scholarly journals Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix

1989 ◽  
Vol 260 (2) ◽  
pp. 413-419 ◽  
Author(s):  
R Kokenyesi ◽  
J F Woessner
Keyword(s):  
Amino Acid ◽  
Core Protein ◽  
Consensus Sequence ◽  
Gel Filtration ◽  
Attachment Site ◽  
Sedimentation Equilibrium ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Core Proteins ◽  
Cscl Density Gradient

A small dermatan sulphate proteoglycan (DSPG) was extracted from rat cervices and was purified by using DEAE-Sephacel ion-exchange chromatography, gel filtration on Sepharose CL-2B and CsCl-density-gradient centrifugation. Sedimentation-equilibrium centrifugation gave a weight-average Mr of 95,000. Amino acid analysis showed a high content of aspartic acid, glutamic acid, glycine and leucine. The glycosaminoglycan chains had Mr 50,000 as determined by gel filtration. Chondroitin AC lyase and chondroitin ABC lyase digestions of these chains showed that they were composed of 75% dermatan sulphate and 25% chondroitin sulphate. Chondroitin ABC lyase digestion produced a core protein of Mr 45,000. The core protein, after treatment with HF, had Mr 37,000. Amino acid sequences of the N-terminus and a CNBr-cleavage peptide showed similarity to the sequences of core proteins of small proteoglycans of bovine and human origin, but the N-terminal glycosaminoglycan-attachment site (Ser-Gly-Ile-Ile) differed from the consensus sequence (Ser-Gly-Xaa-Gly) [Bourdon, Krusius, Campbell, Schwartz & Ruoslahti (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3194-3198]. A polyclonal antibody against the rat cervical DSPG reacted with small proteoglycans from cervices of human, mouse, dog, cow and sheep. DSPG is the major proteoglycan species present in the cervix. The amount of DSPG per cervix increases 4-fold during pregnancy, then falls precipitously within 1 day post partum. A role in cervical dilatation is postulated for this proteoglycan.

scholarly journals Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

1989 ◽  
Vol 262 (3) ◽  
pp. 823-827 ◽  
Author(s):  
P J Roughley ◽  
R J White
Keyword(s):  
Articular Cartilage ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Articular Cartilage ◽  
Amino Acid Residues ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.

scholarly journals Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins

1989 ◽  
Vol 263 (1) ◽  
pp. 137-142 ◽  
Author(s):  
H Hausser ◽  
W Hoppe ◽  
U Rauch ◽  
H Kresse
Keyword(s):  
Binding Proteins ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Scatchard Analysis ◽  
Dermatan Sulphate ◽  
Intact Cells ◽  
Sulphate Proteoglycan ◽  
Human Osteosarcoma Cells ◽  
Antibody Complex ◽  
Triton X

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.

scholarly journals Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA

1996 ◽  
Vol 318 (3) ◽  
pp. 909-914 ◽  
Author(s):  
Kazuhiro KURITA ◽  
Tamayuki SHINOMURA ◽  
Minoru UJITA ◽  
Masahiro ZAKO ◽  
Daihei KIDA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Epiphyseal Cartilage ◽  
Newborn Mouse ◽  
Dermatan Sulphate ◽  
Cdna Fragment ◽  
Sulphate Proteoglycan ◽  
C Terminus ◽  
Degenerate Oligonucleotide

PG-Lb is a chondroitin/dermatan sulphate proteoglycan first isolated from chick embryo limb cartilage. It had been assumed that osteoglycin represents its mammalian homologue. However, partial amino acid sequences of a novel proteoglycan from bovine epiphyseal cartilage showed high identity with those of chick PG-Lb (P. Neame, L. Rosenberg and M. Höök, personal communication). Reverse transcriptase PCR using degenerate oligonucleotide primers gave a cDNA fragment that might correspond to mouse PG-Lb. We isolated a clone from a cDNA library of newborn mouse epiphyseal cartilage using the cDNA fragment as a probe. The cloned cDNA was 1430 bp long and contained a 966 bp open reading frame which encoded the core protein consisting of 322 amino acid residues. The deduced amino acid sequence showed a high overall identity with chick PG-Lb (about 62%, reaching about 80% over the carboxyl two-thirds). In addition, the amino acid sequence contained a signal peptide, six cysteine residues at the invariant relative position to chick PG-Lb, six leucine-rich repeats at the carboxyl two-thirds, three possible glycosaminoglycan-attachment sites (two sites at the N-terminal side and one site at the C-terminus) and two possible Asn-glycosylation sites near the C-terminus. Northern-blot analysis demonstrated the specific expression of a 1.5 kb message in cartilage and testis. These structural features and the characteristic expression suggest that the cloned molecule is mouse PG-Lb.

scholarly journals Expression of a Xenopus counterpart of mammalian syndecan 2 during embryogenesis

1995 ◽  
Vol 309 (1) ◽  
pp. 69-76 ◽  
Author(s):  
N D Rosenblum ◽  
B B Botelho ◽  
M Bernfield
Keyword(s):  
Amino Acid ◽  
Northern Blot Analysis ◽  
Transmembrane Domain ◽  
Heparan Sulphate ◽  
Attachment Site ◽  
Heparan Sulphate Proteoglycan ◽  
Cell Stage ◽  
Reading Frame ◽  
Sulphate Proteoglycan ◽  
Polyclonal Antisera

We have identified a Xenopus cDNA, XS-2, by screening a Xenopus embryonic stage-22-24 cDNA library with a DNA probe encoding the transmembrane and cytoplasmic domains of mouse syndecan 1. The 1.4 kb cDNA consists of an open reading frame of 642 nucleotides encoding a protein of 191 amino acids. The predicted protein of 20869 Da contains a 25-amino acid putative transmembrane domain and a 32-amino acid putative cytoplasmic domain, both of which are highly similar to the corresponding regions of rat syndecan 2 (92% identity) and to a lesser degree those of rat syndecans 1, 3 and 4 (62, 64 and 78% respectively). The putative N-terminal ectodomain contains a possible attachment site for heparan sulphate, identical with the comparable glycosaminoglycan-attachment sequence of rat syndecan 2. Polyclonal antisera raised against recombinant ectodomain of XS-2, expressed as a fusion protein, recognized a heparan sulphate proteoglycan in XTC cell-culture medium. This proteoglycan bound to DEAE-Sephacel and was eluted with 1 M NaCl; digestion with heparitinase but not chondroitinase ABC resulted in the identification of a 46 kDa protein by these antisera. Northern-blot analysis indicated that XS-2 identifies two Xenopus mRNA species approx. 4 and 2 kb in size in embryos ranging in maturation from the 64-cell stage to stage 54. These results demonstrate that a heparan sulphate proteoglycan, similar to syndecan 2, is expressed during Xenopus embryogenesis.

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 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 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 The potential dolichol recognition sequence of β-1,4-mannosyltransferase is not required for enzymic activity using phytanyl-pyrophosphoryl-α-N,N'- diacetylchitobioside as acceptor

1994 ◽  
Vol 299 (1) ◽  
pp. 23-27 ◽  
Author(s):  
L Revers ◽  
I B H Wilson ◽  
M C Webberley ◽  
S L Flitsch
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Consensus Sequence ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Recognition Sequence ◽  
Mannose Residue ◽  
Before And After ◽  
Km Value ◽  
Good Agreement

The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation [Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition. Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain. This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992) J. Chem. Soc., Perkin Trans. 1, 2087-2093]. The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM. These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate. Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1->4GlcNAc beta 1->4GlcNAc. We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate.

scholarly journals Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

1990 ◽  
Vol 272 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Pacifici
Keyword(s):  
Acid Treatment ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Collagen Types ◽  
Keratan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondrocyte Cultures ◽  
Chondroitin Sulphate Proteoglycan ◽  
Core Proteins

The mechanisms regulating the secretion of proteoglycans and collagens in chondrocytes, in particular those operating at the level of the rough endoplasmic reticulum (RER), are largely unknown. To examine these mechanisms, I studied the effects of acute ascorbate treatment on the secretion of two collagen types (types II and IX) and two proteoglycan types (PG-H and PG-Lb, the major keratan sulphate/chondroitin sulphate proteoglycan and the minor chondroitin sulphate proteoglycan respectively in cartilage) in scorbutic cultures of chick vertebral chondrocytes. I found that the scorbutic chondrocytes synthesized underhydroxylated precursors of types II and IX collagen that were secreted very slowly and accumulated in the RER. When the cultures were treated acutely with ascorbate, both macromolecules underwent hydroxylation within 1-1.5 h of treatment, and began to be secreted at normal high rates starting at about 2 h. Proteoglycan synthesis and secretion, however, remained largely unaffected by ascorbate treatment. Both the half-time of newly synthesized PG-H core protein in the RER and its conversion into completed proteoglycan were unchanged during treatment. Similarly, the overall rates of synthesis and secretion of both PG-H and PG-Lb remained at control levels during treatment. The data indicate that secretion of types II and IX collagen is regulated independently of secretion of PG-H and PG-Lb. This may be mediated by the ability of the RER of the chondrocyte to discriminate between procollagens and proteoglycan core proteins.

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.

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