A Chondroitin Sulfate Chain of Urinary Trypsin Inhibitor Enhances Protease Inhibitory Activity of the Core Protein.
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ABSTRACT:
SUBMITTER: Teshigahara Y
PROVIDER: S-EPMC8283410 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Chondroitin sulfate proteoglycans (CSPGs) are upregulated in the CNS after injury and participate in the inhibition of axon regeneration mainly through their glycosaminoglycan (GAG) side chains. In the present study, we have identified a new way to alleviate the inhibition of axonal regeneration by CSPG GAGs. We have successfully decreased the amount of CSPG GAG produced by astrocytes by targeting chondroitin polymerizing factor (ChPF), a key enzyme in the CSPG biosynthetic pathway. Using short interfering RNA (siRNA), we reduced ChPF mRNA levels by 70% in both the Neu7 astrocyte cell line and primary rat astrocytes. This reduction leads to a decrease in ChPF protein levels and a reduced amount of CSPG GAG chains in the conditioned media (CM) of these cells. Secretion of neurocan by primary astrocytes and NG2 core protein by Neu7 cells transfected with ChPF siRNA is not decreased, suggesting that inhibiting GAG chain synthesis does not affect core protein trafficking from these cells. CM from siRNA-treated Neu7 cells is a less repulsive substrate for axons than CM from control cells. In addition, axonal outgrowth from cerebellar granule neurons is increased on or in CM from ChPF siRNA-treated Neu7 cells. These data indicate that targeting the biosynthesis of CSPG GAG is a potentially new therapeutic avenue for decreasing CSPG GAG produced by astrocytes after CNS injury.
| S-EPMC6673453 | biostudies-literature
Project description:BackgroundDiabetes mellitus is a global health problem representing the fifth leading cause of mortality and a major risk factor for cardiovascular diseases. In the last years, we reported an association among urinary trypsin inhibitor (UTI), a small proteoglycan that plays pleiotropic roles in many inflammatory processes, and both type 1 and 2 diabetes and developed a method for its direct quantitation and structural characterization.MethodsUrine from 39 patients affected by type 1 diabetes, 32 patients with type 2 diabetes, and 52 controls were analysed. UTI was separated from the main glycosaminoglycans physiologically present in urine by anion exchange chromatography, treated for chondroitin sulphate (CS) chain complete depolymerisation, and analysed for both UTI content and CS structure. UTI identification was performed by nano-LC-MS/MS analysis.ResultsWe evidenced increased UTI levels, as well as reduced sulphation of its CS moiety in association with diabetes, regardless of both age and medium-term glycaemic control. Furthermore, no association between UTI and albumin excretion rate was found.ConclusionsEvidences suggest that UTI levels are not directly correlated with renal function or, otherwise, that they may increase before the onset of renal impairment in diabetes, representing a potential marker for the underlying inflammatory condition.
| S-EPMC5818928 | biostudies-literature
Project description:Familial members of urolithiasis have high risk for stone development. We observed the low sulfated glycosaminoglycan (GAG) excretion in urolithiasis patients and their descendants. In this study, we investigated urinary excretion of sulfated GAG, chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) in urolithiasis and their children, and explored the effect of CS and HA supplement in urolithic hyperoxaluric rats. The 24-hour urines were collected from urolithiasis patients (28) and their children (40), as well as healthy controls (45) and their children (33) to measure urinary sulfated GAG, CS, HS and HA excretion rate. Our result showed that urinary sulfated GAG and CS were diminished in both urolithiasis patients and their children, while decreased HS and increased HA were observed only in urolithiasis patients. Percentage of HS per sulfated GAG increased in both urolithiasis patients and their children. In hyperoxaluric rats induced by ethylene glycol and vitamin D, we found that CS supplement could prevent stone formation, while HA supplement had no effect on stone formation. Our study revealed that decreased urinary GAG and CS excretion are common in familial members of urolithiasis patients, and CS supplement might be beneficial in calcium oxalate urolithiasis prophylaxis for hyperoxaluric patients.
| S-EPMC6405089 | biostudies-literature
Project description:Inter-?-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-?-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-?-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-?-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-?-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-?-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-?-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-?-inhibitor.
| S-EPMC4813489 | biostudies-literature
Project description:Inter-?-trypsin inhibitor (I?I) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. I?I is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 M NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1-1.0 M NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5-0.8 M NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1-0.5 M NaCl) contained unsulfated and 4-sulfated CS disaccharides. I?I in the 0.5-0.8 M NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1-0.5 M NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.
| S-EPMC3743471 | biostudies-literature
Project description:The primary macrophage growth factor, colony-stimulating factor-1 (CSF-1), is homodimeric and exists in 3 biologically active isoforms: a membrane-spanning, cell-surface glycoprotein (csCSF-1) and secreted glycoprotein (sgCSF-1) and proteoglycan (spCSF-1) isoforms. To investigate the in vivo role of the chondroitin sulfate glycosaminoglycan (GAG) chain of spCSF-1, we created mice that exclusively express, in a normal tissue-specific and developmental manner, either the secreted precursor of spCSF-1 or the corresponding precursor in which the GAG addition site was mutated. The reproductive, hematopoietic tooth eruption and tissue macrophage defects of CSF-1-deficient, osteopetrotic Csf1(op)/Csf1(op) mice were corrected by transgenic expression of the precursors of either sgCSF-1 or spCSF-1. Furthermore, in contrast to the transgene encoding csCSF-1, both failed to completely correct growth retardation, suggesting a role for csCSF-1 in the regulation of body weight. However, spCSF-1, in contrast to sgCSF-1, completely resolved the osteopetrotic phenotype. Furthermore, in transgenic lines expressing different concentrations of sgCSF-1 or spCSF-1, spCSF-1 more efficiently corrected Csf1(op)/Csf1(op) defects of tooth eruption, eyelid opening, macrophage morphology, and B-cell deficiency than sgCSF-1. These results indicate an important role of the CSF-1 chondroitin sulfate proteoglycan in in vivo signaling by secreted CSF-1.
| S-EPMC1895624 | biostudies-literature
Project description:The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.
| S-EPMC4762621 | biostudies-literature
Project description:Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.
| S-EPMC2172406 | biostudies-literature
Project description:Chondroitin sulfate synthases, a family of enzyme involved in chondroitin sulfate (CS) polymerization, are dysregulated in various human malignancies, but their roles in glioma remain unclear. We performed database analysis and immunohistochemistry on human glioma tissue, to demonstrate that the expression of CHSY1 was frequently upregulated in glioma, and that it was associated with adverse clinicopathologic features, including high tumor grade and poor survival. Using a chondroitin sulfate-specific antibody, we showed that the expression of CHSY1 was significantly associated with CS formation in glioma tissue and cells. In addition, overexpression of CHSY1 in glioma cells enhanced cell viability and orthotopic tumor growth, whereas CHSY1 silencing suppressed malignant growth. Mechanistic investigations revealed that CHSY1 selectively regulates PDGFRA activation and PDGF-induced signaling in glioma cells by stabilizing PDGFRA protein levels. Inhibiting PDGFR activity with crenolanib decreased CHSY1-induced malignant characteristics of GL261 cells and prolonged survival in an orthotopic mouse model of glioma, which underlines the critical role of PDGFRA in mediating the effects of CHSY1. Taken together, these results provide information on CHSY1 expression and its role in glioma progression, and highlight novel insights into the significance of CHSY1 in PDGFRA signaling. Thus, our findings point to new molecular targets for glioma treatment.
| S-EPMC7000683 | biostudies-literature
Project description:Remyelination is the generation of new myelin sheaths after injury facilitated by processes of differentiating oligodendrocyte precursor cells (OPCs). Although this repair phenomenon occurs in lesions of multiple sclerosis patients, many lesions fail to completely remyelinate. A number of factors have been identified that contribute to remyelination failure, including the upregulated chondroitin sulfate proteoglycans (CSPGs) that comprise part of the astrogliotic scar. We show that in vitro, OPCs have dramatically reduced process outgrowth in the presence of CSPGs, and a medication library that includes a number of recently reported OPC differentiation drugs failed to rescue this inhibitory phenotype on CSPGs. We introduce a novel CSPG synthesis inhibitor to reduce CSPG content and find rescued process outgrowth from OPCs in vitro and accelerated remyelination following focal demyelination in mice. Preventing CSPG deposition into the lesion microenvironment may be a useful strategy to promote repair in multiple sclerosis and other neurological disorders.
| S-EPMC4853428 | biostudies-literature