tools for mining GAG-protein interaction data very challenging. We give here an overview of the experimental approaches used in glycosaminoglycomics, of the major GAG-protein interactomes characterized so far, and of the computational tools and databases available to analyze and store GAG structures and interactions.     

A structural analysis of glycosaminoglycans from lethal and nonlethal breast cancer tissues: toward a novel class of theragnostics for personalized medicine in oncology?

Cancer is one of the leading noncommunicable diseases that vastly impacts both developed and developing countries. Truly innovative diagnostics that inform disease susceptibility, prognosis, and/or response to treatment (theragnostics) are seriously needed for global public health and personalized medicine for patients with cancer. This study examined the structure and content of glycosaminoglycans (GAGs) in lethal and nonlethal breast cancer tissues from six patients. The glycosaminoglycan content isolated from tissue containing lethal cancer tumors was approximately twice that of other tissues. Molecular weight analysis showed that glycosaminoglycans from cancerous tissue had a longer weight average chain length by an average of five disaccharide units, an increase of approximately 15%. Dissacharide analysis found differences in sulfation patterns between cancerous and normal tissues, as well as sulfation differences in GAG chains isolated from patients with lethal and nonlethal cancer. Specifically, cancerous tissue showed an increase in sulfation at the "6S" position of CS chains and an increase in the levels of the HS disaccharide NSCS. Patients with lethal cancer showed a decrease in HS sulfation, with lower levels of "6S" and higher levels of the unsulfated "0S" disaccharide. Although these findings come from a limited sample size, they indicate that structural changes in GAGs exist between cancerous and noncancerous tissues and between tissues from patients with highly metastatic cancer and cancer that was successfully treated by chemotherapy. Based on these findings, we hypothesize that (1) there are putative changes in the body's construction of GAGs as tissue becomes cancerous; (2) there may be innate structural person-to-person variations in GAG composition that facilitate the metastasis of tumors in some patients when they develop cancer.     

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.     

One-pot analysis of sulfated glycosaminoglycans

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.     

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.     

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae)

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to ∼80% and 20% of the total GAGs, respectively. In the fish eye, KS represents ∼ 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by ΔUA-1→3-GalNAc(4SO₄) (59.4%), ΔUA-1→3-GalNAc(6SO₄) (23.1%), and ΔUA-1→3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism. The contributions of Aline R.C. Souza and Eliene O. Kozlowski should be considered equal.     

Age-related changes in rat myocardium involve altered capacities of glycosaminoglycans to potentiate growth factor functions and heparan sulfate-altered sulfation

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.     

Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow−/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O₂) and hypoxic (5% O₂) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

     

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:     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies

This article describes a simple and selective procedure used for direct measurement of sulfated glycosaminoglycans (GAGs) in biological samples and its application to the determination of GAGs during tissue regeneration and myogenic differentiation. We describe a modified procedure of previous GAG assays that has improved specificity, reproducibility, and sensitivity. The assay is based on the ability of sulfated GAGs to bind the cationic dye 1,9-dimethylmethylene blue. We describe conditions that allow isolation of the GAG-dye complex. This complex was dissociated; the optical density measurement of the dissociated dye permitted quantification of GAGs in biological samples. Applied to the study of myogenic cell differentiation in vitro, muscle repair, and skin ulceration, this method revealed significant modifications in the patterns of expression of different sulfated GAGs in these tissues. In particular, application of the method after nitrous acid treatment revealed that heparan sulfate and chondroitin sulfate ratio changed during muscle regeneration process.