Interaction of bone proteoglycans and proteoglycan components with hydroxyapatite

The small leucine-rich proteoglycans (SLRPs) of bone interact with hydroxyapatite (HAP) and are proposed to play an important role in the regulation of the mineralisation process. The present study has examined the interaction of bone SLRPs, purified, liberated bone glycosaminoglycan (GAG) chains and core proteins, as well as commercial chondroitin 4-sulphate (C4S) with HAP. Isotherm data (0.02 M sodium acetate) revealed that the intact proteoglycans (PGs) and bone GAGs showed greater binding onto HAP with higher adsorption maxima than the constituent core proteins and commercial C4S. Adsorption was dependent on pH and ionic strength, increasing with decreasing pH and in the presence of calcium whilst decreasing in the presence of phosphate, suggesting that electrostatic effects are important. The data indicates that PG/GAG chemistry and conformation in solution are significant determinants in the adsorption process and provides important information concerning interfacial adsorption phenomena between the organic-inorganic phases of mineralised systems.     

Interaction of bone proteoglycans and proteoglycan components with hydroxyapatite.

The small leucine-rich proteoglycans (SLRPs) of bone interact with hydroxyapatite (HAP) and are proposed to play an important role in the regulation of the mineralisation process. The present study has examined the interaction of bone SLRPs, purified, liberated bone glycosaminoglycan (GAG) chains and core proteins, as well as commercial chondroitin 4-sulphate (C4S) with HAP. Isotherm data (0.02 M sodium acetate) revealed that the intact proteoglycans (PGs) and bone GAGs showed greater binding onto HAP with higher adsorption maxima than the constituent core proteins and commercial C4S. Adsorption was dependent on pH and ionic strength, increasing with decreasing pH and in the presence of calcium whilst decreasing in the presence of phosphate, suggesting that electrostatic effects are important. The data indicates that PG/GAG chemistry and conformation in solution are significant determinants in the adsorption process and provides important information concerning interfacial adsorption phenomena between the organic-inorganic phases of mineralised systems.     

The matricellular functions of small leucine-rich proteoglycans (SLRPs)

The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFα), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-β, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Expression of c-Fos in subcutaneous adipose tissue of the fetal pig

Calcium oxalate crystal growth and aggregation leads to the formation of renal calculi. It is known to be inhibited by several compounds both in vitro and in vivo conditions. The present study highlights the inhibitory potential of sodium pentosan polysulphate (SPP), a semi-synthetic glycosaminoglycan (GAG) on calcium oxalate crystal growth in vitro. Its efficacy was compared with those of known inhibitors like pyrophosphate, heparin and chondroitin-4-sulphate. Of the above compounds pyrophosphate was found to be the most potent inhibitor. Among the GAGs, SPP exhibited 80% inhibitory activity as compared to heparin. A lesser degree of inhibition was observed with chondroitin-4-sulphate.     

Glycosaminoglycan-dependent and -independent inhibition of neurite outgrowth by agrin

Agrin is a proteoglycan that can inhibit neurite outgrowth from multiple neuronal types when present as a substrate. Agrin's neurite inhibitory activity is confined to the N-terminal segment of the protein (agrin N150), which contains heparan sulfate (HS) and chondroitin sulfate (CS) side chains. We have examined the activities of various purified recombinant agrin fragments and their glycosaminoglycan (GAG) side chains in neurite outgrowth inhibition. Inhibitory activity was tested using dissociated chick ciliary ganglion neurons or dorsal root ganglion explants growing on laminin or N-cadherin. Initial experiments demonstrated that agrin N150 lacking GAG chains inhibited neurite outgrowth. Both halves of N150, each containing HS and/or CS side chains, could also inhibit neurite growth. Experiments using agrin fragments in which the GAG acceptor residues were mutated, or using agrin fragments purified from cells deficient in GAG synthesis, demonstrated that inhibition by the N-terminal portion of N150 requires GAGs, but that inhibition from the C-terminal part of N150 does not. Thus, the core protein or other types of glycosylation are important for inhibition from the more C-terminal region. Our results suggest that there are two distinct mechanisms for neurite outgrowth inhibition by agrin, one that is GAG-dependent and one that is GAG-independent.     

Carriers for enzymatic attachment of glycosaminoglycan chains to peptide

In the previous study, we have found that the endo-beta-xylosidase from Patinopecten had the attachment activities of glycosaminoglycan (GAG) chains to peptide. As artificial carrier substrates for this reaction, synthesis of various GAG chains having the linkage region tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, between GAG chain and core protein of proteoglycan was investigated. Hyaluronic acid (HA), chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and desulfated dermatan sulfate (desulfated DS) as donors and the 4-metylumbelliferone (MU)-labeled hexasaccharide having the linkage region tetrasaccharide at its reducing terminals (MU-hexasaccharide) as an acceptor were subjected to a transglycosylation reaction of testicular hyaluronidase. The products were analyzed by high-performance liquid chromatography and enzyme digestion, and the results indicated that HA, Ch, Ch4S, Ch6S, and desulfated DS chains elongated by the addition of disaccharide units to the nonreducing terminal of MU-hexasaccharide. It was possible to custom-synthesize various GAG chains having the linkage region tetrasaccharide as carrier substrates for enzymatic attachment of GAG chains to peptide.     

A difference in the specificities of human liver N-acetyl-beta-hexosaminidases A and B detected by their activities towards glycosaminoglycan oligosaccharides.

N-Acetyl-beta-hexosaminidases A and B differ in their activities towards oligosaccharides prepared from glycosaminoglycans. Trisaccharides from hyaluronic acid and desulphated chondroitin 4-sulphate were hydrolysed by N-acetyl-beta-hexosaminidase A, but not by N-acetyl-beta-hexosaminidase B.     

Diagnosis of Maroteaux-Lamy syndrome by the use of radiolabelled oligosaccharides as substrates for the determination of arylsulphatase B activity.

The kinetic parameters (Km and V) of human arylsulphatase B (4-sulpho-N-acetylgalactosamine sulphatase) activity in cultured skin fibroblasts were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo chondroitin 4-sulphate and dermatan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, were desulphated up to 4400 times faster than the minimum arylsulphatase-B-specific substrate, namely the monosaccharide N-acetylgalactosamine 4-sulphate. Aglycone structures that influence substrate binding and/or enzyme activity were an adjacent-residue C-6 carboxy group and a second but internal N-acetylgalactosamine 4-sulphate residue. Arylsulphatase B activity in fibroblast homogenates assayed with O-(beta-N-acetylgalactosamine 4-sulphate)-(1----4)-O-D-(beta-glucuronic acid)-(1----3)-O-D-N-acetyl[1-3H] galactosaminitol 4-sulphate derived from chondroitin 4-sulphate as substrate clearly distinguished Maroteaux-Lamy-syndrome patients from normal controls and other mucopolysaccharidosis patients. We recommend the use of the above trisaccharide substrate for both postnatal and prenatal diagnosis of Maroteaux-Lamy syndrome.     

Transforming growth factor-beta (TGF-beta) receptor proteoglycan. Cell surface expression and ligand binding in the absence of glycosaminoglycan chains

The type III transforming growth factor-beta (TGF-beta) receptor is a cell surface chondroitin/heparan sulfate proteoglycan that binds various forms of TGF-beta with high affinity and specificity. Here, we have used a genetic approach to determine the requirement for glycosaminoglycan (GAG) chains for normal TGF-beta receptor expression and the role that the receptor proteoglycan core and GAG chains play in TGF-beta binding. Chinese hamster ovary (CHO) cells defective in GAG synthesis express on their surface 110-130-kDa type III receptor proteoglycan cores that can bind normal levels of TGF-beta compared to wild type CHO cells. The affinity of the receptor core for TGF-beta 1 and TGF-beta 2 in CHO cell mutants is similar to that of the TGF-beta receptor proteoglycan forms present in wild type CHO cells or in CHO cell mutants that have been allowed to bypass their metabolic defect and express the wild type proteoglycan phenotype. The binding properties of TGF-beta receptor types I and II in CHO cells and the growth-inhibitory response of CHO cell mutants to TGF-beta are not impaired by the absence of GAG chains in the type III receptor. These results show that the GAG chains are dispensable for type III receptor expression on the cell surface, binding of TGF-beta to the receptor core, and growth inhibitory response of the cells to TGF-beta. The evidence also suggests that the type III receptor may act as a multifunctional proteoglycan able to bind TGF-beta via the receptor core while performing another as yet unidentified function(s) via the GAG chains.     

The high molecular weight receptor to transforming growth factor-beta contains glycosaminoglycan chains

Proteoglycans are constituents of the cell surface that may play important roles in the regulation of cell behavior. Here we report that the 250-kDa receptor subunit that binds the multifunctional protein, transforming growth factor-beta 1 (TGF-beta 1), contains chains of heparan sulfate and chondroitin sulfate and thus is a proteoglycan. Digestion of TGF-beta 1-receptor complexes with glycosaminoglycan (GAG)-specific degradative enzymes yield core proteins of 115-140 kDa. Cell monolayers that had been predigested with GAG-specific degradative enzymes were capable of binding high levels of TGF-beta 1, but the size of the binding components was shifted from the high molecular weight species to the lower molecular weight core proteins, indicating that GAG chains are not necessary for TGF-beta 1 binding to the cell. The presence of GAG chains on the receptor subunit indicates that it has the potential for interaction with the extracellular matrix.     

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.     

Electrophoretic and enzymatic analysis of glycosaminoglycans in the blood serum of patients with hyperthyroidism

An effect of hyperthyroidism on the composition and levels of glycosaminoglycans in the blood serum was studied. Glycosaminoglycans isolated from 1-ml blood samples were assayed with the following techniques: carbazole, electrophoretic and enzymatic. Separation and assay of particular GAG were made with bidirectional electrophoresis. Isomers of the remaining chondroitin sulphates were assayed enzymatically. Electrophoretograms of GAG in blood serum of healthy women have shown two fractions: low sulphate chondroitin sulphate and chondroitin-4-sulphate. The same fractions of GAG were found in blood serum of the female patients with hyperthyroidism. Mean concentration of GAG in the blood serum of hyperthyroid patients increased by 51%: low sulphate chondroitin sulphate and chondroitin-4-sulphate concentrations increased by 22% and 190% respectively. Chondroitin sulphates in the blood serum of both groups were degraded to unsaturated disaccharides not containing sulphur and unsaturated 4-sulphate disaccharides. Concentrations of unsaturated 4-sulphate and unsaturated sulphur-free disaccharides increased by 71% and 17% in hyperthyroidism. Observed changes in the blood serum GAG concentrations reflect changes in the connective tissue metabolism in hyperthyroidism.     

Disubstituted naphthyl β-D-xylopyranosides: Synthesis,GAG priming,and histone acetyltransferase (HAT) inhibition

Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)β-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects.     

Glycosaminoglycan (GAG) level and activities of certain enzymes of GAG metabolism in Culex quinquefasciatus and Setaria digitata

Significant differences were observed in GAG metabolism of S. digitata and one of its intermediate vectors, C. quinquefasciatus. Distribution of different components such as hyaluronic acid, heparin-sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate and heparin was comparable in both. However, there were quantitative differences; the difference was marked in the activity of enzymes of GAG metabolism in presence and absence of diethylcarbamazine (DEC) a known antifilarial drug. While the activities of beta-galactosidase and beta-N-acetyl glucosaminidase of S. digitata systems showed an inhibition of 96.5 and 92.6% respectively, in the Culex systems they showed an inhibition of 93.3% and an activation of 18% respectively. The differences clearly indicate the existence of basic differences in GAG metabolism of vector and parasite.     

Protein–polysaccharide of chicken cartilage and chondrocyte cell cultures

The nature of the matrix produced by embryonic chicken chondrocytes in cell culture was studied, and compared with adult and embryonic chicken cartilage. Adult chicken cartilage contains a protein-polysaccharide easily extracted with EDTA-sodium chloride at 4 degrees C. Purification of this macromolecule on Bio-Gel P-300 and Bio-Gel A-50m yielded a progressively more homogeneous species in the ultracentrifuge. It contained mostly chondroitin 4-sulphate, some chondroitin 6-sulphate, and keratan sulphate. Embryonic chicken cartilage was previously shown to contain mostly chondroitin 4-sulphate, some chondroitin 6-sulphate and essentially no keratan sulphate. The matrix produced in chondrocyte cell cultures was shown to contain a protein-polysaccharide with alkali-labile linkages of chondroitin 4-sulphate to the protein core. A fraction was isolated from the matrix with many properties of keratan sulphate.     

Purification of the transforming growth factor-beta (TGF-beta) binding proteoglycan betaglycan.

We report the purification of betaglycan, a low-abundance membrane proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). Betaglycan solubilized from rat embryo membrane preparations was purified to near-homogeneity by sequential chromatography through DEAE-Trisacryl, wheat germ lectin-Sepharose, and TGF-beta 1-agarose. Purified betaglycan has properties similar to betaglycan affinity-labeled in intact cells: it binds TGF-beta 1 and TGF-beta 2 with KD approximately 0.2 nM, contains heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains and N-linked glycans attached to a 110-kDa core protein, and can spontaneously associate with phosphatidylcholine liposomes. The betaglycan core obtained by enzymatic removal of the GAG chains has high affinity for TGF-beta and associates with artificial liposomes, indicating that the core protein binds TGF-beta and anchors to membranes independently of the GAG chains present on the native protein or of any ancillary protein.     

The influence of fluoride administration on the structure of proteoglycans in the developing rat incisor.

1. 35S-labelled chondroitin 4-sulphate proteoglycan was isolated from the mineralized elements of the developing incisor teeth of Harvard rats receiving intraperitoneal administration of Na235SO4. 2. The chondroitin 4-sulphate proteoglycan underwent a decrease in molecular size in fluorotic teeth as judged by gel filtration on Sepharose 2B. 3. When examined by anion-exchange chromatography on DEAE cellulose-52, the proteoglycan from fluorotic teeth resolved into four peaks in comparison with the material from non-fluorotic teeth, which exhibited only a single major peak. 4. Both the single peak from non-fluoridated teeth and the four peaks from the fluorotic teeth were further resolved on cellulose acetate electrophoresis. 5. Isolated chondroitin 4-sulphate chains obtained from fluorotic teeth also were of smaller molecular size as judged by gel filtration on Sephadex G-150. 6. Some possible influences of fluoride on the metabolism of these connective-tissue components in the developing rat incisor are discussed.     

The influence of the type of sulphate bond and degree of sulphation of glycosaminoglycans on their interaction with lysosomal enzymes.

Significant differences occur between the interaction of several sulphated glycosaminoglycans with a particular lysosomal protein, leading to inhibition in the case of lysosomal enzymes. The order of strength of inhibition at pH4 was: heparin greater than chondroitin 4-sulphate = chondroitin 6-sulphate greater than dermatan sulphate.