Enzymatic comparison of two homologous enzymes reveals N-terminal domain of chondroitinase ABC I regulates substrate selection and product generation.

Chondroitinase ABC-type I (CSase ABC I), which can digest both chondroitin sulfate (CS) and dermatan sulfate (DS) in an endolytic manner, is an essential tool in structural and functional studies of CS/DS. Although a few CSase ABC I have been identified from bacteria, the substrate-degrading pattern and regulatory mechanisms of them have rarely been investigated. Herein, two CSase ABC I, IM3796 and IM1634, were identified from the intestinal metagenome of CS-fed mice. They show high sequence homology (query coverage: 88.00%, percent identity: 90.10%) except for an extra peptide (Met1-His109) at the N-terminus in IM1634, but their enzymatic properties are very different. IM3796 prefers to degrade 6-O-sulfated GalNAc residue-enriched CS into tetra- and disaccharides. In contrast, IM1634 exhibits nearly a thousand times more activity than IM3796 and can completely digest CS/DS with various sulfation patterns to produce disaccharides, unlike most CSase ABC I. Structure modeling showed that IM3796 did not contain an N-terminal domain composed of two ß-sheets, which is found in IM1634 and other CSase ABC I. Furthermore, deletion of the N-terminal domain (Met1-His109) from IM1634 caused the enzymatic properties of the variant IM1634-T109 to be similar to those of IM3796, and conversely, grafting this domain to IM3796 increased the similarity of the variant IM3796-A109 to IM1634. In conclusion, the comparative study of the new CSase ABC I provides two unique tools for CS/DS-related studies and applications and, more importantly, reveals the critical role of the N-terminal domain in regulating the substrate binding and degradation of these enzymes.

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.

Glycosaminoglycans from the Starfish Lethasterias fusca: Structures and Influence on Hematopoiesis.

Crude anionic polysaccharides extracted from the Pacific starfish Lethasterias fusca were purified by anion-exchange chromatography. The main fraction LF, having MW 14.5 kDa and dispersity 1.28 (data of gel-permeation chromatography), was solvolytically desulfated and giving rise to preparation LF-deS with a structure of dermatan core [→3)-ß-d-GalNAc-(1→4)-α-l-IdoA-(1→]n, which was identified according to NMR spectroscopy data. Analysis of the NMR spectra of the parent fraction LF led to identification of the main component as dermatan sulfate LF-Derm →3)-ß-d-GalNAc4R-(1→4)-α-l-IdoA2R3S-(1→ (where R was SO3 or H), bearing sulfate groups at O-3 or both at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor signals in NMR spectra of LF were assigned as resonances of heparinoid LF-Hep composed of the fragments →4)-α-d-GlcNS3S6S-(1→4)-α-l-IdoA2S3S-(1→. The 3-O-sulfated and 2,3-di-O-sulfated iduronic acid residues are very unusual for natural glycosaminoglycans, and further studies are needed to elucidate their possible specific influence on the biological activity of the corresponding polysaccharides. To confirm the presence of these units in LF-Derm and LF-Hep, a series of variously sulfated model 3-aminopropyl iduronosides were synthesized and their NMR spectra were compared with those of the polysaccharides. Preparations LF and LF-deS were studied as stimulators of hematopoiesis in vitro. Surprisingly, it was found that both preparations were active in these tests, and hence, the high level of sulfation is not necessary for hematopoiesis stimulation in this particular case.

Clinical and pathophysiological delineation of musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE): A detailed and comprehensive glycobiological and pathological investigation in a novel patient.

Musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE) is a rare connective tissue disorder. This is the first report describing the detailed and comprehensive clinical and pathophysiological features of mcEDS-DSE. The patient, with a novel homozygous nonsense variant (NM_013352.4:c.2601C>A:p.(Tyr867*)), exhibited mild skin hyperextensibility without fragility and small joint hypermobility, but developed recurrent large subcutaneous hematomas. Dermatan sulfate (DS) moieties on chondroitin sulfate/DS proteoglycans were significantly decreased, but remained present, in skin fibroblasts. Electron microscopy examination of skin specimens, including cupromeronic blue-staining to visualize glycosaminoglycan (GAG) chains, revealed coexistence of normally assembled collagen fibrils with attached curved GAG chains and dispersed collagen fibrils with linear GAG chains from attached collagen fibrils across interfibrillar spaces to adjacent fibrils. Residual activity of DS-epi1, encoded by DSE, and/or compensation by DS-epi2, a minor homolog of DS-epi1, may contribute to the mild skin involvement through this "mosaic" pattern of collagen fibril assembly.

Compositional analysis of chondroitin/dermatan sulfate in rhesus monkeys (Macaca mulatta).

Glycosaminoglycans (GAGs) are found in various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of this study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A and C types. The E and iE types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.

Effects of Dermatan Sulfate from Marine Invertebrate Styela plicata in the Wound Healing Pathway: A Natural Resource Applied to Regenerative Therapy.

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.

The Specific Role of Dermatan Sulfate as an Instructive Glycosaminoglycan in Tissue Development.

The crucial roles of dermatan sulfate (DS) have been demonstrated in tissue development of the cutis, blood vessels, and bone through construction of the extracellular matrix and cell signaling. Although DS classically exerts physiological functions via interaction with collagens, growth factors, and heparin cofactor-II, new functions have been revealed through analyses of human genetic disorders as well as of knockout mice with loss of DS-synthesizing enzymes. Mutations in human genes encoding the epimerase and sulfotransferase responsible for the biosynthesis of DS chains cause connective tissue disorders including spondylodysplastic type Ehlers-Danlos syndrome, characterized by skin hyperextensibility, joint hypermobility, and tissue fragility. DS-deficient mice show perinatal lethality, skin fragility, vascular abnormalities, thoracic kyphosis, myopathy-related phenotypes, acceleration of nerve regeneration, and impairments in self-renewal and proliferation of neural stem cells. These findings suggest that DS is essential for tissue development in addition to the assembly of collagen fibrils in the skin, and that DS-deficient knockout mice can be utilized as models of human genetic disorders that involve impairment of DS biosynthesis. This review highlights a novel role of DS in tissue development studies from the past decade.

Profound Impact of Decline in N-Acetylgalactosamine-4-Sulfatase (Arylsulfatase B) on Molecular Pathophysiology and Human Diseases.

The enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) was originally identified as a lysosomal enzyme which was deficient in Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy Syndrome). The newly directed attention to the impact of ARSB in human pathobiology indicates a broader, more pervasive effect, encompassing roles as a tumor suppressor, transcriptional mediator, redox switch, and regulator of intracellular and extracellular-cell signaling. By controlling the degradation of chondroitin 4-sulfate and dermatan sulfate by removal or failure to remove the 4-sulfate residue at the non-reducing end of the sulfated glycosaminoglycan chain, ARSB modifies the binding or release of critical molecules into the cell milieu. These molecules, such as galectin-3 and SHP-2, in turn, influence crucial cellular processes and events which determine cell fate. Identification of ARSB at the cell membrane and in the nucleus expands perception of the potential impact of decline in ARSB activity. The regulation of availability of sulfate from chondroitin 4-sulfate and dermatan sulfate may also affect sulfate assimilation and production of vital molecules, including glutathione and cysteine. Increased attention to ARSB in mammalian cells may help to integrate and deepen our understanding of diverse biological phenomenon and to approach human diseases with new insights.

Kinetic and Structural Aspects of Glycosaminoglycan-Monkeypox Virus Protein A29 Interactions Using Surface Plasmon Resonance.

Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western Africa is well-known, the recent rise in the number of cases spread through intimate personal contact, particularly in the United States, poses a grave international threat. Previous studies have shown that cell-surface heparan sulfate (HS) is important for vaccinia virus (VACV) infection, particularly the binding of VACV A27, which appears to mediate the binding of virus to cellular HS. Some other glycosaminoglycans (GAGs) also bind to proteins on Orthopoxviruses. In this study, by using surface plasmon resonance, we demonstrated that MPXV A29 protein (a homolog of VACV A27) binds to GAGs including heparin and chondroitin sulfate/dermatan sulfate. The negative charges on GAGs are important for GAG-MPXV A29 interaction. GAG analogs, pentosan polysulfate and mucopolysaccharide polysulfate, show strong inhibition of MPXV A29-heparin interaction. A detailed understanding on the molecular interactions involved in this disease should accelerate the development of therapeutics and drugs for the treatment of MPXV.

Identification and Structural Characterization of Novel Chondroitin/Dermatan Sulfate Hexassacharide Domains in Human Decorin by Ion Mobility Tandem Mass Spectrometry.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where they are highly expressed, CS/DS are involved in fundamental processes and various pathologies. The structural diversity of CS/DS domains gave rise to efforts for the development of efficient analytical methods, among which is mass spectrometry (MS), one of the most resourceful techniques for the identification of novel species and their structure elucidation. In this context, we report here on the introduction of a fast, sensitive, and reliable approach based on ion mobility separation (IMS) MS and MS/MS by collision-induced dissociation (CID), for the profiling and structural analysis of CS/DS hexasaccharide domains in human embryonic kidney HEK293 cells decorin (DCN), obtained after CS/DS chain releasing by ß-elimination, depolymerization using chondroitin AC I lyase, and fractionation by size-exclusion chromatography. By IMS MS, we were able to find novel CS/DS species, i.e., under- and oversulfated hexasaccharide domains in the released CS/DS chain. In the last stage of analysis, the optimized IMS CID MS/MS provided a series of diagnostic fragment ions crucial for the characterization of the misregulations, which occurred in the sulfation code of the trisulfated-4,5-Δ-GlcAGalNAc[IdoAGalNAc]2 sequence, due to the unusual sulfation sites.

Inhibition of iduronic acid biosynthesis by ebselen reduces glycosaminoglycan accumulation in mucopolysaccharidosis type I fibroblasts.

Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

Beware, commercial chondroitinases vary in activity and substrate specificity.

Chondroitin sulfate (CS)and dermatan sulfate (DS) are negatively charged polysaccharides found abundantly in animal tissue and have been extensively described to play key roles in health and disease. The most common method to analyze their structure is by digestion into disaccharides with bacterial chondroitinases, followed by chromatography and/or mass spectrometry. While studying the structure of oncofetal CS, we noted a large variation in the activity and specificity of commercially available chondroitinases. Here studied the kinetics of the enzymes and used high-performance liquid chromatography-mass spectrometry to determine the di- and oligosaccharide products resulting from the digestion of commercially available bovine CS A, shark CS C and porcine DS, focusing on chondroitinases ABC, AC and B from different vendors. Application of a standardized assay setup demonstrated large variations in the enzyme-specific activity compared to the values provided by vendors, large variation in enzyme specific activity of similar enzymes from different vendors and differences in the extent of cleavage of the substrates and the generated products. The high variability of different chondroitinases highlights the importance of testing enzyme activity and monitoring product formation in assessing the content and composition of chondroitin and DSs in cells and tissues.

Systematic investigation of the skin in Chst14-/- mice: A model for skin fragility in musculocontractural Ehlers-Danlos syndrome caused by CHST14 variants (mcEDS-CHST14).

Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14-/-) mice. We used skin samples of wild-type (Chst14+/+) and Chst14-/- mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14-/- mice, skin tensile strength was significantly decreased compared with that in Chst14+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14-/- mice by anion-exchange chromatography. Chst14-/- mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.

Structural analysis of glycosaminoglycans from Oviductus ranae.

Oviductus ranae (O.ran.) has been widely used as a tonic and a traditional animal-based Chinese medicine. O.ran. extracts have been reported to have numerous biological activities, including activities that are often associated with mammalian glycosaminoglycans such as anti-inflammatory, antiosteoperotic, and anti-asthmatic. Glycosaminoglycans are complex linear polysaccharides ubiquitous in mammals that possess a wide range of biological activities. However, their presence and possible structural characteristics within O.ran. were previously unknown. In this study, glycosaminoglycans were isolated from O.ran. and their disaccharide compositions were analyzed by liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). Heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate (DS) and hyaluronic acid (HA) were detected in O.ran. with varied disaccharide compositions. HS species contain highly acetylated disaccharides, and have various structures in their constituent chains. CS/DS chains also possess a heterogeneous structure with different sulfation patterns and densities. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of O.ran..

Differences in MPS I and MPS II Disease Manifestations.

Mucopolysaccharidosis (MPS) type I and II are two closely related lysosomal storage diseases associated with disrupted glycosaminoglycan catabolism. In MPS II, the first step of degradation of heparan sulfate (HS) and dermatan sulfate (DS) is blocked by a deficiency in the lysosomal enzyme iduronate 2-sulfatase (IDS), while, in MPS I, blockage of the second step is caused by a deficiency in iduronidase (IDUA). The subsequent accumulation of HS and DS causes lysosomal hypertrophy and an increase in the number of lysosomes in cells, and impacts cellular functions, like cell adhesion, endocytosis, intracellular trafficking of different molecules, intracellular ionic balance, and inflammation. Characteristic phenotypical manifestations of both MPS I and II include skeletal disease, reflected in short stature, inguinal and umbilical hernias, hydrocephalus, hearing loss, coarse facial features, protruded abdomen with hepatosplenomegaly, and neurological involvement with varying functional concerns. However, a few manifestations are disease-specific, including corneal clouding in MPS I, epidermal manifestations in MPS II, and differences in the severity and nature of behavioral concerns. These phenotypic differences appear to be related to different ratios between DS and HS, and their sulfation levels. MPS I is characterized by higher DS/HS levels and lower sulfation levels, while HS levels dominate over DS levels in MPS II and sulfation levels are higher. The high presence of DS in the cornea and its involvement in the arrangement of collagen fibrils potentially causes corneal clouding to be prevalent in MPS I, but not in MPS II. The differences in neurological involvement may be due to the increased HS levels in MPS II, because of the involvement of HS in neuronal development. Current treatment options for patients with MPS II are often restricted to enzyme replacement therapy (ERT). While ERT has beneficial effects on respiratory and cardiopulmonary function and extends the lifespan of the patients, it does not significantly affect CNS manifestations, probably because the enzyme cannot pass the blood-brain barrier at sufficient levels. Many experimental therapies, therefore, aim at delivery of IDS to the CNS in an attempt to prevent neurocognitive decline in the patients.

Mucopolysaccharidosis Type VI, an Updated Overview of the Disease.

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is a rare, autosomal recessive genetic disease, mainly affecting the pediatric age group. The disease is due to pathogenic variants of the ARSB gene, coding for the lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). The enzyme deficit causes a pathological accumulation of the undegraded glycosaminoglycans dermatan-sulphate and chondroitin-sulphate, natural substrates of ASB activity. Intracellular and extracellular deposits progressively take to a pathological scenario, often severe, involving most organ-systems and generally starting from the osteoarticular apparatus. Neurocognitive and behavioral abilities, commonly described as maintained, have been actually investigated by few studies. The disease, first described in 1963, has a reported prevalence between 0.36 and 1.3 per 100,000 live births across the continents. With this paper, we wish to contribute an updated overview of the disease from the clinical, diagnostic, and therapeutic sides. The numerous in vitro and in vivo preclinical studies conducted in the last 10-15 years to dissect the disease pathogenesis, the efficacy of the available therapeutic treatment (enzyme replacement therapy), as well as new therapies under study are here described. This review also highlights the need to identify new disease biomarkers, potentially speeding up the diagnostic process and the monitoring of therapeutic efficacy.

Structural basis of chemokine interactions with heparan sulfate, chondroitin sulfate, and dermatan sulfate.

Chemokines play diverse roles in human pathophysiology, ranging from trafficking leukocytes and immunosurveillance to the regulation of metabolism and neural function. Chemokine function is intimately coupled to binding tissue glycosaminoglycans (GAGs), heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS). Currently, very little is known about how the structural features and sequences of a given chemokine, the structure and sulfation pattern of a given GAG, and structural differences among GAGs and among chemokines impact binding interactions. In this study, we used solution NMR spectroscopy to characterize the binding interactions of two related neutrophil-activating chemokines, CXCL1 and CXCL5, with HS, CS, and DS. For both chemokines, the dimer bound all three GAGs with higher affinity than did the monomer, and affinities of the chemokines for CS and DS were lower than for HS. NMR-based structural models reveal diverse binding geometries and show that the binding surfaces for each of the three GAGs were different between the two chemokines. However, a given chemokine had similar binding interactions with CS and DS that were different from HS. Considering the fact that CXCL1 and CXCL5 activate the same CXCR2 receptor, we conclude that GAG interactions play a role in determining the nature of chemokine gradients, levels of free chemokine available for receptor activation, how chemokines bind their receptors, and that differences in these interactions determine chemokine-specific function.

Glycosaminoglycans from Co-Products of «Scyliorhinus canicula¼: Extraction and Purification in Reference to the European Pharmacopoeia Requirement.

BACKGROUND: Glycosaminoglycans (GAGs), including hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS) are essential components of the bone and cartilage tissues. CS isolated from the cartilage tissue of various animals has found application in pharmaceuticals, cosmetics and food industries. In the first part of the present work, three methods were used and compared to extract and purify glycosaminoglycans (GAGs) from the cartilage powder of a local cartilaginous marine species «Scyliorhinus canicula¼. One of these GAGs, chondroitin sulfate (CS), will be exploited for the development of an anti-osteoarthritis generic at the request of a collaborative pharmaceutical industry. Thus this active ingredient must meet the requirements and tests described by the European Pharmacopoeia (Ph. Eur.). These tests are treated in the second part of this work. RESULTS: Among the three methods that have been applied in the present work, in order to optimize the best process for GAGs preparation, enzymatic hydrolysis with papain followed by deproteinisation using trichloroacetic acid (TCA) was found the best one. The separation of the extracted GAGs using agarose gel electrophoresis, and the identification of bands by Fourier Transform Infrared (FT-IR) Spectroscopy, revealed that the cartilage GAGs of « Scyliorhinus canicula¼ are exclusively chondroitin sulfate (CS) and dermatane sulfate (DS), with proportions of 12.889 and 87.111% respectively, and that CS is of type C. The extraction technique with papain provides a product with GAGs content of around 90%. The TCA deproteinisation yielded the lowest level of protein (2.8%) in the extracted GAGs, less than 3%, which is the standard required by the European Pharmacopoeia (Ph. Eur.).Cetylpyridinium chloride (CPC) assay suggests that the titration technique, although is introduced by the Ph. Eur. for the determination of CS content, is not an accurate method, and that the values obtained by the optimized and validated HPLC method, described in this work, are more exact. CONCLUSION: The extracted and purified active ingredient is perfectly conform to the tests described by the Ph. Eur. The results suggest that the co-product of Scyliorhinus canicula would be a perfect source of molecules of pharmacological interest, obtained by a simple and non-agressive process.

Advances in glycosaminoglycan detection.

BACKGROUND: Glycosaminoglycans (GAGs) are negatively charged long linear (highly sulfated) polysaccharides consisting of repeating disaccharide units that are expressed on the surfaces of all nucleated cells. The expression of GAGs is required for embryogenesis, regulation of cell growth and proliferation, maintenance of tissue hydration, and interactions of the cells via receptors. Mucopolysaccharidoses (MPS) are caused by deficiency of specific lysosomal enzymes that result in the accumulation of GAGs in multiple tissues leading to organ dysfunction. Therefore, GAGs are important biomarkers for MPS. Without any treatment, patients with severe forms of MPS die within the first two decades of life. SCOPE OF REVIEW: Accurate measurement of GAGs is important to understand the diagnosis and pathogenesis of MPS and to monitor therapeutic efficacy before, during, and after treatment of the disease. This review covers various qualitative and quantitative methods for measurement of GAGs, including dye specific, thin layer chromatography (TLC), capillary electrophoresis, high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), gas chromatography, ELISA, and automated high-throughput mass spectrometry. Major conclusion: There are several methods for GAG detection however, specific GAG detection in the various biological systems requires rapid, sensitive, specific, and cost-effective methods such as LC-MS/MS. GENERAL SIGNIFICANCE: This review will describe different methods for GAG detection and analysis, including their advantages and limitation.

A proteomic repertoire of autoantigens identified from the classic autoantibody clinical test substrate HEp-2 cells.

BACKGROUND: Autoantibodies are a hallmark of autoimmune diseases. Autoantibody screening by indirect immunofluorescence staining of HEp-2 cells with patient sera is a current standard in clinical practice. Differential diagnosis of autoimmune disorders is based on commonly recognizable nuclear and cytoplasmic staining patterns. In this study, we attempted to identify as many autoantigens as possible from HEp-2 cells using a unique proteomic DS-affinity enrichment strategy. METHODS: HEp-2 cells were cultured and lysed. Total proteins were extracted from cell lysate and fractionated with DS-Sepharose resins. Proteins were eluted with salt gradients, and fractions with low to high affinity were collected and sequenced by mass spectrometry. Literature text mining was conducted to verify the autoantigenicity of each protein. Protein interaction network and pathway analyses were performed on all identified proteins. RESULTS: This study identified 107 proteins from fractions with low to high DS-affinity. Of these, 78 are verified autoantigens with previous reports as targets of autoantibodies, whereas 29 might be potential autoantigens yet to be verified. Among the 107 proteins, 82 can be located to nucleus and 15 to the mitotic cell cycle, which may correspond to the dominance of nuclear and mitotic staining patterns in HEp-2 test. There are 55 vesicle-associated proteins and 12 ribonucleoprotein granule proteins, which may contribute to the diverse speckled patterns in HEp-2 stains. There are also 32 proteins related to the cytoskeleton. Protein network analysis indicates that these proteins have significantly more interactions among themselves than would be expected of a random set, with the top 3 networks being mRNA metabolic process regulation, apoptosis, and DNA conformation change. CONCLUSIONS: This study provides a proteomic repertoire of confirmed and potential autoantigens for future studies, and the findings are consistent with a mechanism for autoantigenicity: how self-molecules may form molecular complexes with DS to elicit autoimmunity. Our data contribute to the molecular etiology of autoimmunity and may deepen our understanding of autoimmune diseases.