Isolation and characterization of two sialoproteins present only in bone calcified matrix

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

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Extraction and purification of proteoglycans from mature bovine bone

Biochemical Journal ◽

10.1042/bj2240047 ◽

1984 ◽

Vol 224 (1) ◽

pp. 47-58 ◽

Cited By ~ 49

Author(s):

A Franzén ◽

D Heinegård

Keyword(s):

Core Protein ◽

Proteinase Inhibitors ◽

Extraction Procedure ◽

Deae Cellulose ◽

Bovine Bone ◽

Guanidinium Chloride ◽

Extraction Solvent ◽

Extraction And Purification ◽

Cscl Density Gradient ◽

Mineralized Matrix

Proteoglycans were extracted in good yields from the mineralized matrix of ground bovine bone, by using a two-step extraction procedure. Proteoglycans (8% of total), not associated with the bone mineral, were extracted at − 20 degrees C with 4M-guanidinium chloride containing proteinase inhibitors. Proteoglycans associated with the mineral, which accounted for 60% of the total, were then solubilized when EDTA was added to the extraction solvent. They were fractionated and purified in the presence of 4M-guanidinium chloride by CsCl-density-gradient centrifugations followed by chromatography on Sepharose CL-4B. Further purification was obtained by chromatography on DEAE-cellulose and hydroxyapatite in the presence of 7 M-urea. Three populations of proteoglycans and additional glycosaminoglycan peptides were obtained. The molecular dimensions of both intact molecules and of their side chains as well as their amino acid composition were different, indicating that they represent separate molecular entities. The main proteoglycan self-aggregated in the absence of 4M-guanidinium chloride or 7 M-urea, a property that was abolished when the proteoglycan core protein was fragmented.

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Characterization of proteoglycans from the calcified matrix of bovine bone

Biochemical Journal ◽

10.1042/bj2240059 ◽

1984 ◽

Vol 224 (1) ◽

pp. 59-66 ◽

Cited By ~ 55

Author(s):

A Franzén ◽

D Heinegård

Keyword(s):

Molecular Mass ◽

Core Protein ◽

Chondroitin Sulphate ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sedimentation Coefficient ◽

Sedimentation Equilibrium ◽

Assay Method ◽

Enzyme Linked Immunosorbent Assay ◽

Bovine Bone

The proteoglycans characterized were those isolated from the calcified matrix of mature bovine bone [Franzén & Heinegård (1984) Biochem. J. 224, 47-58]. The average molecular mass of the bone proteoglycan is 74 600 Da, determined by sedimentation-equilibrium centrifugation in 4M-guanidinium chloride. Its sedimentation coefficient (s0(20),w) is 3.04 S. The apparent Mr of its core protein is 46 000, estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chondroitinase ABC-digested proteoglycan. A more likely molecular mass of the core protein is 30 000 Da, as calculated from the molecular mass and the protein content (40%) of the proteoglycan. The bone proteoglycan contains one or probably two chondroitin sulphate chains each with a molecular mass (weight-average) of 33 700 Da and several oligosaccharides both of the N-glycosidically and the O-glycosidically linked type. Antibodies against the homogeneous bone proteoglycans were raised in rabbits. An e.l.i.s.a. (enzyme-linked immunosorbent assay) method was developed that allowed specific quantification of bone proteoglycans at nanogram levels. The specificity of the antibodies was tested by using the e.l.i.s.a. method. The bone proteoglycan showed partial cross-reactivity with the small proteoglycan of cartilage. The antibodies were used to localize immunoreactivity of bone proteoglycans by indirect immunofluorescence in frozen sections of foetal bovine epiphysial growth plate. The fluorescence was entirely found in the primary spongiosa, and no fluorescence was found among the hypertrophied chondrocytes or in the region of provisional calcification.

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Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta

Biochemical Journal ◽

10.1042/bj2400575 ◽

1986 ◽

Vol 240 (2) ◽

pp. 575-583 ◽

Cited By ~ 21

Author(s):

R Kapoor ◽

C F Phelps ◽

T N Wight

Keyword(s):

Uronic Acid ◽

Glucuronic Acid ◽

Core Protein ◽

Chondroitin Sulphate ◽

Polymer Concentration ◽

Dermatan Sulphate ◽

Guanidinium Chloride ◽

Structural Units ◽

The Core ◽

Covalently Linked

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 × 10(6) (PG-25), 0.30 × 10(6) (PG-35) and 0.88 × 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.

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Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro

Biochemical Journal ◽

10.1042/bj2250493 ◽

1985 ◽

Vol 225 (2) ◽

pp. 493-507 ◽

Cited By ~ 111

Author(s):

J A Tyler

Keyword(s):

Articular Cartilage ◽

Core Protein ◽

Fold Increase ◽

Acid Proteinase ◽

Binding Region ◽

Guanidinium Chloride ◽

The Core ◽

The Matrix ◽

Average Size

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The ‘clipped’ monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

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Isolation and characterization of syringacin W-1, a bacteriocin produced by Pseudomonas syringae pv. syringae

Canadian Journal of Microbiology ◽

10.1139/m86-046 ◽

1986 ◽

Vol 32 (3) ◽

pp. 231-236 ◽

Cited By ~ 12

Author(s):

Mary L. Smidt ◽

Anne K. Vidaver

Keyword(s):

Pseudomonas Syringae ◽

Inner Core ◽

Deae Cellulose ◽

Sedimentation Coefficient ◽

Temperature Stability ◽

Ultrafiltration Rate ◽

Isolation And Characterization ◽

Outer Sheath ◽

Shaped Particle

Syringacin W-1, a bacteriocin produced by Pseudomonas syringae pathovar syringae strain PsW-1, is a 20 × 75 nm rod-shaped particle composed of an inner core and outer sheath. Production of syringacin W-1 in broth was induced with 0.1 μLg/mL mitomycin C. The bacteriocin was purified from culture lysates using ultrafiltration, rate zonal centrifugation in sucrose gradients, and DEAE-cellulose chromatography. Purity was evaluated by subjecting syringacin W-1 preparations to electrophoresis in polyacrylamide gels under nondenaturing and denaturing conditions. The chemical composition was principally protein (67.2%), and also comigrating nonessential carbohydrate (10–35%). The physical properties of purified syringacin W-1 were a sedimentation coefficient of 104 for rod-shaped particles, pH stability of 5.2–8.2, and temperature stability from −20 to 40 °C. The bacteriocin was resistant to proteases and to 12 of 13 surfactants tested.

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ISOLATION AND CHARACTERIZATION OF THE MAJOR ANTIGEN OF RAT KIDNEY

Canadian Journal of Biochemistry ◽

10.1139/o67-089 ◽

1967 ◽

Vol 45 (6) ◽

pp. 781-789 ◽

Cited By ~ 5

Author(s):

Bao-Linh Dinh

Keyword(s):

Rat Kidney ◽

Gel Filtration ◽

Deae Cellulose ◽

Sedimentation Coefficient ◽

Molecular Weights ◽

Isolation And Characterization ◽

Antigenic Properties ◽

Major Antigen ◽

Purified Antigen

The major antigen of rat kidney (KMA) was isolated by chromatography on DEAE-cellulose and Sephadex G-25 gel filtration. The sedimentation coefficient of the purified antigen was about 1.2 S and its electrophoretic mobility corresponded to that of α2-globulins. Its diffusion coefficient was estimated by the method of Allison and Humphrey as 11.4 × 10−7 cm2 sec−1. On gel filtration through Sephadex G-100, the purified preparation was resolved into two components with identical antigenic properties but of different molecular weights, estimated as 16,600 and 4,100. The concentrations of KMA were determined in different tissues, urine, and serum by immunodiffusion. The probable relationship between physical and immunochemical properties and the possible physio-pathological role of KMA are discussed.

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Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

Biochemical Journal ◽

10.1042/bj2090497 ◽

1983 ◽

Vol 209 (2) ◽

pp. 497-503 ◽

Cited By ~ 63

Author(s):

N Uldbjerg ◽

A Malmström ◽

G Ekman ◽

J Sheehan ◽

U Ulmsten ◽

...

Keyword(s):

Molecular Weight ◽

Uterine Cervix ◽

Proteinase Inhibitors ◽

Gradient Centrifugation ◽

Average Molecular Weight ◽

Deae Cellulose ◽

Sedimentation Equilibrium ◽

Dermatan Sulphate ◽

Guanidinium Chloride ◽

Isolation And Characterization

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

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Extracellular matrix microfibrils are composed of core proteins coated with fibronectin.

Journal of Histochemistry & Cytochemistry ◽

10.1177/33.4.3980980 ◽

1985 ◽

Vol 33 (4) ◽

pp. 268-274 ◽

Cited By ~ 48

Author(s):

E Schwartz ◽

S Goldfischer ◽

B Coltoff-Schiller ◽

O O Blumenfeld

Keyword(s):

Extracellular Matrix ◽

Cultured Cells ◽

Core Protein ◽

Extracellular Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

The Core ◽

Core Proteins ◽

Double Label ◽

Active Fragments ◽

Partial Dissociation

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.

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Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone

The Journal of Cell Biology ◽

10.1083/jcb.141.3.839 ◽

1998 ◽

Vol 141 (3) ◽

pp. 839-847 ◽

Cited By ~ 105

Author(s):

Mikael Wendel ◽

Yngve Sommarin ◽

Dick Heinegård

Keyword(s):

Guanidine Hydrochloride ◽

Bone Matrix ◽

Keratan Sulfate ◽

Apparent Size ◽

Enzyme Linked Immunosorbent Assay ◽

Bovine Bone ◽

Metabolic Labeling ◽

Cell Binding ◽

Isolation And Characterization

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride–containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mr of the proteoglycan was 85,000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin αvβ3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts.

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Isolation and characterization of proteoglycans synthesized by mouse osteoblastic cells in culture during the mineralization process

Biochemical Journal ◽

10.1042/bj2660015 ◽

1990 ◽

Vol 266 (1) ◽

pp. 15-24 ◽

Cited By ~ 40

Author(s):

Y Takeuchi ◽

T Matsumoto ◽

E Ogata ◽

Y Shishiba

Keyword(s):

Core Protein ◽

Chondroitin Sulphate ◽

Heparan Sulphate ◽

Osteoblastic Cell ◽

Guanidinium Chloride ◽

Mineralization Process ◽

Osteoblastic Cell Line ◽

Isolation And Characterization

Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.

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