Intact Proteoglycan Research Articles

Tribology of enzymatically degraded cartilage mimicking early osteoarthritis

Healthy cartilage is a water-filled super lubricious tissue. Collagen type II provides it structural stability, and proteoglycans absorb water to keep the cartilage in a swollen condition, providing it the ability to creep and provide weeping lubrication. Osteoarthritis (OA) is a degenerative and debilitating disorder of diarthrodial joints, where articular cartilage damage originates from enzymatic degradation and mechanical damage (wear). The objective of this research is to observe the level of cartilage damage present in knee arthroplasty patients and to understand the friction and creep behavior of enzymatically degraded bovine cartilage in vitro. Lateral (Lat) and medial (Med) condylar cartilages from OA patients undergoing total knee arthroplasty showed signs of enzymatic degradation and mechanical damage. Bovine cartilages were exposed to collagenase III and chondroitinase ABC to degrade collagen and proteoglycans, respectively. The loss of proteoglycans or collagen network and morphological changes were observed through histology and the atomic force microscope (AFM), respectively. A significant effect on creep due to enzymatic treatment was not observed. But the enzymatic treatment was found to significantly decrease the coefficient of friction (COF) at 4 N, while higher COF was shown from chondroitinase ABC degraded cartilage at 40 N. Collagenase III treatment leads to the release of intact proteoglycans at the sliding interface, while chondroitinase ABC treatment leads to the loss of chondroitin sulfate (CS) from the proteoglycans. Chondroitinase ABC-digested bovine cartilage mimicked patient samples the best because of the similar distributions of proteoglycans, collagen network, and friction behavior.

Decellularized extracellular matrix particle-based biomaterials for cartilage repair applications

Cartilage Decellularized ExtraCellular Matrix (dECM) materials have shown promising cartilage regeneration capacity due to their chondrogenic bioactivity. However, the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes impair the biomimicry of these materials. The current study aims to fabricate biomimetic materials containing decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair. For this, we established a novel two-fraction decellularization strategy for the preparation of reconstituted dECM (rdECM) particles by mixing the two-fraction components, as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM (bdECM) particles. Hyaluronic acid-tyramine (THA) hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test, swelling and stability evaluation, and compression test. The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of decellularization. The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties. bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70% and high retention of glycosaminoglycans and collagen after long-term incubation, while rdECM particle-embedded THA hydrogels showed unsatisfactory stability as self-standing biomaterials. Compared to pure THA hydrogels, the addition of bdECM particles significantly enhanced the compression moduli. In summary, our decellularization methods are successful in the retention of functional and intact cartilage components with high yield. Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing. Hydrogels with cartilage bdECM particles possess the functional structure and the natural composition of cartilage ECM, long-term stability, and enhanced mechanical properties, and are promising biomaterials for cartilage repair.

The non-aggregated aggrecan in the human intervertebral disc can arise by a non-proteolytic mechanism.

Analysis of both the aggregated and non-aggregated fractions of aggrecan isolated from adult human intervertebral disc using immunoblotting with antibodies specific for the different domains constituting the aggrecan core protein or atomic force microscopy revealed that many components contained the G1 domain. However, little of the disc aggrecan was able to reform aggregates with hyaluronan, as determined by gel filtration chromatography, suggesting that the G1 domains had been rendered non-functional. Since previous studies have shown that disc aggrecan undergoes non-enzymatic glycation with age, the functional effect of such modification was investigated in vitro using bovine aggrecan isolated from young animals. Incubation of monomeric aggrecan with ribose to induce glycation rendered it unable to form complexes with hyaluronan stable to agarose gel electrophoresis or gel filtration chromatography. Similarly, extended treatment of intact proteoglycan aggregate with ribose resulted in destabilisation of the complex with separation of the aggrecan from the hyaluronan. Although it is clear that proteolysis occurs in the intervertebral disc and gives rise to some non-aggregating molecules, a different mechanism is required to explain the presence of many non-aggregating molecules bearing the G1 domain. The products of non-enzymatic glycation of the globular domains of aggrecan would account for this phenomenon and explain why some of the non-aggregating molecules are still large proteoglycans. While such molecules may be retained in the nucleus pulposus, they may be able to diffuse within it, reducing the ability of the tissue to resist compression under asymmetric loading such as bending and ultimately contributing to disc degeneration.

Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction.

The endothelial surface glycocalyx is a hydrated mesh in which proteoglycans are prominent. It is damaged in diseases associated with elevated levels of tumor necrosis factor α (TNF-α). We investigated the mechanism of TNF-α-induced disruption of the glomerular endothelial glycocalyx. We used conditionally immortalized human glomerular endothelial cells (GEnCs), quantitative PCR arrays, Western blotting, immunoprecipitation, immunofluorescence, and dot blots to examine the effects of TNF-α. TNF-α induced syndecan 4 (SDC4) mRNA up-regulation by 2.5-fold, whereas cell surface SDC4 and heparan sulfate (HS) were reduced by 36 and 30%, respectively, and SDC4 and sulfated glycosaminoglycan in the culture medium were increased by 52 and 65%, respectively, indicating TNF-α-induced shedding. Small interfering (siRNA) knockdown of SDC4 (by 52%) caused a corresponding loss of cell surface HS of similar magnitude (38%), and immunoprecipitation demonstrated that SDC4 and HS are shed as intact proteoglycan ectodomains. All of the effects of TNF-α on SDC4 and HS were abrogated by the metalloproteinase (MMP) inhibitor batimastat. Also abrogated was the associated 37% increase in albumin passage across GEnC monolayers. Specific MMP9 knockdown by siRNA similarly blocked TNF-α effects. SDC4 is the predominant HS proteoglycan in the GEnC glycocalyx. TNF-α-induced MMP9-mediated shedding of SDC4 is likely to contribute to the endothelial glycocalyx disruption observed in diabetes and inflammatory states.

Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury.

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2(Δ3/Δ3) (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type (P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB (P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

Mapping of matrix metalloproteinase cleavage sites on syndecan‐1 and syndecan‐4 ectodomains

Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine or autocrine effectors, or as competitive inhibitors of the intact proteoglycan. Tumour-associated MMPs are shown here to cleave the ectodomains of human syndecan-1 and syndecan-4. Two membrane proximal regions of both syndecan-1 and syndecan-4 are favoured MMP cleavage sites, six and 15 residues from the transmembrane domain. Other sites are 35-40 residues C-terminal from the heparan sulfate chain substitution sites in both syndecans. The MT1-MMP cleavage sites in syndecan-1 and syndecan-4 were confirmed by site-directed mutagenesis. These findings provide insights into the characteristics of syndecan shedding.

Preventing root caries development under oral biofilm challenge in an artificial mouth

Objectives: To study the preventive effects of chlorhexidine against root caries under oral biofilm in an artificial mouth. Study Design: Sixteen human tooth-root disks were inoculated with a salivary sample that was produced by mixing the unstimulated saliva of three adults who had no untreated caries. The disks were incubated in an artificial mouth fed with a 5% sucrose solution three times daily for one week. Eight disks received a twice daily rinse of 0.12% chlorhexidine (test group). The other eight disks were rinsed in distilled water (control). The biofilm was then studied with three techniques: colony forming unit (CFU) counting, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The changes in the chemical structure of the root surface were studied using Fourier transform infra-Red spectroscopy. Type-I collagen and proteoglycans on the root surface were quantified using immunocytochemical staining. Results: The log CFU for the test and control groups were 4.21 and 8.27, respectively (p<0.001). The CFU count of Streptococci and Lactobacilli were negligible. Both the SEM and the CLSM showed suppressed bacteria growth in the test group. The log [amide-I: HPO42-] of the test and control groups were 1.11 and 1.93, respectively (p=0.02). The mean counts of sound type-I collagen in the test and control groups were 16.8/?m2 and 13.0/?m2, respectively (p<0.001), whereas the mean counts of intact proteoglycans were 5.6/?m2 and 3.5/?m2, respectively (P<0.001). Conclusions: Chlorhexidine suppressed the growth of selected cariogenic bacteria in oral biofilm on the root surface and thus protected tooth-root from cariogenic challenge. Key words:Chlorhexidine, biofilm, caries risk, root, caries, artificial mouth, demineralization, streptococci, lactobacilli, proteoglycans, collagen I.

Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production

To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-d-xyloside (PNPX) to inhibit GAG incorporation into newly formed proteoglycans. As expected, GAG deposition in alginate beads decreased with increasing PNPX concentration. Next to GAGs, collagen deposition and cross-linking also decreased. In the presence of PNPX, GAGs and collagen were deposited further away from the chondrocyte than in the control and increased amounts were found in the culture medium. These changes resulted in decreased functional properties of the construct. We conclude that in our culture system, intact proteoglycans play a role in deposition of collagen and thus the formation of a functional matrix. The effect of less proteoglycans on the collagen network could explain why cartilage repair is ineffective in osteoarthritis and help us with development of new therapies.

Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.

Cartilage proteoglycans.

The structure of the protein core of the high molecular weight aggregating proteoglycan from pig laryngeal cartilage has been investigated. Mild trypsin digestion of proteoglycan aggregates released a large (Mr approximately equal to 150K) protein-rich fragment that contained the hyaluronate-binding region (Mr 66K). Rotary-shadowing electron microscopy of this preparation showed it to contain 'double globe' structures, similar to those seen with intact proteoglycans. Interaction studies and immunochemical evidence showed that one of the globular domains was the binding region. The second globular domain did not interact with hyaluronate or share any major antigenic determinants with the binding region and its function remains unknown. Further evidence from rotary shadowing also suggested that the protein core contained a third globular domain at the C-terminal end. The complete protein core sequence thus contains long folded globular protein regions, in addition to the extended regions bearing glycosaminoglycan chains. Studies of proteoglycan turnover in explants of pig articular cartilage showed that proteoglycan fragments were continuously released into the medium during culture. These included large non-aggregating proteoglycan fragments, free binding region and also link protein. Proteoglycans retained within the cartilage matrix remained intact and able to aggregate. Only in the presence of interleukin 1 was there evidence of more extensive proteolytic digestion. The results suggest normal turnover to be a conservative mechanism involving the selective cleavage of proteoglycan close to the hyaluronate-binding region. This releases the major glycosaminoglycan-bearing domain and enables it to diffuse out of the matrix. The site of the initial cleavage appears to be in the region of the N-terminal globular domains.

HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells

Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake-deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with alpha-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer.

Identification of a Drosophila Gene Encoding Xylosylprotein β4-Galactosyltransferase That Is Essential for the Synthesis of Glycosaminoglycans and for Morphogenesis

In mammals, the xylosylprotein beta4-galactosyltransferase termed beta4GalT7 (XgalT-1, EC ) participates in proteoglycan biosynthesis through the transfer of galactose to the xylose that initiates each glycosaminoglycan chain. A Drosophila cDNA homologous to mammalian beta4-galactosyltransferases was identified using a human beta4GalT7 cDNA as a probe in a BLAST analysis of expressed sequence tags. The Drosophila cDNA encodes a type II membrane protein with 322 amino acids and shows 49% identity to human beta4GalT7. Extracts from L cells transfected with the cDNA exhibited marked galactosyltransferase activity specific for a xylopyranoside acceptor. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis in beta4GalT7-deficient Chinese hamster ovary cells. In transfectant lysates the properties of Drosophila and human beta4GalT7 resembled each other, except that Drosophila beta4GalT7 showed a less restricted specificity and was active at a wider range of temperatures. Drosophila beta4GalT7 is expressed throughout development, with higher expression levels in adults. Reduction of Drosophila beta4GalT7 levels using expressed RNA interference (RNAi) in imaginal discs resulted in an abnormal wing and leg morphology similar to that of flies with defective Hedgehog and Decapentaplegic signaling, which are known to depend on intact proteoglycan biosynthesis. Immunohistochemical analysis of tissues confirmed that both heparan sulfate and chondroitin sulfate biosynthesis were impaired. Our results demonstrate that Drosophila beta4GalT7 has the in vitro and in vivo properties predicted for an ortholog of human beta4GalT7 and is essential for normal animal development through its role in proteoglycan biosynthesis.

Cleavage of the Xylosyl Serine Linkage between a Core Peptide and a Glycosaminoglycan Chain by Cellulases

We previously found that endo-beta-xylosidase from Patinopecten is an endo-type glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265, 854-860). Screening for endo-beta-xylosidase activity in several cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei, Trichoderma viride, and Irpex lacteus. The cellulase derived from A. niger was purified, and its molecular weight was determined to be 26,000 by SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain; 2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily hydrolyzes the linkage in glycosaminoglycan peptides, but intact proteoglycan is cleaved only slowly; and 4) the activity is unaffected by the glycosaminoglycan component (chondroitin sulfate, dermatan sulfate, and heparan sulfate). Judging from these enzymatic characteristics, this cellulase is different from the endo-beta-xylosidase of Patinopecten. We believe that this cellulase will become a useful tool in the further development of glycotechnology, because, like the endo-beta-xylosidase of Patinopecten, it enables the release of intact glycosaminoglycans from glycosaminoglycan peptides.

Identification of the metalloproteinase stromelysin in the physis

For long bone growth to occur, calcification of the matrix must commence in the lower hypertrophic zone of the growth plate. It is generally accepted that physeal proteoglycans help regulate mineralization, and that at least in vitro, intact proteoglycans can inhibit mineralization. Thus degradation of proteoglycan may be a necessary step prior to calcification. Previous work in our laboratory has demonstrated the presence of neutral metallo-proteases in the growth plate with highest levels in the hypertrophic zone, where calcification occurs. Stromelysin (MMP-3) is a connective tissue matrix-degrading enzyme. It was formerly known as proteoglycanase and is generally considered to be one of the major proteoglycan degrading enzymes in cartilage. Stromelysin is implicated in cartilage destruction in osteoarthritis and may also be involved in tissue remodeling in the physis. Our goal was to determine if the neutral protease previously reported by the authors in the physis was stromelysin. In this study we used Western blots and antibodies to stromelysin and to the stromelysin cleavage site in aggrecan, the most common form of proteoglycan, to demonstrate the presence of stromelysin in the bovine physis. When an antibody raised against the stromelysin cleavage site of aggrecan (FVDIPEN) was incubated with a Western blot, which had been run with aggrecan extracted from bovine physes, a positive reaction resulted. This suggests that there is stromelysin degradation in vivo in the physis. Two different polyclonal antibodies to stromelysin gave positive results on Western blots of purified media from growth plate cultures indicating that stromelysin is produced in vitro in culture. These antibodies also reacted with active stromelysin. The presence of stromelysin in the physis implicates it in physeal physiology. The concentration of its activity in the lower hypertrophic zone and zone of provisional calcification suggests that it may be particularly important in mineralization.

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.

Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1.

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K(d) values estimated as 1.95 x 10(-7) m and 5.3 x 10(-7) m, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of elastic fiber formation.

A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells.

We show here that the endothelial cell-line ECV 304 expresses the heparan sulfate proteoglycan glypican-1. The predominant cellular glycoform carries truncated side-chains and is accompanied by heparan sulfate oligosaccharides. Treatment with brefeldin A results in accumulation of a glypican proteoglycan with full-size side-chains while the oligosaccharides disappear. During chase the glypican proteoglycan is converted to partially degraded heparan sulfate chains and chain-truncated proteoglycan, both of which can be captured by treatment with suramin. The heparan sulfate chains in the intact proteoglycan can be depolymerized by nitrite-dependent cleavage at internally located N-unsubstituted glucosamine moieties. Inhibition of NO-synthase or nitrite-deprivation prevents regeneration of intact proteoglycan from truncated precursors as well as formation of oligosaccharides. In nitrite-deprived cells, formation of glypican proteoglycan is restored when NO-donor is supplied. We propose that, in recycling glypican-1, heparan sulfate chains are cleaved at or near glucosamines with unsubstituted amino groups. NO-derived nitrite is then required for the removal of short, nonreducing terminal saccharides containing these N-unsubstituted glucosamine residues from the core protein stubs, facilitating re-synthesis of heparan sulfate chains.

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.

Molecular Characterization of a Novel Basement Membrane-associated Proteoglycan, Leprecan

A monoclonal antibody was used in early studies to identify a novel chondroitin sulfate proteoglycan, secreted by L-2 cells, the core protein of which was approximately 100 kDa. To characterize this proteoglycan core protein at the molecular level, an L-2 cell cDNA library was probed by expression screening and solution hybridization. Northern blot analysis assigned transcript size to approximately 3.1 kilobases and, after contig assembly, the coding region of the mRNA corresponded to 2.18 kilobases. Immunoassays were performed to confirm the identity of this sequence, using a polyclonal antibody raised against an expressed fusion protein encoded by sequence representing the carboxyl half of the molecule. The antibody recognized the core protein in Western blots after prior digestion of the intact proteoglycan with chondroitinase ABC. Immunostaining tissue sections with the same antibody localized the proteoglycan to basement membranes, and expression of the entire sequence in Chinese hamster ovary K-1 cells showed that the protein encoded by the sequence secreted as a chondroitin sulfate proteoglycan. The core protein not only has motifs permitting glycosylation as a proteoglycan, but also possesses the endoplasmic reticulum retrieval signal, KDEL, which suggests that, in addition to its role as a basement membrane component, it may also participate in the secretory pathway of cells.