Class Of Proteoglycans Research Articles

Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain.

Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro cell proliferation and proteoglycan synthesis of rabbit meniscal fibrochondrocytes as a function of age and sex.

Meniscal fibrochondrocytes from male and female New Zealand white rabbits, ages 6, 12, and 24 months, were grown in primary and secondary monolayer cell culture. Neither age nor sex affected the majority of their cell culture characteristics. Cells from young males (6 months old) synthesized greater amounts of sulfated proteoglycans than did those from young females, but by 2 years of age, this result was reversed. All age groups synthesized 2 classes of proteoglycans, based on hydrodynamic size, but the ratio of the 2 classes changed as a function of age. Overall, the meniscal fibrochondrocytes from both skeletally immature and skeletally mature rabbits of both sexes were capable of proliferation and matrix synthesis in vitro.

Arabinogalactan-proteins are localized extracellularly in the transmitting tissue of Nicotiana alata link and otto, an ornamental tobacco

The localization of a class of proteoglycans, the arabinogalactan-proteins (AGPs), within mature styles of N. alata has been examined using three probes: (1) β-glycosyl Yariv reagent, a red dye which is used as a cytochemical stain for light microscopy, (2) monoclonal antibodies to terminal α- L-arabinofuranosyl residues which bind to the terminal α- L-arabinofuranosyl residues of AGPs, (3) monoclonal antibodies to terminal β- D- galactopyranosyl residues which bind to style glycoconjugates including the AGPs. The monoclonal antibodies were used in both immunofluorescence and gold immunocytochemical studies. Staining with all probes indicates that the AGPs are present mainly in the extracellular matrix of the transmitting tissue.

Identification of a sulfate-bearing molecule associated with HLA class II antigens.

The human Ia antigens (DR, DS, and SB), determined by genes contained within the HLA complex on chromosome 6, are glycoprotein heterodimers consisting of a Mr approximately equal to 34,000 alpha chain and a Mr approximately equal to 28,000 beta chain. As a result of studies exploring the possibility that alpha or beta (or both) might be sulfated, a unique component of the oligomeric Ia antigen complex was discovered. When anti-Ia immunoprecipitates from Nonidet P-40 lysates of [35S]sulfate-labeled lymphoid cells were analyzed by NaDodSO4/PAGE, a molecule of considerable size heterogeneity (Mr 40,000-70,000) was observed. This component was present in both anti-DR and anti-DS immunoprecipitates prepared from both human tonsil cells and lymphoblastoid B-cell lines but was not observed in control precipitates or in association with immunoglobulin or class I HLA molecules. Preliminary biochemical studies indicate that this Mr 40,000-70,000 molecule is polyanionic, disperse in molecular weight, and sensitive to protease digestion. The sulfate-bearing moiety of this component was resistant to Pronase but sensitive to chondroitinase ABC, indicating that this molecule belongs to the chondroitin sulfate class of proteoglycans.

Effect of dissociative extraction with 1.5 M calcium chloride on proteoglycans in rat-incisor predentine visualized with cuprolinic blue

Treatment of rat-incisor predentine for 48 h in a 1.5 M CaCl 2 solution halved the number and size of the proteoglycan precipitates visualized using a critical MgCl 2 concentration and cuprolinic blue staining. About 75 per cent of the aggregable proteoglycans were extracted; 25 per cent resisted extraction. Treatment with CaCl 2 also completely abolished the electron density of the amorphous ground substance, an observation consistent with the possible existence of two classes of proteoglycans in predentine.

Analysis of perinotochordal materials. 2. Studies on the influence of proteoglycans in somite chondrogenesis

Notochordal proteoglycans influence somite chondrogenesis. In earlier work we learned that notochord cultured under improved conditions for 8 days contains three different classes of perinotochordal proteoglycan molecules, are necessary for inducing somite chondrogenesis. When the notochords were exposed to substances causing metabolic alterations (BUdR, excess vitamin A, and hyaluronic acid), only small proteoglycans accumulated in the perinotochordal sheath. Such treated notochords or the proteoglycans extracted from them failed to support somite chondrogenesis, suggesting that the large aggregate in the perinotochordal matrix may be necessary for somite induction. Adding a crude proteoglycan prepared from embryonic sternal cartilage increased the induction of chondrogenesis in somites, but papain treatment of the proteoglycan diminished its inductive capacity. This result further suggests that large aggregate proteoglycan may be necessary for the tissue interactions that promote chondrogenesis.

Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture.

Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[35S]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCl (dissociative solvent) or 0.5 M guanidine HCl (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population (Kav = 0.31) contained predominantly chondroitin sulfate chains with Mr = approximately 40,000. The smaller population (Kav = 0.61) contained dermatan sulfate chains of similar Mr (approximately 40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kav = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans.

Isolation and characterization of proteoglycans synthesized by human colon and colon carcinoma.

The biosynthesis of proteoglycans in short term organ culture of human colon and colon carcinoma was studied. Proteoglycans, labeled with [35S]sulfate and [3H]serine, were extracted with either 4 M or 0.5 M guanidine HCl in the presence of protease inhibitors and sequentially purified by associative and dissociative CsCl density gradient ultracentrifugation. Normal colon synthesized two polydisperse classes of proteoglycans: a large heparan sulfate-containing monomer, with a Kav of 0.48 on Sepharose CL-2B and a small dermatan sulfate-containing monomer with a Kav of 0.65. A portion (25%) of the proteoglycans was found as aggregate when chromatographed under associative conditions, and the larger monomers interacted with hyaluronic acid to an extent greater than the smaller proteoglycans. Following papain or alkali treatment, the free glycosaminoglycan side chains of both monomers eluted as a single broad peak (Kav = 0.5) from Sepharose CL-6B, with an estimated Mr of 20 X 10(3). In contrast, colon carcinoma synthesized only one proteoglycan monomer, which aggregated to a limited extent (12%). This proteoglycan population, with a Kav of 0.7 on CL-2B, contained chondroitin sulfate as the major glycosaminoglycan (greater than 81%), with small amounts of dermatan sulfate. The glycosaminoglycans had an estimated Mr of 9 X 10(3), and the disaccharides released by chondroitinase ABC consisted of 32% 4-sulfate and 68% 6-sulfate. Electron microscopy of mixed proteoglycancytochrome c monolayers from the associative fractions of normal and neoplastic colon revealed aggregated complexes which were similar in over structure, although smaller than the proteoglycan aggregates from cartilage.

45] Proteoglycans: Isolation and Characterization

This chapter presents procedure for extraction and characterization of proteoglycans. In general, tissue or cultures should be processed for extraction quickly and using procedures that minimize endogenous degradation. Fresh tissue should be chilled on ice, cleaned, and minced quickly. Extraction efficiency is often improved when finer tissue pieces are used to expose more surface area to the solvent. Once the proteoglycans are in solution, different fractionation procedures (ion exchange in 7 M urea, rate zonal-velocity, centrifugation molecular-sieve chromatography, electrophoresis in composite polyacrylamide-agarose gels), which take advantage of their macromolecular properties, are used to purify them from other macromolecules in the extract and to begin to separate them from each other. Application of the various techniques described above has identified the different classes of proteoglycans in a starting extract based upon differences in charge density, hydrodynamic size, and buoyant density. Such analyses not only provide information about the physical properties of the proteoglycans but help design effective strategies for purifying at least some of the subpopulations of proteoglycans identified.

The acidic macromolecules in rabbit cortical bone tissue

Rabbit cortical bone powder was extracted with EDTA solutions at neutral pH and the soluble constituents fractionated by DEAE-cellulose chromatography. The acidic fractions obtained were further investigated by gel chromatography and chemical analysis. Two classes of proteoglycans were present; one containing chondroitin sulphate and the other containing material resembling keratan sulphate mixed with a smaller amount of chondroitin sulphate. Sialoglycoproteins were detected by specific chemical introduction of tritium label into the sialic acid residues of the acidic glycoprotein fraction. Following sodium dodecyl sulphatepolyacrylamide gel electrophoresis and fluorography, the presence of numerous distinct sialoglycoprotein components was demonstrated.

Differences in the sulfated macromolecules synthesized by normal and transformed hamster fibroblasts

Analysis of the 35SO 4-labelled macromolecules synthesized by cultures of normal (NIL8) and transformed (NIL8-HSV) hamster fibroblasts has revealed the following differences between the two cell lines: 1. (1) The proportion of sulfate incorporated into cell-associated macromolecules is three times higher in normal than in transformed cells. In addition, normal fibroblasts incorporate more sulfate into extracellular, middle and low molecular weight species than do transformed cells. Transformed cells, however, incorporate more sulfate into extracellular, very high molecular weight species than do normal cells. 2. (2) Normal fibroblasts, which synthesize much more extracellular dermatan sulfate than do transformed cells, produce a class of extracellular heterogeneous sulfate proteoglycans absent from transformed cultures. This macromolecular species consists largely of dermatan sulfate. The transformed cells instead release a lower molecular weight class of proteoglycans which consist of chondroitin sulfates A and C. 3. (3) The large, external, transformation-sensitive glycoprotein is sulfated in NIL8 cultures. This macromolecular species is present on the surface membrane of normal cells, but absent from transformed cells. Sulfated large, external transformation-sensitive protein is also present in the conditioned medium from normal cultures. A similar species is present in the conditioned medium from transformed cultures, but has a slightly higher apparent molecular weight and differs in other properties from the large, external, transformation-sensitive protein of normal cells.

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells

Balb/c 3T3 cells synthesize 5--10 times more 35SO2/4- -labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular 35SO2/4- -labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70--80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65-75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular 35SO2/4- -labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 35 SO2/4- -labeled proteoglycans and contains chondroitin sulfate extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5-10-fold decreased synthesis of 35SO2/4- -labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.