Showing papers on "Keratan sulfate published in 2006"

Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

Abstract: Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.

The structure and degradation of aggrecan in human intervertebral disc.

Abstract: The ability of the intervertebral disc to resist compression is dependent on its high proteoglycan concentration. The disc proteoglycans are classified as aggregating or non-aggregating depending on their ability to interact with hyaluronan. The majority of the aggregating proteoglycans are derived from aggrecan, though their glycosaminoglycan substitution pattern has not been determined. In contrast, the origin of the non-aggregating proteoglycans is unclear, though it has been postulated that they are derived from aggrecan by proteolysis. The present work demonstrates that keratan sulfate (KS) in the glycosaminoglycan-binding region of disc aggrecan is confined to the KS-rich domain of the core protein and is not present in association with chondroitin sulfate (CS) in the CS1 and CS2 domains. It also shows that the non-aggregating disc proteoglycans are derived from aggrecan, with the large molecules possessing both the KS-rich and CS1 domains and the smaller molecules being generated from either the KS-rich or CS2 domain. The origin and spectrum of disc proteoglycan heterogeneity is the same in both the annulus fibrosus and nucleus pulposus.

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase.

Abstract: Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Identification of Glycosaminoglycans in Human Airway Secretions

Abstract: Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated ...

N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury.

Abstract: Keratan sulfate (KS) is a glycosaminoglycan composed of repeating disaccharide units with sulfate residues at the C6 positions of galactose and N-acetylglucosamine (GlcNAc). The N-acetylglucosamine 6-O-sulfotransferase(s) (GlcNAc6ST) involved in the synthesis of KS in the central nervous system (CNS) has long been unidentified. Here, we report that a deficiency of GlcNAc6ST-1 leads to loss of 5D4-reactive brain KS and reduction of glial scar formation after cortical stab injury in mice. During the development of mice deficient in GlcNAc6ST-1, KS expression in the brain was barely detectable with the KS-specific antibody 5D4. The reactivity of 5D4 antibody with protein tyrosine phosphatase zeta (PTPzeta), a KS proteoglycan (KSPG), was abolished in the deficient mice. In adults, brain injury induced 5D4-reactive KS synthesis in the wounded area in wild-type (WT) mice but not in the deficient mice. Glial scar is formed via the accumulation of reactive astrocytes and is a major obstacle to axonal regeneration by injured neurons. Reactive astrocytes appeared to similar extents in the two genotypes, but they accumulated in the wounded area to a lesser extent in the deficient mice. Consequently, the deficient mice exhibited a marked reduction of scarring and enhanced neuronal regeneration after brain injury. These findings highlight the indispensable role of GlcNAc6ST-1 in brain KS biosynthesis and glial scar formation after brain injury.

Brain keratan sulfate and glial scar formation.

Abstract: In response to injury to the central nervous system (CNS), reactive astrocytes appear and accumulate in the wounded area, leading to glial scar formation. Glial scar is the physical barrier to axonal regeneration of injured neurons. Chondroitin sulfate proteoglycans are inhibitory to axon outgrowth and are upregulated in reactive astrocytes upon CNS injury. It is known that keratan sulfate proteoglycans (KSPGs) are also augmented after CNS injury and act as inhibitory cues. We give a brief overview of CNS injury and cover our recent data regarding the relationship between glial scar formation and KS. KS expression in the developing brain is detectable with 5D4, a KS-specific monoclonal antibody. These 5D4 immunoreactivities are eliminated in mice deficient in N-acetylglucosamine 6-O-sulfotransferase-1. In adult mice, brain injury apparently upregulates mRNA expression of N-acetylglucosamine 6-O-sulfotransferase-1 as well as 5D4-reactive KS in the wounded area. Intriguingly, the expression of 5D4-reactive KS and reactive astrocyte accumulation in the wounded area are dramatically diminished in the sulfotransferase-deficient mice. Consequently, the deficient mice exhibit a marked reduction in scar formation and enhancement of neuronal regeneration after brain injury. Thus, N-acetylglucosamine 6-O-sulfotransferase-1 plays indispensable roles in brain KS biosynthesis and glial scar formation after brain injury.

Thyroxine affects expression of KSPG-related genes, the carbonic anhydrase II gene, and KS sulfation in the embryonic chicken cornea.

Abstract: Purpose Opaque chick corneas become thin and transparent from embryonic day (E)9 to E20 of incubation. Thyroxine (T4) injected in ovo on E9 induces precocious transparency by E12. The present study was conducted to determine whether corneal cells differentially express genes for T4 regulation, keratan sulfate proteoglycan (KSPG) synthesis, crystallins, and endothelial cell ion transporters during transparency development and whether these expressions are altered when E9 embryos are treated with T4. Methods E9 eggs received T4 or buffer; corneas were dissected on E12. Corneal transparency was measured digitally and thickness was determined from cryostat cross sections. mRNA expressions were determined by real-time PCR using cDNA synthesized from whole-cell RNA, cells expressing T4 receptor mRNAs assessed by in situ hybridization, and KS disaccharide sulfation measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Results All corneal layers expressed T4 receptor alpha (THRA) mRNA; keratocytes and endothelial cells expressed T4 receptor beta (THRB) mRNA. During normal development, THRB expression increased 20-fold from E12 to E20; THRA expression remained constant. Expressions of most genes involved in KS synthesis increased from E9 to E16, and then decreased from E16 to E20. From E9 to E20, expressions of crystallin genes increased; T4/3-deiodinase DIII (DIO3) increased 10-fold; and sodium-potassium ATPase transporter (ATP1A1), sodium-bicarbonate transporter (NBC), and carbonic anhydrase II (CA2) increased 5- to 10-fold. E9 T4 administration decreased corneal thickness by E12; increased DIO3, THRB, and CA2 expressions 5- to 20-fold; decreased KSPG core protein genes and galactose sulfotransferase CHST1 expressions 2-fold; and reduced KS disulfated/monosulfated disaccharide (DSD/MSD) ratios. Conclusions Thyroxine modifies expressions of KSPG synthesis and carbonic anhydrase genes.

Stimulation of collagen synthesis by insulin and proteoglycan accumulation by ascorbate in bovine keratocytes in vitro.

Abstract: PURPOSE. Ascorbate is required for the hydroxylation of collagen that is present in the corneal stroma. The keratan sulfate proteoglycans (KSPGs) lumican and keratocan are also present, and they interact with collagen and modulate its assembly into fibrils. In this study, ascorbate was added to a defined medium containing insulin, and its effects on the synthesis of collagen and KSPGs by keratocytes were determined. METHODS. Collagenase-isolated keratocytes were cultured with or without insulin with or without ascorbate. Collagen and glycosaminoglycan synthesis was determined by collagenase digestion of incorporated 3 H-glycine and by chondroitinase ABC or endo-β-galactosidase digestion of incorporated 35 SO 4 . KSPGs were detected by Western blot. Collagen stability was determined by pepsin digestion. Ethyl-3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation. RESULTS. Insulin stimulated the synthesis of collagen but did not affect the accumulation of lumican and keratocan. Insulin plus ascorbate, however, stimulated the synthesis of collagen and increased the accumulation of these proteoglycans. The accumulation of PGDS, a KSPG that does not interact with collagen, was not affected by ascorbate. Only the collagen synthesized in the presence of ascorbate was pepsin resistant. EDB overrode the effects of ascorbate on pepsin resistance and proteoglycan accumulation. CONCLUSIONS. The results of this study indicate that the accumulation of lumican and keratocan depends in part on the level of collagen synthesis and its hydroxylation. The interaction of lumican and keratocan with the stably folded triple helix provided by hydroxylation may also serve to stabilize these proteoglycans.

Lumican, a small leucine-rich proteoglycan substituted with keratan sulfate chains is expressed and secreted by human melanoma cells and not normal melanocytes

Abstract: Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.

Phage display-derived human antibodies against specific glycosaminoglycan epitopes.

Abstract: Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hyaluronic acid, and keratan sulfate). Modification of the backbone by sulfation, deacetylation, and epimerization results in unique sequences within GAG molecules, which are instrumental in the binding of a large number of proteins. Investigating the exact roles of GAGs has long been hampered by the lack of appropriate tools, but we have successfully implemented phage display technology to generate a large panel of antibodies against CS, DS, HS, and heparin epitopes. These antibodies provide unique and highly versatile tools to study the topography, structure, and function of specific GAG domains. In this chapter, we describe the selection, characterization, and application of antibodies against specific GAG epitopes.

Small leucine rich repeat proteoglycans (SLRPs) in the human sclera: identification of abundant levels of PRELP.

Abstract: PURPOSE The small leucine rich proteoglycan (SLRP) family is made up of several members which are thought to guide matrix assembly and organization through protein:protein and/or protein:carbohydrate interactions. In order to better characterize the composition of the scleral extracellular matrix, gene and protein expression of several members of the SLRP family were evaluated in the human sclera from donors aged 2-93 years of age. METHODS Semi-quantitative and quantitative RT-PCR analyses were performed on RNA isolated from human donor sclera using primers for decorin, fibromodulin, PRELP (proline arginine rich end leucine-rich protein), biglycan, chondroadherin, and lumican. Additionally, the protein expression and distribution of the SLRP family member, PRELP, was determined in the human sclera through western blot detection and immunohistochemistry. RESULTS Semi-quantitative and quantitative PCR analysis showed that all six SLRPs were expressed in the human sclera, with PRELP exhibiting the highest steady state mRNA levels, relative to that of the other SLRPs (p<0.001, ANOVA). Further analysis of PRELP in the human sclera by western blot analysis indicated that PRELP contained a 45 kDa core protein with short unsulfated keratan sulfate side chains and appeared in greatest abundance in sclera during the fourth decade of life. CONCLUSIONS These results suggest that several SLRP proteoglycans are expressed in the human sclera and provide the first description of the PRELP protein in the human sclera. The relative abundance of PRELP mRNA and protein in the human sclera, and the observed age-related variation in scleral PRELP expression suggests that PRELP may play a critical role in regulating the biomechanical properties of scleral extracellular matrix.

The effects of glycosaminoglycans on thrombopoietin-induced megakaryocytopoiesis

Abstract: BACKGROUND AND OBJECTIVES: The extracellular matrix plays an essential role in normal hematopoiesis. Proteoglycans and glycosaminoglycans (GAG) are major components of the matrix. In this study, the effects of various GAG on the proliferation and differentiation of CD34+ megakaryocytic progenitor cells (CFU-Meg) were evaluated in vitro. DESIGN AND METHODS: CD34+ cells were highly purified from steady-state human peripheral blood. The GAG tested were hyaluronic acid (from humans, pigs and roosters), keratan sulfate, heparan sulfate, chondroitin sulfate (from whale, shark or squid cartilage) and dermatan sulfate (DS). RESULTS: When used alone, none of the GAG supported the clonal growth of CFU-Meg; however, in cultures stimulated by recombinant human thrombopoietin, human hyaluronic acid, whale chondroitin sulfate and DS significantly enhanced such growth. In particular, the addition of DS resulted in increases of about 1.3-fold, 1.6-fold and 2.0-fold in the numbers of total cells, megakaryocytes and CFU-Meg, respectively, compared with the control culture stimulated by thrombopoietin alone after 9-12 days of serum-free liquid culture. Furthermore, DS induced the generation of hyperploid megakaryocytes and promoted pro-platelet formation. Chemical fragmentation and desulfation of DS showed that a chain of at least 12 saccharides is required for colony-promoting activity and that the sulfate groups play an essential role. INTERPRETATION AND CONCLUSIONS: DS acts on an immature population of CD34+ cells, stimulates the proliferation of CFU-Meg, and enhances the terminal maturation of megakaryocytes and thrombopoiesis. These results suggest that DS has a wide spectrum of action in promoting megakaryocytopoiesis and thrombopoiesis.

Differential immunogold localisation of sulphated and unsulphated keratan sulphate proteoglycans in normal and macular dystrophy cornea using sulphation motif-specific antibodies.

Abstract: Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease.

Cell surface glycoconjugate abnormalities and corneal epithelial wound healing in the pax6+/- mouse model of aniridia-related keratopathy.

Abstract: When an epithelium is wounded, a complex series of signaling pathways are initiated in cells radiating from the wound site that promote immediate cell migration into the wound.1-4 Epithelial proliferation is initiated within a few hours, and stem cells or stemlike cells within the epithelium may be activated.5 Heterozygous deficiency in human PAX6 leads to ocular surface disease characterized by corneal opacification (aniridia-related keratopathy [ARK]), and the Pax6+/− mouse models all the morphologic defects of human ARK.6-9 In vitro, Pax6+/− corneal epithelial cells do not respond to wounding within the first 1 to 2 hours, in contrast to Pax6+/+ cells that start to migrate into the wound within a few minutes.10 However, when Pax6+/− cells overcome their block, they migrate as fast as wild-type, and so there is no intrinsic defect in cell migration, only in their ability to respond quickly to wounding. ARK may be due to corneal epithelial fragility combined with an abnormal wound-healing response.11 Impaired re-epithelialization after wounding increases the risk of infection, exacerbates inflammation, and undermines normal stromal remodeling.12 In vivo studies have suggested that changes in the adhesive properties of mutant cells may underlie many of the observed morphologic abnormalities, and Pax6 controls expression of cell adhesion molecules, most of which are glycoproteins.8,13-15 Before Pax6 was identified as the mutant gene in small-eye (Pax6Sey/+) mice, Pritchard16 and Pritchard et al.17 biochemically identified multiple glycoconjugate abnormalities in Pax6+/− eyes and suggested that the defective gene may be involved in synthesis or processing of glycoproteins. Glycosaminoglycans (GAGs) are linear polymers of amino sugar uronic acid disaccharides, usually covalently attached to a small protein core as components of proteoglycans, which may be anchored to the plasma membrane or released into the extracellular matrix (ECM).18 GAG families are classed according to the disaccharides from which they are polymerized: chondroitin/dermatan sulfate, keratan sulfate, heparin/heparan sulfate, or hyaluronic acid.19 GAGs are necessary for cell adhesion and directed cell migration.19,20 They act as coreceptors for growth factor ligands in the ECM or on the cell surface.20-22 Sulfated GAG chains are regulators of cell– cell and cell–ECM interactions.23,24 Mutations in keratocan, a keratan sulfate proteoglycan, lead to cornea plana, whereas repair of damaged adult corneal epithelium is retarded in lumican (a keratan sulfate proteoglycan) and syndecan-1 (a heparan sulfate proteoglycan)– deficient mice (Gala PH et al. IOVS 2000; 41:ARVO Abstract 4846).25-27

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

Abstract: PURPOSE To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas METHODS Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) RESULTS Concentrations of both monosulfated GlcNAc(6S)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls The concentration of DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-0S CS/DS disaccharides demonstrated no significant differences from normal corneas CONCLUSIONS The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS

Determination of substrate specificity of sulfotransferases and glycosyltransferases (proteoglycans).

Abstract: Proteoglycans have sulfated linear polysaccharide chains, that is, heparan sulfate, heparin, chondroitin sulfates, dermatan sulfate, and keratan sulfate. Many glycosyltransferases and sulfotransferases are involved in biosynthesis of the polysaccharides. Specificities of these enzymes have been mainly determined by evaluating their activities to various acceptor carbohydrates and by analyzing the structure of the products. For the latter purpose, enzymatic hydrolysis using heparitinases, heparinase, and chondroitinases or chemical degradation employing nitrous acid deamination has been effectively used in combination with high‐performance liquid chromatography (HPLC) of the degraded products. As examples, we describe methods for assays and product characterization of sulfotransferases involved in biosynthesis of these polysaccharides, namely heparan sulfate 2‐sulfotransferase, heparan sulfate 6‐sulfotransferases, chondroitin 4‐sulfotransferases, chondroitin 6‐sulfotransferase, N‐acetylgalactosamine 4‐sulfate 6‐sulfotransferase, and N‐acetylglucosamine 6‐sulfotransferases.

Sulphation patterns of keratan sulphate proteoglycan in sclerocornea resemble cornea rather than sclera

Abstract: Sclerocornea is one of the most frequent causes of congenital blindness,1 in which the cornea is opaque and resembles sclera at birth.2 The stromal matrix in sclera and in cornea is composed of collagen fibrils, with proteoglycans (PGs) in the interfibrillar space fulfilling important roles in relation to tissue structure and function. Sclera and cornea contain distinct PG populations. Whereas the predominant corneal PG carries highly sulphated keratan sulphate (KS) glycosaminoglycans (GAGs), sulphated KSPGs are not major scleral components.3 Here, we investigate if sclerocornea is more like sclera or cornea in terms of its KSPG molecular profile. An 8 month old girl, born at 39 weeks after an uncomplicated pregnancy and delivery, had bilateral corneal opacification and was referred to Kyoto Prefectural University of Medicine. There was no family history of eye disease. Slit lamp examination disclosed diffuse, full thickness opacification, which was more severe in her left eye (fig 1A), preventing examination of the anterior chamber. Ultrasound biomicroscopy detected synechiae between …

CHAPTER 5 – Proteoglycans and Glycosaminoglycans

Abstract: Protoglycans are important components of extracellular matrices (ECM) and have multiple functions that depend on both their protein and carbohydrate constituents. They form a special class of glycoproteins with attached long unbranched and highly charged glycosaminoglycan chains. These chains are strongly hydrophilic and dominate the physical properties of protoglycans. But the proteins to which these chains are attached are quite diverse in structure and form several distinct protein families. Protoglycans are most abundant in those tissues where the ECM is highly hydrated. Cartilage and bone are tissues that both contain large expanded ECM, although the composition of these two tissues is strikingly different. In cartilage the major protoglycan is aggrecan, which forms supramolecular aggregates and is important in expanding and hydrating the matrix. It contains mainly chondroitin sulfate and increasing amounts of keratan sulfate with age. There are also lesser amounts of the lower-molecular-weight leucine-rich protoglycans, including decorin, fibromodulin, and lumican, which are collagen fibril-associated, and also biglycan. These also contain chondroitin sulfate, but with varying degrees of epimerization to dermatan sulfate. There is no aggrecan present in bones, and the collagen fibril-associated protoglycans and biglycan are the predominant forms. The cells of bone and cartilage also contain some cell surface integral membrane protoglycans, which predominantly contain heparan sulfate. These are very important in cell–matrix interactions and cell signaling, and control many aspects of chondrocyte and ostoblast function. But as the cell density is low, they do not contribute to a large fraction of the tissue content of protoglycans.

[The metabolism of glycosaminoglycans in the course of Graves' disease].

Abstract: UNLABELLED Glycosaminoglycans (GAGs), which include chondroitin sulfate (CS), dermatan sulfates (DS), heparan sulfate (HS), heparin (H), keratan sulfate (KS), and hyaluronic acid (HA), are a group of linear, polyanionic heteropolysaccharides. The GAGs chains, except for those of hyaluronic acid, are covalently attached to core proteins, forming proteoglycans (PGs). PGs/GAGs are present at the cellular level as elements of the cell membrane and intracellular granules. They are also components of the ground substance of the extracellular matrix. These macromolecules are involved in cell adhesion, migration, and proliferation. Alterations in GAGs metabolism may influence the pathogenesis of many disorders, including Graves' disease. Graves' disease is an autoimmune thyroid pathology characterized by hyperthyroidism, thyroid hyperplasia, as well as ophthalmopathy and/or pretibial myxedema. The pathogenesis of these extrathyroidal manifestations involves fibroblast activation and increased glycosaminoglycan synthesis and accumulation. Disturbances in GAGs metabolism in tissue are associated with qualitative and quantitative GAGs alterations in Graves' patients' serum and urine. Although the mechanisms leading to the development of orbital and/or skin complications in the course of Graves' disease have not been fully elucidated, it is postulated that they depend on both immunological disturbances and the hyperthyroid state. SUMMARY The alterations in GAGs metabolism connected with Graves' disease could lead to systemic changes in the proprieties of the extracellular matrix.

Metabolizm glikozoaminoglikanów w przebiegu choroby Gravesa-Basedowa The metabolism of glycosaminoglycans in the course of Graves' disease

Abstract: Slowa kluczowe: choroba Gravesa-Basedowaglikozoaminoglikanycytokinyhipertyreoza Summary Glycosaminoglycans (GAGs), which include chondroitin sulfate (CS), dermatan sulfates (DS), heparan sulfate (HS), heparin (H), keratan sulfate (KS), and hyaluronic acid (HA), are a group of linear, polyanionic heteropolysaccharides. The GAGs chains, except for those of hyaluronic acid, are covalently attached to core proteins, forming proteoglycans (PGs). PGs/GAGs are present at the cellular level as elements of the cell membrane and intracellular granules. They are also com- ponents of the ground substance of the extracellular matrix. These macromolecules are involved in cell adhesion, migration, and proliferation. Alterations in GAGs metabolism may infl uence the pathogenesis of many disorders, including Graves' disease. Graves' disease is an autoimmune thy- roid pathology characterized by hyperthyroidism, thyroid hyperplasia, as well as ophthalmopat- hy and/or pretibial myxedema. The pathogenesis of these extrathyroidal manifestations involves

A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation

Abstract: ["The proteoglycan aggregate is a major component of the articular cartilage extracellular matrix comprising hyaluronan (HA), aggrecan, and link protein. Aggrecan, heavily substituted with chondroitin sulfate (CS) and keratan sulfate (KS) glycosaminoglycans, contributes to cartilage hydration by binding water, thus lending articular cartilage its resistance to compressive deformation. A loss of aggrecan is observed in the early stages of osteoarthritis, which may relate to age-dependent changes in biochemical properties of proteoglycan aggregates. The research reported in this thesis was focused on producing recombinant molecules for modeling proteoglycan aggregate assembly and aggrecan proteolytic degradation. First, the proteoglycan tandem repeat (PTR) domains of link protein, which are responsible for HA binding, were expressed in E. coli to develop a tool for studying the PTR-HA interaction. Soluble monomeric PTR domains were obtained by a novel refolding procedure. Second, full-length recombinant link protein and aggrecan were expressed in a mammalian expression system as functional molecules capable of forming a ternary complex with HA. Furthermore, the expression of recombinant aggrecan in various mammalian cell lines allows the production of differently glycosylated aggrecans for comparison of glycosylation-specific functional differences. Last, both cartilage-derived and recombinant wild-type and mutagenized aggrecans were used to study proteinase-dependent aggrecan degradation observed in the cartilage extracellular matrix. The present work demonstrated that sulfated glycosaminoglycans covalently attached to both cartilage-derived and recombinant aggrecans regulate the susceptibility of aggrecan to ADAMTS4. A study using mutagenized recombinant aggrecan suggested that potentially glycosylated threonine and serine residues N-terminal and C-terminal to the ADAMTS4 cleavage site within the interglobular domain of aggrecan influence the rate of cleavage by ADAMTS4. The degree of hydrophobicity N-terminal to the ADAMTS4 cleavage site also affects aggrecan s susceptibility to ADAMTS4. Recombinant aggrecan was substituted with KS by co-transfecting aggrecan with KS-sulfotransferases. KS-substituted aggrecan, which also had altered CS components, was found to be differentially susceptible to different isoforms of ADAMTS4. This work demonstrates the usefulness of the recombinant system for studies of molecular interactions with HA, aggrecan, and link protein, as well as for in vitro analyses of matrix proteinase-mediated degradation of aggrecan."]