scholarly journals Identification of the core proteins in proteoglycans synthesized by vascular endothelial cells

1989 ◽  
Vol 261 (1) ◽  
pp. 145-153 ◽  
Author(s):  
A Lindblom ◽  
I Carlstedt ◽  
L Å Fransson
Keyword(s):  
Endothelial Cells ◽  
Molecular Mass ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Buoyant Density ◽  
Cell Extract ◽  
Apparent Molecular Mass ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Proteoglycans, metabolically labelled with [3H]leucine and 35SO4(2-), were isolated from the spent media and from guanidinium chloride extracts of cultured human umbilical-vein endothelial cells by using isopycnic density-gradient centrifugation, gel filtration and ion-exchange h.p.l.c. The major proteoglycan species were subjected to SDS/polyacrylamide-gel electrophoresis before and after enzymic degradation of the polysaccharide chains. The cell extract contained mainly a heparan sulphate proteoglycan that has a buoyant density of 1.31 g/ml and a protein core with apparent molecular mass 300 kDa. The latter was heterogeneous and migrated as one major and one minor band. After reduction, the apparent molecular mass of the major band increased to approx. 350 kDa, indicating the presence of intrachain disulphide bonds. The proteoglycan binds to octyl-Sepharose and its polysaccharide chains are extensively degraded by heparan sulphate lyase. The proteoglycans of the medium contained 90% of all the incorporated 35SO4(2-). Here the predominant heparan sulphate proteoglycan was similar to that of the cell extract, but was more heterogeneous and contained an additional core protein with apparent molecular mass 210 kDa. Furthermore, two different chondroitin sulphate proteoglycans were found: one 200 kDa species with a high buoyant density (approx. 1.45 g/ml) and one 100 kDa species with low buoyant density (approx. 1.3 g/ml). Both these proteoglycans have a core protein of molecular mass approx. 47 kDa.

scholarly journals Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane

1989 ◽  
Vol 264 (2) ◽  
pp. 457-465 ◽  
Author(s):  
L P W J van den Heuvel ◽  
J van den Born ◽  
T J A M van de Velden ◽  
J H Veerkamp ◽  
L A H Monnens ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Molecular Mass ◽  
Core Protein ◽  
Mammalian Species ◽  
Heparan Sulphate ◽  
Basement Membranes ◽  
Heparan Sulphate Proteoglycan ◽  
Average Molecular Mass ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.

scholarly journals Purification and characterization of heparan sulphate proteoglycan from bovine brain

1999 ◽  
Vol 344 (3) ◽  
pp. 723-730 ◽  
Author(s):  
Youmie PARK ◽  
Guyong YU ◽  
Nur Sibel GUNAY ◽  
Robert J. LINHARDT
Keyword(s):  
Core Protein ◽  
Sequence Similarity ◽  
Heparan Sulphate ◽  
Compositional Analysis ◽  
Bovine Brain ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Protein Size ◽  
Core Proteins ◽  
Internal Sequence

A heparan sulphate proteoglycan was purified from adult bovine brain tissues and its structure was characterized. The major heparan sulphate proteoglycan from whole bovine brain had a molecular mass of > 200 kDa on denaturing SDS/PAGE and a core protein size of 66 kDa following the removal of glycosaminoglycan chains. Fractionation on DEAE-Sephacel showed that this proteoglycan consisted of three major forms having high, intermediate and low overall charge. All core proteins were identical in size and reacted with heparan sulphate proteoglycan-stub antibody and an antibody made to a synthetic peptide based on rat glypican. The three forms of proteoglycans had identical peptide maps and their amino acid compositional analysis did not match any of the known glypicans. The internal sequence of a major peptide showed only 37.5% sequence similarity with human glypican 5. The glycosaminoglycan chain sizes of the three forms of this proteoglycan, determined after β-elimination by PAGE, were identical. The disaccharide compositional analysis on the heparan sulphate chains from the three forms of the proteoglycan, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that they differed primarily by degree of sulphation. The most highly sulphated proteoglycan isolated had a disaccharide composition similar to heparan sulphate glycosaminoglycans found in brain tissue. Based on their sensitivity to low pH nitrous acid treatment, the N-sulphate groups in these proteoglycans were found to be primarily in the smaller glycosaminoglycan chains. The heparan sulphate proteoglycans were also heavily glycosylated with O-linked glycans and no glycosylphosphatidylinositol anchor could be detected.

scholarly journals Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody

1987 ◽  
Vol 247 (3) ◽  
pp. 765-771 ◽  
Author(s):  
H de Boeck ◽  
V Lories ◽  
G David ◽  
J J Cassiman ◽  
H van den Berghe
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Human Lung ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Mass ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts ◽  
The Core

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).

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 Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture

1991 ◽  
Vol 277 (1) ◽  
pp. 81-88 ◽  
Author(s):  
G J Thomas ◽  
R M Mason ◽  
M Davies
Keyword(s):  
Culture Medium ◽  
Mesangial Cells ◽  
Cell Layer ◽  
Heparan Sulphate ◽  
Glomerular Mesangial Cells ◽  
Heparan Sulphate Proteoglycan ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Human Adult ◽  
Core Proteins

1. The newly synthesized proteoglycans from human adult glomerular mesangial cells labelled in vitro for 24 h with [35S]sulphate have been characterized using biochemical and immunological techniques. 2. The following proteoglycans were identified (% of total synthesized). (i) A large chondroitin sulphate proteoglycan, CSPG-I, Mr approximately 1 x 10(6) (10.6%). This proteoglycan consisted of a protein core of Mr approximately 4 x 10(5) and glycosaminoglycan chains of Mr 2.5 x 10(4), and was present in both the cell layer and the culture medium. (ii) A major small dermatan sulphate proteoglycan, DSPG-I, Mr 3.5 x 10(5) (46%), which was mainly located in the culture medium. (iii) A second minor small dermatan sulphate, DSPG-II, Mr approximately 2 x 10(5) (9.8%). This molecule was exclusively located in the culture medium. (iv) A large heparan sulphate proteoglycan, HSPG-I, Mr 8 x 10(5) (3.3%). (v) A second large heparan sulphate proteoglycan HSPG-II, Mr approximately 6 x 10(5) (23%). HSPG-I and HSPG-II were extracted from both the culture medium and the cell layer. 3. Western blot analysis of the core proteins released by chondroitin ABC lyase treatment of DSPG-I and DSPG-II identified these dermatan sulphate proteoglycans as biglycan and decorin respectively. Both DSPG-I and DSPG-II had core proteins of Mr 45,000. 4. The cell-layer-associated forms of CSPG-I, HSPG-I and HSPG-II were accessible to limited trypsin treatment, bound to octyl-Sepharose and could be inserted into liposomes, indicating a possible cell membrane location. 5. Pulse-chase experiments indicated that the cell-layer-associated [35S]proteoglycans undergo limited metabolism to inorganic [35S]sulphate, the majority of which is accounted for by the degradation of HSPG-II and to a lesser extent DSPG-I.

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 Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans

1990 ◽  
Vol 265 (1) ◽  
pp. 289-300 ◽  
Author(s):  
A Schmidtchen ◽  
I Carlstedt ◽  
A Malmström ◽  
L Å Fransson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Heparan Sulphate ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblast ◽  
Heparan Sulphate Proteoglycan ◽  
Dermatan Sulphate ◽  
The Core ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ‘matrix’, and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).

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