Showing papers on "Keratan sulfate published in 2020"

Brain Pathology in Mucopolysaccharidoses (MPS) Patients with Neurological Forms.

Abstract: Mucopolysaccharidoses (MPS) are the group of lysosomal storage disorders caused by deficiencies of enzymes involved in the stepwise degradation of glycosaminoglycans. To identify brain pathology common for neurological MPS, we conducted a comprehensive analysis of brain cortex tissues from post-mortem autopsy materials of eight patients affected with MPS I, II, IIIA, IIIC, and IIID, and age-matched controls. Frozen brain tissues were analyzed for the abundance of glycosaminoglycans (heparan, dermatan, and keratan sulfates) by LC-MS/MS, glycosphingolipids by normal phase HPLC, and presence of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor superfamily member 10 (TNFSF10) by Western blotting. Fixed tissues were stained for the markers for microgliosis, astrogliosis, misfolded proteins, impaired autophagy, and GM2 ganglioside. Our results demonstrate that increase of heparan sulfate, decrease of keratan sulfate, and storage of simple monosialogangliosides 2 and 3 (GM2 and GM3) as well as the neutral glycosphingolipid, LacCer, together with neuroinflammation and neuronal accumulation of misfolded proteins are the hallmarks of brain pathology in MPS patients. These biomarkers are similar to those reported in the corresponding mouse models, suggesting that the pathological mechanism is common for all neurological MPS in humans and mice.

Impairment of the autophagy-lysosomal pathway and activation of pyroptosis in macular corneal dystrophy.

Abstract: Macular corneal dystrophy (MCD) is ascribed to mutations in the carbohydrate sulfotransferase (CHST6) gene affecting keratan sulfate (KS) hydrophilicity and causing non-sulfated KS to precipitate in keratocytes and the corneal stroma. We investigated roles for inflammatory responses in MCD pathogenesis by examining the lysosomal-autophagy pathway and activation of pyroptosis in MCD keratocytes. Normal and lesioned keratocytes were obtained from MCD patients undergoing corneal transplantation. The keratocytes were subjected to gene sequencing, RT-PCR, western blotting, transmission electron microscopy, histological staining, induction and inhibition assays of autophagy and pyroptosis, CCK-8 and LysoTracker Green DND-26 labeling, and flow cytometry. A novel homozygous MCD mutation was identified in a family from Northeast China; the mutation was distinguished by cytoplasmic vacuolation, cell membrane disruption, electron dense deposits, and deposition of a band of Periodic acid-Schiff and Alcian blue-positive material in the keratocytes and stroma layer. KS protein levels were decreased, expression of p62 and LC3-II proteins was enhanced, cathepsin D expression was declined and the LysoTracker Green DND-26 signal was dramatically reduced in MCD keratocytes. Bafilomycin-A1 treatment significantly increased caspase-1 and Pro-IL-1β expression in normal and MCD keratocytes. Nod-like receptors pyrins-3 (NLRP3), caspase-1, Pro-IL-1β, and IL-1β levels were pronouncedly elevated in cells exposed to H2O2. Ac-YVAD-CMK treatment reversed this expression in normal and MCD keratocytes. Suppression of the autophagic degradation of non-sulfated KS by impaired autophagic flux in MCD keratocytes triggers pyroptosis. Amelioration of impaired autophagy and restraint of pyroptosis may, therefore, have therapeutic efficacy in the treatment of MCD.

Structural insight into the binding of human galectins to corneal keratan sulfate, its desulfated form and related saccharides.

Abstract: Glycosaminoglycan chains of keratan sulfate proteoglycans appear to be physiologically significant by pairing with tissue lectins. Here, we used NMR spectroscopy and molecular dynamics (MD) simulations to characterize interactions of corneal keratan sulfate (KS), its desulfated form, as well as di-, tetra- (N-acetyllactosamine and lacto-N-tetraose) and octasaccharides with adhesion/growth-regulatory galectins, in particular galectin-3 (Gal-3). The KS contact region involves the lectin canonical binding site, with estimated KD values in the low µM range and stoichiometry of ~ 8 to ~ 20 galectin molecules binding per polysaccharide chain. Compared to Gal-3, the affinity to Gal-7 is relatively low, signaling preferences among galectins. The importance of the sulfate groups was delineated by using desulfated analogs that exhibit relatively reduced affinity. Binding studies with two related di- and tetrasaccharides revealed a similar decrease that underscores affinity enhancement by repetitive arrangement of disaccharide units. MD-based binding energies of KS oligosaccharide-loaded galectins support experimental data on Gal-3 and -7, and extend the scope of KS binding to Gal-1 and -9N. Overall, our results provide strong incentive to further probe the relevance of molecular recognition of KS by galectins in terms of physiological processes in situ, e.g. maintaining integrity of mucosal barriers, intermolecular (lattice-like) gluing within the extracellular meshwork or synaptogenesis.

Electro-Stimulation, a Promising Therapeutic Treatment Modality for Tissue Repair: Emerging Roles of Sulfated Glycosaminoglycans as Electro-Regulatory Mediators of Intrinsic Repair Processes

Abstract: Glycosaminoglycans (GAGs) are a family of diverse biomolecules that decorate proteoglycans in the glycocalyx and extracellular matrix of all cells. They exist as linear polysaccharide chains consisting of repeating disaccharide units that can be variably sulfated and carboxylated along their GAG chain length. These ionizable carboxyl and sulfate groups on GAGs create charged interactive motifs that convey cell regulatory properties important in tissue homeostasis and the maintenance of optimal tissue function. GAGs participate in a number of essential physiological processes including coagulation‐fibrinolysis, matrix assembly and stabilization, immune regulation, and the complement system. The high fixed charge density and the counter‐ions of GAGs is central to their role in the hydration of various connective tissues within the body. Charge transfer properties of GAGs make them amenable to electro‐stimulation and offers a potential mechanism for promoting or enhancing cellular tissue repair processes. This review is undertaken to illustrate these properties and to gain a better understanding of how these processes might be manipulated through electro‐stimulation to help improve tissue repair and the recovery of normal function in traumatized tissues. Weight‐bearing and tension‐bearing, collagen‐rich, avascular tissues have intrinsically poor repair properties and represent difficult clinical challenges. Electro‐stimulation represents a novel approach with significant potential in the stimulation of repair in these most intransigent of tissues.

Contributions of Chondroitin Sulfate, Keratan Sulfate and N-linked Oligosaccharides to Inhibition of Neurite Outgrowth by Aggrecan.

Abstract: The role of proteoglycans in the central nervous system (CNS) is a rapidly evolving field and has major implications in the field of CNS injury. Chondroitin sulfate proteoglycans (CSPGs) increase in abundance following damage to the spinal cord and inhibit neurite outgrowth. Major advances in understanding the interaction between outgrowing neurites and CSPGs has created a need for more robust and quantitative analyses to further our understanding of this interaction. We report the use of a high-throughput assay to determine the effect of various post-translational modifications of aggrecan upon neurite outgrowth from NS-1 cells (a PC12 cell line derivative). Aggrecan contains chondroitin sulfate, keratan sulfate, and N-linked oligosaccharides (N-glycans), each susceptible to removal through different enzymatic digestions. Using a sequential digestion approach, we found that chondroitin sulfate and N-glycans, but not keratan sulfate, contribute to inhibition of neurite outgrowth by substrate-bound aggrecan. For the first time, we have shown that N-linked oligosaccharides on aggrecan contribute to its inhibition of neuritogenesis.

The expression of keratan sulfate reveals a unique subset of microglia in the mouse hippocampus after pilocarpine-induced status epileptics

Abstract: Induction of keratan sulfate in microglia has been found in several animal models of neurological disorders. However, the significance of keratan sulfate-expressing microglia is not fully understood. To address this issue, we analyzed the characteristics of microglia labeled by the 5D4 epitope, a marker of high-sulfated keratan sulfate, in the mouse hippocampus during the latent period after pilocarpine-induced status epilepticus (SE). Only 5D4-negative (5D4- ) microglia were found in the CA1 region of vehicle-treated controls and pilocarpine-treated mice at 1 day after SE onset. A few 5D4+ microglia appeared in the strata oriens and radiatum at 5 days post-SE, and they were distributed into the stratum pyramidale at 14 days post-SE. The expressions of genes related to both anti- and pro-inflammatory cytokines were higher in 5D4+ cells than in 5D4- cells at 5 but not 14 days post-SE. The expressions of genes related to phagocytosis were higher in 5D4+ cells than in 5D4- cells throughout the latent period. The phagocytic activity of microglia, as measured by engulfment of the zymosan bioparticles, was higher in 5D4+ cells than in 5D4- cells. The contact ratios between excitatory synaptic boutons and microglia were also higher in 5D4+ cells than in 5D4- cells at 5 and 14 days post-SE. The excitatory/inhibitory ratios of synaptic boutons within the microglial domain were lower in 5D4+ cells than in 5D4- cells at 14 days post-SE. Our findings indicate that 5D4+ microglia may play some role in epileptogenesis via pruning of excitatory synapses during the latent period after SE.

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Abstract: Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using the Keratanase Generated BKS-1(+) Neoepitope: Correlation of Expression Patterns with the Class II SLRPs, Lumican and Keratocan

Abstract: This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development.

Novel viscoelastic solution and use thereof in rhumatology

Abstract: A viscoelastic solution including a polysaccharide selected from hyaluronic acid, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, chitosan, xanthans, alginates and carrageenans, or one of the salts thereof; and an antifibrinolytic agent selected from tranexamic acid, epsilon-aminocaproic acid, protamine and desmopressin; and its use in rheumatology.