A glycomic workflow for LC-MS/MS analysis of urine glycosaminoglycan biomarkers in mucopolysaccharidoses.

In recent years, several rational designed therapies have been developed for treatment of mucopolysaccharidoses (MPS), a group of inherited metabolic disorders in which glycosaminoglycans (GAGs) are accumulated in various tissues and organs. Thus, improved disease-specific biomarkers for diagnosis and monitoring treatment efficacy are of paramount importance. Specific non-reducing end GAG structures (GAG-NREs) have become promising biomarkers for MPS, as the compositions of the GAG-NREs depend on the nature of the lysosomal enzyme deficiency, thereby creating a specific pattern for each subgroup. However, there is yet no straightforward clinical laboratory platform which can assay all MPS-related GAG-NREs in one single analysis. Here, we developed and applied a GAG domain mapping approach for analyses of urine samples of ten MPS patients with various MPS diagnoses and corresponding aged-matched controls. We describe a nano-LC-MS/MS method of GAG-NRE profiling, utilizing 2-aminobenzamide reductive amination labeling to improve the sensitivity and the chromatographic resolution. Diagnostic urinary GAG-NREs were identified for MPS types IH/IS, II, IIIc, IVa and VI, corroborating GAG-NRE as biomarkers for these known enzyme deficiencies. Furthermore, a significant reduction of diagnostic urinary GAG-NREs in MPS IH (n = 2) and MPS VI (n = 1) patients under treatment was demonstrated. We argue that this straightforward glycomic workflow, designed for the clinical analysis of MPS-related GAG-NREs in one single analysis, will be of value for expanding the use of GAG-NREs as biomarkers for MPS diagnosis and treatment monitoring.

A Glycoproteomic Approach to Identify Novel Proteoglycans.

In this chapter, we describe a glycoproteomic approach for the identification of novel chondroitin sulfate proteoglycans (CSPGs) using a combination of biochemical enrichments, enzymatic digestions, and nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. The identification is achieved by trypsin digestion of CSPG-containing samples, followed by enrichment of chondroitin sulfate (CS) glycopeptides by strong anion exchange chromatography (SAX). The enriched CS glycopeptides are then digested with chondroitinase ABC to depolymerize the CS polysaccharides, generating a residual hexasaccharide structure, composed of the linkage region tetrasaccharide extended with a terminal dehydrated disaccharide, still attached to the peptide. The obtained CS glycopeptides are analyzed by nLC-MS/MS, and the generated data sets are evaluated through proteomic software with adjustment in the settings to allow for glycopeptide identification. This approach has enabled the identification of several novel core proteins in human samples and in Caenorhabditis elegans. Here we specifically describe the procedure for the enrichment and characterization of CS glycopeptides from human cerebrospinal fluid (CSF).

Production and HPLC-Based Disaccharide Analysis of Xyloside-Primed Glycosaminoglycans.

Although glycosaminoglycans (GAGs) are known to be involved in a variety of physiological and pathological processes, knowledge about their expression by cells or tissues, the GAGome, is limited. Xylosides can be used to induce the formation of GAGs without the presence of a proteoglycan core protein. The administration of xylosides to living cells tends to result in a considerable amplification in GAG production, and the xylosides can, therefore, be used as analytical tools to study the GAG produced by a certain cell type. One of the most common ways to analyze the GAGs structurally is by disaccharide analysis, which involves depolymerization of the GAGs into disaccharides, fluorescent labeling of the disaccharides with 2-aminoacridone, and quantification using high-pressure liquid chromatography (HPLC). Here, we describe the procedure of producing xyloside-primed GAGs and how to study them structurally by disaccharide analysis.

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

ß-1,4-Galactosyltransferase 7 (ß4GalT7) 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 ß4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl ß-D-xylopyranoside. We have cloned and expressed ß4GalT7, 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 ß4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of ß4GalT7.

Expanding the Chondroitin Sulfate Glycoproteome - But How Far?

Chondroitin sulfate proteoglycans (CSPGs) are found at cell surfaces and in connective tissues, where they interact with a multitude of proteins involved in various pathophysiological processes. From a methodological perspective, the identification of CSPGs is challenging, as the identification requires the combined sequencing of specific core proteins, together with the characterization of the CS polysaccharide modification(s). According to the current notion of CSPGs, they are often considered in relation to a functional role in which a given proteoglycan regulates a specific function in cellular physiology. Recent advances in glycoproteomic methods have, however, enabled the identification of numerous novel chondroitin sulfate core proteins, and their glycosaminoglycan attachment sites, in humans and in various animal models. In addition, these methods have revealed unexpected structural complexity even in the linkage regions. These findings indicate that the number and structural complexity of CSPGs are much greater than previously perceived. In light of these findings, the prospect of finding additional CSPGs, using improved methods for structural and functional characterizations, and studying novel sample matrices in humans and in animal models is discussed. Further, as many of the novel CSPGs are found in low abundance and with not yet assigned functions, these findings may challenge the traditional notion of defining proteoglycans. Therefore, the concept of proteoglycans is considered, discussing whether "a proteoglycan" should be defined mainly on the basis of an assigned function or on the structural evidence of its existence.

Proteoglycan profiling of human, rat and mouse insulin-secreting cells.

Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.

Domain Mapping of Chondroitin/Dermatan Sulfate Glycosaminoglycans Enables Structural Characterization of Proteoglycans.

Of all posttranslational modifications known, glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs. Chondroitin/dermatan sulfate conjugates from the rat insulinoma cell line, INS-1832/13, known to produce primarily the PG chromogranin-A, were enriched by anion-exchange chromatography after pronase digestion. Following benzonase and hyaluronidase digestions, included in the sample preparation due to the apparent interference from oligonucleotides and hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive glucuronic-acid-containing disaccharide units, or CS motifs, of which the N-acetylgalactosamine residues were 4-O-sulfated, interspersed by single iduronic-acid-containing disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions.

An affinity chromatography and glycoproteomics workflow to profile the chondroitin sulfate proteoglycans that interact with malarial VAR2CSA in the placenta and in cancer.

Chondroitin sulfate (CS) is the placental receptor for the VAR2CSA malaria protein, expressed at the surface of infected erythrocytes during Plasmodium falciparum infection. Infected cells adhere to syncytiotrophoblasts or get trapped within the intervillous space by binding to a determinant in a 4-O-sulfated CS chains. However, the exact structure of these glycan sequences remains unclear. VAR2CSA-reactive CS is also expressed by tumor cells, making it an attractive target for cancer diagnosis and therapeutics. The identities of the proteoglycans carrying these modifications in placental and cancer tissues remain poorly characterized. This information is clinically relevant since presentation of the glycan chains may be mediated by novel core proteins or by a limited subset of established proteoglycans. To address this question, VAR2CSA-binding proteoglycans were affinity-purified from the human placenta, tumor tissues and cancer cells and analyzed through a specialized glycoproteomics workflow. We show that VAR2CSA-reactive CS chains associate with a heterogenous group of proteoglycans, including novel core proteins. Additionally, this work demonstrates how affinity purification in combination with glycoproteomics analysis can facilitate the characterization of CSPGs with distinct CS epitopes. A similar workflow can be applied to investigate the interaction of CSPGs with other CS binding lectins as well.

Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates.

Glycosaminoglycans (GAGs) are polysaccharides produced by most mammalian cells and involved in a variety of biological processes. However, due to the size and complexity of GAGs, detailed knowledge about the structure and expression of GAGs by cells, the glycosaminoglycome, is lacking. Here we report a straightforward and versatile approach for structural domain mapping of complex mixtures of GAGs, GAGDoMa. The approach is based on orthogonal enzymatic depolymerization of the GAGs to generate internal, terminating, and initiating domains, and nanoflow reversed-phase ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) tandem mass spectrometry (MS/MS) for structural characterization of the individual domains. GAGDoMa provides a detailed structural insight into the glycosaminoglycome, and offers an important tool for deciphering the complexity of GAGs in cellular physiology and pathology.

Identification of a non-canonical chondroitin sulfate linkage region trisaccharide.

It is generally accepted that the biosynthesis of chondroitin sulfate and heparan sulfate is proceeding from a common linkage region tetrasaccharide comprising GlcA-Gal-Gal-Xyl-O-. The linkage region can undergo various modifications such as sulfation, phosphorylation and sialylation, and as the methods for studying glycosaminoglycan structure have been developed and refined, the number of discovered modifications has increased. Previous studies on the linkage region and the glycosyltransferases involved in the biosynthesis suggest that variants of the linkage region tetrasaccharide may also be possible. Here, using LC-MS/MS, we describe a non-canonical linkage region trisaccharide comprising GlcA-Gal-Xyl-O-. The trisaccharide was identified as a minor constituent in the proteoglycan bikunin from urine of human healthy donors present as a disulfated pentasaccharide, ΔHexA-GalNAc(S)-GlcA-Gal(S)-Xyl-O-, after chondroitinase ABC degradation. Furthermore, it was present as the corresponding disulfated pentasaccharide after chondroitinase ABC degradation in chondroitin sulfate primed on xylosides isolated from human cell lines. This linkage region trisaccharide may serve as an alternative point of entry for glycosaminoglycan biosynthesis.

The GAGOme: a cell-based library of displayed glycosaminoglycans.

Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.

LC-MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications.

Structural characterization of glycosaminoglycans remains a challenge but is essential for determining structure-function relationships between glycosaminoglycans and the biomolecules with which they interact and for gaining insight into the biosynthesis of glycosaminoglycans. We have recently reported that xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, has cytotoxic effects and shown that it differs in disaccharide composition from nontoxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we developed a novel LC-MS/MS approach based on reversed-phase dibutylamine ion-pairing chromatography and negative-mode higher-energy collision dissociation and used it in combination with cell growth studies and disaccharide fingerprinting. This strategy enabled detailed structural characterization of linkage regions, internal oligosaccharides, and nonreducing ends, revealing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also sialylation of the linkage region and previously undescribed methylation and sulfation of the nonreducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared to be essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.

Fine-tuning the structure of glycosaminoglycans in living cells using xylosides.

Xylosides can induce the formation and secretion of xyloside-primed glycosaminoglycans when administered to living cells; however, their impact on the detailed glycosaminoglycan structure remains unknown. Here, we have systematically investigated how the xyloside concentration and the type of xyloside, as well as the cell type, influenced the structure of xyloside-primed glycosaminoglycans in terms of the heparan sulfate and chondroitin/dermatan sulfate proportion and disaccharide composition. We found that although greatest influence was exerted by the cell type, both the xyloside concentration and type of xyloside impacted the proportion of heparan sulfate and the complexity of chondroitin/dermatan sulfate. The disaccharide composition of the chondroitin/dermatan sulfate was influenced by the xyloside concentration and type of xyloside to a higher extent than that of the heparan sulfate; the proportion of 4S-sulfated disaccharides in the chondroitin/dermatan sulfate decreased and the proportions of 6S-sulfated and/or nonsulfated disaccharides increased both with increasing concentrations of xyloside and with increasing xyloside hydrophobicity, whereas the proportion of nonsulfated disaccharides was primarily altered in the heparan sulfate with increasing concentrations of xyloside. Our results indicate that it is feasible to not only produce large amounts of glycosaminoglycans in living cells but also to fine-tune their structures by using xylosides of different types and at different concentrations.

Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro.

We previously reported that the xyloside 2-(6-hydroxynaphthyl) ß-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl ß-d-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH- and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH- and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/dermatan sulfate nor the heparan sulfate derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH- and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate.

Disubstituted naphthyl ß-D-xylopyranosides: Synthesis, GAG priming, and histone acetyltransferase (HAT) inhibition.

Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)ß-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects.

Exploration of the active site of ß4GalT7: modifications of the aglycon of aromatic xylosides.

Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase ß4GalT7. Some ß-d-xylosides, such as 2-naphthyl ß-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for ß4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for ß4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with ß-glycosidic linkages function as good substrates for ß4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by ß4GalT7 is dependent on subtle differences in orientation of the xylose moiety.

Synthesis and biology of oligoethylene glycol linked naphthoxylosides.

Proteoglycans (PGs) are important macromolecules in mammalian cells, consisting of a core protein substituted with carbohydrate chains, known as glycosaminoglycans (GAGs). Simple xylosides carrying hydrophobic aglycons can enter cells and act as primers for GAG chain synthesis, independent of the core protein. Previously it has been shown that aromatic aglycons can be separated from the sugar residue by short linkers without affecting the GAG priming ability. To further investigate the effects of the xylose-aglycon distance on the GAG priming ability, we have synthesized xyloside derivatives with 2-naphthyl and 2-(6-hydroxynaphthyl) moieties connected to xylose, directly, via a methylene bridge, or with oligoethylene glycol linkers of three different lengths. The GAG priming ability and the antiproliferative activity of the xylosides, as well as the composition of the xyloside-primed GAG chains were investigated in a matched pair of human breast fibroblasts and human breast carcinoma cells. An increase of the xylose-aglycon distance from 0.24 to 0.37 nm resulted in an increased GAG priming ability in both cell lines. Further increase of the xylose-aglycon distance did not result in any pronounced effects. We speculate that by increasing the xylose-aglycon distance, and thereby the surface area of the xyloside, to a certain level would make it more accessible for enzymes involved in the GAG synthesis. The compositions of the primed GAG chains varied with different xylosides, independent of the xylose-aglycon distance, probably due to various affinities for enzymes and/or different cellular uptake. Furthermore, no correlations between the antiproliferative activities, the xylose-aglycon distances, and the amounts or compositions of the GAG chains were detected suggesting involvement of other factors such as fine structure of the GAG chains, effects on endogenous PG synthesis, or other unknown factors for the antiproliferative activity.