The core protein of fibroblast proteoheparan sulphate consists of disulphide-bonded subunits

Cöster, L; Malström, A; Carlstedt, Ingemar; Fransson, Lars‐Âke

doi:10.1042/bj2150417

Cited by 36 publications

(11 citation statements)

References 14 publications

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“…Analysis of the ECM-HSPG through size fractionation indicates the presence of two different PGs (Kavs of 0.25 and 0.41 on Sepharose CL-4B). The sizes of these two species are in good agreement with those reported for other HSPGs isolated from different sources (Coster et al, 1983;Rapraeger and Bernfield, 1985;Mehta et al, 1985), including basement membrane PGs (Hassell et al, 1980;. The two HSPGs present in the ECM of rat skeletal muscles possess two different chain lengths, and preliminary results in our laboratory indicate that the HSPG of Kav = 0.25, which is concentrated in synaptic regions, contains only the GAG chain of 20,000 daltons (Brandan and Inestrosa, unpublished results).…”

Section: Discussionsupporting

confidence: 87%

“…3(A). Two polydisperse peaks of P5S)-material with elution positions of Kav = 0.25 and 0.41 were found [corresponding to Mr = 4-6 x lo5 and 1.5-2.5 x lo5, respectively ;Coster et al (1983) andHassell et al (1985)l. Treatment of these PGs with nitrous acid, broke them down to small fragments that eluted close to the total volume of the column [Fig.…”

Section: Resultsmentioning

confidence: 99%

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Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

Brandan

Inestrosa

1987

J. Neurobiol.

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We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.

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Section: Discussionsupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

Brandan

Inestrosa

1987

J. Neurobiol.

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“…Reduction and alkylation of the cell-derived macromolecule gave rise to a component with an apparent Mr of 140,000, while the medium-derived form was not affected. Heparan-sulfate lyase treatment of the cell-derived proteoglycan yielded a core protein of apparent Mr 150,000, which, upon reduction, afforded polypeptides of Mr 80,000-100,000 (4 proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer in the presence of proteinase inhibitors (3). Proteoheparan sulfate was purified by isopycnic density-gradient centrifugation in CsCl/4 M guanidinium chloride followed by gel-permeation and ionexchange chromatography after degrading contaminating proteodermatan sulfate with chondroitin ABC lyase in the presence of proteinase inhibitors (3).…”

mentioning

confidence: 99%

“…The gel was first eluted with 0.5 ml each of buffer A and then with 50 mM KPO4 buffer (pH 5.0) containing 0.5 M NaCl and 0.5% (vol/vol) Triton X-100. Bound material was finally displaced with 4 M guanidinium chloride containing 1% (vol/vol) Triton X-100 or with 1% (wt/vol) NaDodSO4.…”

mentioning

confidence: 99%

Binding of transferrin to the core protein of fibroblast proteoheparan sulfate.

Fransson¹,

Carlstedt²,

Cöster³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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Cell-surface-associated proteoheparan sulfate from confluent human skin fibroblasts appears to consist of two disulfide-bonded polypeptides of Mr =90,000. The transferrin receptor, a ubiquitous cell-surface component of proliferating cells, also consists of two subunits of Mr 90,000 linked by S-S bonds. Radiolabeled proteoheparan sulfate mixed with holotransferrin or apotransferrin at pH 4-5 followed by rabbit anti-human transferrin was adsorbed onto protein A-Sepharose to -80-90%. At pH 7.5 apotransferrin bound :40% of the proteoglycan, whereas 80% was bound to holotransferrin. Trypsin digestion of the proteoglycan markedly lowered its ability to bind transferrin. However, binding was essentially unaffected by heparan-sulfate lyase treatment and after reduction and alkylation. Over 90% of the 3H activity of an L-[3H]leucine-labeled proteoglycan was recovered by immunoprecipitation (transferrin antitransferrin) of a heparan-sulfate lyase digest of the proteoglycan. The immunoprecipitated core protein had an apparent Mr of 150,000 before reduction and Mr of 90,000 after reduction of disulfide bonds. The core protein of the proteoglycan was recognized by the monoclonal antibody B3/25, which is known to be receptor specific. The present findings suggest that the core polypeptides of proteoheparan sulfate and the transferrin receptor may be identical or closely similar.Cell surface glycoproteins and proteoglycans of animal cells have been proposed to play a role in cell-cell and cell-matrix interactions, growth regulation, and uptake of nutrients (1 proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer in the presence of proteinase inhibitors (3). Proteoheparan sulfate was purified by isopycnic density-gradient centrifugation in CsCl/4 M guanidinium chloride followed by gel-permeation and ionexchange chromatography after degrading contaminating proteodermatan sulfate with chondroitin ABC lyase in the presence of proteinase inhibitors (3). Proteodermatan sulfate (type II) was from bovine sclera (5). Heparan-sulfate Iyase (heparitinsulfate lyase; EC 4.2.2.8) from Flavobacterium heparinum was a product of Seikagaku Kogyo (Tokyo, Japan). Ovalbumin, ovomucoid, human albumin, transferrin (substantially iron-free), a,-acid glycoprotein, a1-antitrypsin, and a2-macroglobulin were from Sigma. Rabbit immunoglobulin fractions against the human serum proteins and against mouse immunoglobulins were from Dakopatts (Copenhagen), and an antiserum against the proteodermatan sulfate was raised in this laboratory. The following monoclonal antibodies against the transferrin receptor were used: B3/25 (Hybritech, San Diego, CA), OKT9 (Ortho), and L5/1 (gift from T. Kalland, Dept. of Anatomy, Univ. of Lund). Highly purified human lactoferrin and an antiserum against it were a generous gift of A. Franzdn of this department. Protein ASepharose, Sepharose gels, as well as proteins for calibration were purchased from Pharmacia. Desferrioxamine was from CIBA Pharmaceutical. Other chemicals ...

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“…Extraction with lipid solvents has demonstrated that the large size of these molecules is not due to the presence of a lipid subunit as is found on some vertebrate proteoglycans (Norling et al 1981;Schafer et al 1984). Further, failure of reduction and alkylation to separate these sulfated molecules into smaller subunits suggests that the large size does not occur as a result of a protein subunit structure held together by disulfide bonds, which occurs in some vertebrate proteoglycans (Coster et al 1983) and in a nonproteoglycan sulfated glycoprotein from Drosophila (Campbell et al 1987). The presence of sugars in the Dl and DlCL2Bex fractions has been demonstrated by GLC, and sulfation of the polysaccharide chains has been shown by electrophoresis and radioautography.…”

Section: Discussionmentioning

confidence: 97%

Characterization and localization of large sulfated glycoproteins in the extracellular matrix of the developing asteroid Pisaster ochraceus

Crawford¹,

Crawford²

1992

Biochem. Cell Biol.

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In this study techniques commonly used to extract and purify proteoglycans of vertebrates were applied to the embryo of the asteroid Pisaster ochraceus at the early bipinnaria larva stage, a stage in which extensive cell migration is occurring within the extracellular matrix of the blastocoel. Several large sulfated glycoproteins were isolated and shown to consist of protein cores covalently bound to sulfated polysaccharide chains. The polysaccharide chains consisted primarily of neutral sugars and were not susceptible to glycosaminoglycan-degrading enzymes, suggesting that these were not glycosaminoglycans. The sulfated glycoproteins could be fractionated by electrophoresis on sodium dodecyl sulfate-agarose-acrylamide composite gels. Two types of monoclonal antibodies prepared against isolated extracellular matrix of these embryos reacted with two subsets of bands on Western blots of the composite gels. Staining of sections of the embryos with the antibodies showed that the epitopes that they recognized were located throughout the extracellular matrices of the embryos. That these high molecular weight glycoproteins were located within the extracellular matrix of the embryos suggests that they may be involved in the control of morphogenesis and cellular movement.

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The core protein of fibroblast proteoheparan sulphate consists of disulphide-bonded subunits

Cited by 36 publications

References 14 publications

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

Binding of transferrin to the core protein of fibroblast proteoheparan sulfate.

Characterization and localization of large sulfated glycoproteins in the extracellular matrix of the developing asteroid Pisaster ochraceus

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