Composition of glycosaminoglycans in human pancreatic cancer

Five glycosaminoglycans were isolated from tryptic digestion of both cancerous and normal tissues of the human pancreas and were assayed by determining the carbohydrate content of materials. Separation of these five polymers was achieved by Dowex 1-X2 column chromatography and fractionation with Benedict's solution. They were identified as hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate, respectively. The total amount of glycosaminoglycans in cancer tissue increased in comparison to the controls. The increase in tissue content of glycosaminoglycans was accompanied by increases in chondroitin-4-sulfate and chondroitin-6-sulfate levels.     

Aggregation of LDL with chondroitin-4-sulfate makes LDL oxidizable in the presence of water-soluble antioxidants

The content of plasma and arterial interstitial fluid in water-soluble antioxidants makes it unlikely for low-density lipoprotein (LDL) to oxidize by the oxidation mechanisms most frequently discussed. By aggregation of LDL in the presence of chondroitin-4-sulfate (C-4-S), but not with chondroitin-6-sulfate or sphingomyelinase, a complex arises which can oxidize in the presence of 20 microM ascorbate and 300 microM urate. This oxidation sensitivity even persists after the gel-filtration of an LDL/C-4-S/Cu(2+) complex, indicating entrapment of Cu(2+) within. This corresponds well to the known ability of C-4-S to bind copper ions and is a potential mechanism by which LDL oxidation in the arterial intima is facilitated after prolonged retention by the extracellular matrix.     

Analyses of glycosaminoglycans in human lung cancer

Studies were conducted on the total amount of glycosaminoglycans and glycosaminoglycan composition in adenocarcinoma tissue of human lung. The glycosaminoglycans were prepared by exhaustive proteinase digestion of adenocarcinoma tissue from human lungs and of lung tissue without pulmonary diseases taken at autopsy as a control. The glycosaminoglycan classes were characterized by biochemical, enzymatic, and electrophoretic methods. The presence of heparin, which has until now not been found in lung cancer tissue, was demonstrated on both carcinoma and control tissues. The levels of whole glycosaminoglycans were markedly increased in cancer tissue compared to the controls. The classes of glycosaminoglycans which increased in lung carcinoma tissue were predominantly chondroitin-4-sulfate and chondroitin-6-sulfate. Both hyaluronic acid and heparin were slightly increased in cancer tissue.     

Differential expression of glycosaminoglycans and proteoglycans in the migratory pathway of the primordial germ cells of the mouse

Primordial germ cells are an embryonic cell line that give rise to gametes in vertebrates. They originate outside the embryo proper and migrate by a well-defined route to the genital ridges. Proteoglycans and glycosaminoglycans have distinctive properties that affect many of the characteristics of the extracellular microenvironment of migratory pathways in a variety of developmental systems. The purpose of this work was to identify the proteoglycans and glycosaminoglycans that are spatially and temporally expressed in the migratory pathway of primordial germ cells. We showed that the expression of proteoglycans and glycosaminoglycans in the primordial germ cells migratory pathway changes according to the different phases of the migratory process. Some molecules such as chondroitin-0-sulfate, decorin, and biglycan are present only in certain phases of the migratory process of primordial germ cells. Heparan sulfate, chondroitin-6-sulfate, versican, perlecan, and syndecan-4, although exhibiting some variation in expression were detected during all phases of the migratory process. Our results indicate that the successive steps of primordial germ cell migration require a coordinated expression of proteoglycans and glycosaminoglycans, that should be present in appropriate levels and in specific areas of the embryo, and that the sequential expression of these extracellular matrix molecules is under a genetic program that appears to be common to a variety of cell types during embryonic development.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: Relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Disaccharide analysis of glycosaminoglycans synthesized by cardiac myxoma cells in tumor tissues and in cell culture

We analyzed the main disaccharide units of glycosaminoglycans synthesized by cardiac myxoma cells in vivo and in cell culture using high-performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labeling. Cardiac myxoma tissues contained large amounts of chondroitin-6-sulfate (46%) and hyaluronic acid (32%), along with some chondroitin-4-sulfate (13%), chondroitin (6%), and much less dermatan sulfate (3%). Cultured cardiac myxoma cells synthesized mainly chondroitin-6-sulfate. The abundant glycosaminoglycans in myxoma tissues may make up the characteristic friable gelatinous matrix which is favorable for embolism and tumor cell growth.     

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

The hydration of proteoglycans of bovine cornea

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a disaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl^?) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.     

Binding of hyaluronate to the surface of cultured cells

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.     

The hydration of proteoglycans of bovine cornea.

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

The preparation of heparan sulfate from the mitral valve of the human heart

A study has been made of the glycosaminoglycan composition of the mitral valve of the normal human heart. Five glycosaminoglycans were isolated from tryptic digest of the material and were assayed by determining the carbohydrate content. Separation of these five polymers was achieved by Dowex 1 X 2 column chromatography. They were identified as hyaluronic acid, heparan sulfate, chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively. As far as the authors are aware, this is the first isolation of heparan sulfate from the preparation of the mitral valve of the normal human heart.     

Inhibition by 5-bromo-2'-deoxyuridine of differentiation-dependent changes in glycosaminoglycans of the retina.

Embryonic chick neural retinas incorporated radio-labeled precursors into glycosaminoglycans in the same relative amounts whether cultured as intact tissues, cell aggregates, or monolayers. Incubation with 5-bromo-2′-deoxyuridine inhibited histogenesis and caused the pattern of synthesis to remain more like that in undifferentiated tissue, when compared with controls without this nucleoside analog. This was determined by the level of incorporation and the ratios of chondroitin sulfate to heparan sulfate and chondroitin-4-sulfate to chondroitin-6-sulfate incorporation. Incubation with 4-methylumbelliferyl-β-D-xylopyranoside stimulated synthesis and release of chondroitin sulfate and heparan sulfate into the medium. The results taken together imply that the production of specific glycosaminoglycans during the course of differentiation in the retina is regulated at the gene level in parallel with histogenesis in this tissue.     

Arylsulfatase B regulates interaction of chondroitin-4-sulfate and kininogen in renal epithelial cells

The enzyme arylsulfatase B (N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen–bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen–C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen–bradykinin axis and, thereby, may influence blood pressure.Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation.     

Chemical modification of low-density lipoprotein enhances the number of binding sites for divalent cations.

The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

Glycosaminoglycan synthesis in endotoxin-induced lung injury

Endotoxin-induced lung injury has previously been shown to produce lesions that resemble emphysema morphologically and biochemically as demonstrated by the reduction in the content of lung elastin. The purpose of this study was to define the changes in one other connective tissue component, glycosaminoglycans, during the acute phase of the lung injury. Intravenous administration of a single dose of endotoxin in rats resulted in an increase in the total synthesis of glycosaminoglycans by the pulmonary parenchyma. There was a significant increase in the proportion of dermatan sulfate synthesized during the first 48 hr and a concomitant decrease in heparin/heparan sulfate synthesis. At 48 hr the increased synthesis of dermatan sulfate had reached 7.3 times control values and began to decline, whereas the synthesis of chondroitin-4-sulfate rose from 4.1 to 10.7 times control values between 48 and 72 hr. Analysis of the rates of synthesis revealed that the total amount of heparin/heparan sulfate remained constant while the synthesis of chondroitin-6-sulfate increased proportionally to the overall synthesis of glycosaminoglycans. These findings indicate that dramatic changes in glycosaminoglycan synthesis are an integral part of endotoxin lung injury.     

Glycosaminoglycans of the plasma membranes of renal tubule and liver cells

Glycosaminoglycans were isolated from plasma membranes of hepatic and renal tubule cells of guinea pig. Plasmalemma of renal tubule cells contained more total glycosaminoglycans, hyaluronic acid, chondroitin-4 sulfates and chondroitin-6 sulfates, and less dermatan sulfates and heparin sulfates than liver plasma membranes. These glycocalyx components, owing to their polyanionic properties, may have a role in the transport of water, ions, and macromolecules across the cell membrane.     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.