scholarly journals Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns

1986 ◽  
Vol 235 (2) ◽  
pp. 469-479 ◽  
Author(s):  
B C Wightman ◽  
E A Weltman ◽  
L A Culp
Keyword(s):  
Extracellular Matrix ◽  
Affinity Chromatography ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Heparan Sulphate Proteoglycan ◽  
3T3 Cells ◽  
Platelet Factor ◽  
Sulphate Proteoglycan ◽  
Adhesion Sites ◽  
Chondroitin Sulphate Proteoglycan

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.

scholarly journals Proteoglycans synthesized by an osteoblast-like cell line (UMR 106-01)

1991 ◽  
Vol 277 (1) ◽  
pp. 199-206 ◽  
Author(s):  
D J McQuillan ◽  
D M Findlay ◽  
A M Hocking ◽  
M Yanagishita ◽  
R J Midura ◽  
...  
Keyword(s):  
Cell Line ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Cell Types ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Umr 106

The proteoglycans synthesized by an osteoblast-like cell line of rat origin (UMR 106-01) were defined after biosynthetic labelling with [35S]sulphate and [3H]glucosamine. Newly synthesized labelled proteoglycans were characterized by differential enzymic digestion in combination with analytical gel filtration and SDS/PAGE. UMR 106-01 cells were found to synthesize three major species of proteoglycan: a large chondroitin sulphate proteoglycan of Mr approximately 1 x 10(6), with a core protein of Mr approximately 350,000-400,000; a small chondroitin sulphate-containing species of Mr approximately 120,000 with a core protein of Mr 43,000; and a heparan sulphate proteoglycan of Mr approximately 150,000, with a core protein of Mr approximately 80,000. Over 70% of the newly synthesized intact proteoglycan species are associated with the cell layer of near-confluent cells; however, accessibility to trypsin digestion suggests an extracellular location. Chemical characteristics of the proteoglycans and preliminary mRNA hybridization indicate that the small chondroitin sulphate proteoglycan is probably PG II (decorin). The large chondroitin sulphate proteoglycan is most likely related to a hyaluronate-aggregating species from fibroblasts (versican), and the heparan sulphate proteoglycan bears striking similarities to cell-membrane-intercalated species described for a number of cell types.

scholarly journals Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts

1985 ◽  
Vol 232 (1) ◽  
pp. 161-168 ◽  
Author(s):  
S Johansson ◽  
K Hedman ◽  
L Kjellén ◽  
J Christner ◽  
A Vaheri ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Human Articular Cartilage ◽  
Sulphate Proteoglycan ◽  
Fibroblast Cultures ◽  
Core Proteins ◽  
The Matrix

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called ‘link proteins’ were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.

scholarly journals 3D distribution of perlecan within intervertebral disc chondrons suggests novel regulatory roles for this multifunctional modular heparan sulphate proteoglycan

2021 ◽  
Vol 41 ◽  
pp. 73-89
Author(s):  
AJ Hayes ◽  
◽  
J Melrose
Keyword(s):  
Extracellular Matrix ◽  
Intervertebral Disc ◽  
Cell Nucleus ◽  
Close Association ◽  
Heparan Sulphate ◽  
Regulatory System ◽  
Distribution Patterns ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
And Function

Perlecan is a modular, multifunctional heparan sulphate-proteoglycan (HS-PG) that is present in the pericellular and wider extracellular matrix of connective tissues. In the present study, confocal microscopy was used to study perlecan distribution within intervertebral disc chondrons. Perlecan immunolabel was demonstrated intracellularly and in close association with the cell nucleus within chondrons of both the annulus fibrosus (AF) and nucleus pulposus (NP). This observation is consistent with earlier studies that have localised HS-PGs with nuclear cytoskeletal components. Nuclear HS-PGs have been proposed to transport fibroblast growth factor (FGF)-1, FGF-2 and FGFR-1 into the cell nucleus, influencing cell proliferation and the cell-cycle. Perlecan has well-known interactive properties with FGF family members in the pericellular and extracellular matrix. Perinuclear perlecan may also participate in translocation events with FGFs. The glycosaminoglycan side chains of HS-PGs can modulate chromatin structure by regulating the access of transcription factors to DNA. These mechanisms are consistent with the distribution patterns identified here and previously reported for other HS-PGs, introducing a potentially-novel arena for perlecan in gene regulation. Whilst much is known of the structure and function of perlecan in the pericellular and extracellular matrix, very little is known of any intracellular forms of perlecan. The perlecan labelling patterns described here suggest the possibility of involvement of this HS-PG in an intracrine regulatory system. Future studies should further explore this possibility and the potential for this HS-PG as a novel therapeutic target.

Influence of testosterone on chondroitin sulphate proteoglycan in the rat prostate

1996 ◽  
Vol 74 (5) ◽  
pp. 645-651 ◽  
Author(s):  
Doris E. Terry ◽  
Albert F. Clark
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Testosterone Propionate ◽  
Chondroitin Sulphate ◽  
Fluorescein Isothiocyanate ◽  
Ventral Lobe ◽  
Adult Rat ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Rat Prostate

There are recognized interactions between prostatic stromal and epithelial cells. These interactions may be influenced by the composition of the extracellular matrix, which is composed of proteins such as collagen, laminin, fibronectin, and proteoglycans (PGs) such as chondroitin sulphate proteoglycan (CSPG). In our continuing studies on prostate biology, we examined the three lobes of the normal adult rat prostate, i.e., ventral, dorsal, and lateral, for CSPG by indirect immunofluorescence, using an immunospecific monoclonal antibody (CS-56) for the chondroitin sulphate (CS) moiety of the PG. Staining of the prostate sections with the CS-56 antibody followed by labelling with IgG fluorescein isothiocyanate conjugate indicated strong fluorescent signals associated with the ventral lobe basement membrane. The signal was stronger and more continuous in the distal acini than in the proximal acini. The staining of the dorsal and lateral lobes was less intense than that of the ventral lobe. Following castration of the rats, the basement membrane staining became discontinuous. Androgen replacement by administration of testosterone propionate (TP) reversed the effects of castration. Quantification of the total CS content showed decreases of about 60% in the ventral and lateral lobes after castration. TP administration for 14 days increased the total CS content several fold above the values for castrated rats in all the lobes. The results demonstrated that CS content was significantly higher for TP-treated animals, suggesting mat the expression of prostate CSPG is regulated by androgens. This approach should be useful in the study of the extracellular matrix in prostate biology.Key words: androgen, basement membrane, extracellular matrix, glycosaminoglycan, prostate.

scholarly journals Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate

1990 ◽  
Vol 265 (1) ◽  
pp. 61-68 ◽  
Author(s):  
H Morita ◽  
T Takeuchi ◽  
S Suzuki ◽  
K Maeda ◽  
K Yamada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans ◽  
Cell Layers ◽  
Aortic Endothelial

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.

Hepatic heparan sulphate proteoglycan and the recycling of transferrin

1992 ◽  
Vol 70 (7) ◽  
pp. 535-538 ◽  
Author(s):  
Wei-Li Hu ◽  
Erwin Regoeczi
Keyword(s):  
Affinity Chromatography ◽  
Iron Metabolism ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Hepatic Iron ◽  
Transferrin Receptors ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Bona Fide ◽  
Rat Livers

Heparan sulphate proteoglycan, labelled with [35S]sulphate, was prepared from rat livers for studies of its interaction with purified rat transferrin. Affinity chromatography of the preparation on columns of immobilized differic transferrin and apotransferrin showed that the proteoglycan possessed affinity for both types of matrices at pH 7.3 and that this affinity significantly increased at pH 5.6. The glycosaminoglycan chains liberated from the proteoglycan by heparan sulphate lyase also bound to apotransferrin, albeit less strongly, whereas the deglycosylated core protein exhibited virtually no interaction with this matrix. In the presence of the proteoglycan at pH 5.6, the release of iron from the N-lobe of transferrin was accelerated. These observations suggest that heparan sulphate proteoglycan from the liver can mimick some of the known functions of bona fide transferrin receptors and, hence, interaction with the proteoglycan may provide an alternative nondegradative pathway for transferrin through hepatic cells.Key words: heparan sulphate proteoglycan, hepatic iron metabolism, receptor-mediated endocytosis, transferrin.

scholarly journals An IKLLI-containing peptide derived from the laminin α1 chain mediating heparin-binding, cell adhesion, neurite outgrowth and proliferation, represents a binding site for integrin α3β1 and heparan sulphate proteoglycan

1999 ◽  
Vol 340 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Ken-ichiro TASHIRO ◽  
Akira MONJI ◽  
Ichiro YOSHIDA ◽  
Yoshihito HAYASHI ◽  
Kazunori MATSUDA ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Neurite Outgrowth ◽  
Pc12 Cell ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Integrin Α3β1

We synthesized and characterized several peptides containing the IKLLI sequence in the α1 chain of laminin-1. The IKLLI-containing peptides, such as LA4 (CSRNLSEIKLLISRARK), LA5 (EIKLLIS) and LA5L (SEIKLLIS), were found to mediate heparin binding and cell adhesion, while also promoting neurite outgrowth in PC12 cells. Furthermore, peptides LA4 and LA5 also mediated proliferation. However, a scrambled peptide, LA5S (ILEKSLI), did not show any of these activities. Anti-LA4 antibodies inhibited laminin- and LA5-mediated cell adhesion and neurite outgrowth, and anti-(integrin α3) and anti-(integrin β1) antibodies inhibited LA5-mediated cell adhesion and neurite outgrowth. Heparin and heparan sulphate inhibited LA5-mediated heparin binding and PC12 cell adhesion in a dose- dependent manner. The IC50 for inhibition of heparin binding and cell adhesion was observed with 9 μM and 8 μM heparin/heparan sulphate respectively. Furthermore, heparan sulphate proteoglycan also inhibited LA5-mediated PC12 cell adhesion with an IC50 of 100 μg/ml. However, chondroitin sulphate (dermatan sulphate) did not inhibit cell adhesion. These data suggest that an IKLLI-containing peptide derived from the laminin α1 chain may be an active site of laminin and that its cell adhesion may thus interact with both integrin α3β1 and cell- surface heparan sulphate proteoglycan.

Glucocorticoid ameliorates altered gene expression of extracellular matrix components in kidneys of New Zealand Black/White F1 mice

1992 ◽  
Vol 83 (6) ◽  
pp. 701-709 ◽  
Author(s):  
Tsukasa Nakamura ◽  
Isao Ebihara ◽  
Yasuhiko Tomino ◽  
Hikaru Koide
Keyword(s):  
Extracellular Matrix ◽  
New Zealand ◽  
Messenger Rna ◽  
Renal Cortex ◽  
Heparan Sulphate ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Extracellular Matrix Components ◽  
Methylprednisolone Treatment ◽  
Collagen Chain

1. We examined the effects of methylprednisolone on the levels of messenger RNA encoding for extracellular matrix components, including α1(IV) collagen chain, laminin B1 and B2 chains, heparan sulphate proteoglycan and α1(I) and α1(III) collagen chains, and on the accumulation of these proteins in the renal cortex of New Zealand Black/White F1 mice. 2. At the onset of nephritis, at about 5 months of age, New Zealand Black/White F1 mice were divided in two groups that received either methylprednisolone or saline injections for 5 months. 3. The development of histological lesions and the glomerular deposition of IgG, IgM and C3 were suppressed by methylprednisolone treatment from 5 to 10 months of age. 4. The distribution and intensity of type IV collagen, laminin, heparan sulphate proteoglycan, type I collagen and type III collagen in renal cortex were decreased by the administration of methylprednisolone. 5. Levels of messenger RNA encoding for α1(IV) collagen chain, laminin B1 and B2 chains, heparan sulphate proteoglycan and α1(I) and α1(III) collagen chains in the renal cortex of New Zealand Black/White F1 mice were significantly ameliorated at 8–10 months of age by methylprednisolone administration. 6. These results indicate that methylprednisolone treatment can serve as an effective therapeutic approach to abnormal extracellular matrix regulation in murine lupus nephritis.

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