Characterization of proteoglycans from adult bovine tendon

Proteoglycans were extracted in good yield from the proximal, fibrous portion of adult bovine tendon with 4 m guanidine HCl. They comprise less than 1% of the dry weight of the tissue. Using CsCl density gradient centrifugation, gel chromatography, and ion exchange chromatography, two populations of proteoglycans were separated and purified from other tissue proteins. One was a large, chondroitin sulfate proteoglycan with high buoyant density in CsCl. This component appeared to be composed of two or three subpopulations as detected by agarose/polyacrylamide electrophoresis, although they could not be effectively separated from one another for individual characterization. As a group, the large proteoglycans eluted from Sepharose CL-2B with Kav from 0.1-0.5 and their core protein had Mr greater than 200,000 with high contents of glutamic acid, serine, and glycine. The glycosaminoglycan chains had a weight average Mr of 17,000 and more than 98% of the uronic acid was glucuronic acid. This group comprised only 12% of the total proteoglycan of the tissue. The other 88% of the proteoglycans appeared to represent one group of small molecules that eluted from Sepharose CL-2B at Kav = 0.70. They demonstrated buoyant densities in a CsCl gradient ranging from greater than or equal to 1.51 to 1.30 g/ml. Their core protein had an apparent Mr = 48,000 following removal of the glycosaminoglycan chains by digestion with chondroitinase ABC. This core protein had a particularly high content of aspartic acid/asparagine and leucine. The glycosaminoglycan chains had a weight average Mr of 37,000 and were dermatan sulfate containing 73% iduronic acid. Those molecules found at highest buoyant density appeared to have additional glycosaminoglycan chains that were shorter. Proteoglycans were also extracted from the pressure-bearing distal region of this tendon, where contents of proteoglycan per wet weight of tissue were 3-fold higher and as much as 50% of this was as large as the large proteoglycans from the proximal tissue. Preparations of large proteoglycans from both tendon regions contained molecules capable of interacting with hyaluronic acid.     

Characterization of the keratan sulphate proteoglycans from bovine corneal stroma.

The keratan sulphate proteoglycans that can be prepared from bovine corneal stroma [Axelsson & Heineg?rd (1975) Biochem. J. 145, 491-500] were characterized by gel chromatography, gel electrophoresis and analytical ultracentrifugation in associative (0.6 M-NaCl) and dissociative (6M-guanidinum chloride) solvents. The proteoglycans aggreagated at low salt concentrations and pH. The weight-average molecular weight of the monomer proteoglycans was established. Keratan sulphate peptides and oligosaccharide peptides were isolated after proteolysis. Their composition indicated that both are linked to protein via asparagine residues. A tentative model for corneal keratan sulphate proteoglycans is suggested.     

Extraction and purification of proteoglycans from mature bovine bone.

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.     

Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

Fractionation of proteoglycans from bovine corneal stroma.

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.     

Characterization of proteoglycans and glycosaminoglycans in bovine renal AA-type amyloidosis.

Highly sulfated glycosaminoglycans (GAG) or proteoglycans (PG), especially heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG), are considered to be intimately associated with amyloid deposits in different types of amyloidosis. Based on this relationship an important role for HS has been suggested in amyloidogenesis. The present immunohistological and ultrastructural study shows that in bovine renal AA-amyloidosis, sulfated GAG/PG was not restricted to amyloid deposits proper and that areas without GAP/PG were also present within the amyloid. Both glomerular and papillary amyloid contained HS (PG), and the latter also contained chondroitin sulfate (CS) and dermatan sulfate (DS), suggesting a correlation between the location of the amyloid and the type of GAG/PG deposited. Amyloid P component (AP) had a distribution similar to that of HSPG, confirming their affinity-based relationship. The GAG types found ultrastructurally in amyloid fibril preparations of glomerular and papillary amyloid isolated from the same kidney, reflected the immunohistological findings. HS was shown to be the predominant GAG in all papillary amyloid fibril extracts. Taking into account the chemico-physical properties of HS, it cannot be excluded that this predominance is introduced by the purification procedure. These results suggest that the association of GAG/PG and amyloid is not necessarily mutually obligatory and that the proposed importance of GAG in amyloidogenesis is disputable.     

Purification of bone morphogenetic protein derived from bovine bone matrix

Bone morphogenetic protein (BMP) was extracted from the bovine bone matrix and purified by liquid chromatography. The molecular weight of the BMP was 18 kDa by SDS-PAGE, and its pI value was 4.9. Amino acid analysis suggested that the BMP is a polypeptide containing 163 amino acids. In the present study, telopeptide-free type I collagen was used as a carrier of BMP.     

Self-association of dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate fractions (I and II) from bovine sclera were studied by gel chromatography, light-scattering and ultracentrifugation under various conditions. 2. Gel chromatography of proteoglycans in the absence or presence of hyaluronate was performed under associative conditions. No effect on the elution profile was noted. 3. Ultracentrifugation experiments (sedimentation-velocity and sedimentation-equilibrium) with proteoglycan I and II in 6 M-guanidine hydrochloride gave molecular weights (Mw) of 160000-220000 and 70000-100000 respectively. As the protein contents were 45% and 60% respectively, it may be calculated that proteoglycan I contained four to five side chains, whereas proteoglycan II contained one or two. Sedimentation-equilibrium runs performed in 0.15 M-NaCl gave an apparent molecular weight (Mw) of 500000-800000 for proteoglycan I and 90000-110000 for proteoglycan II. 4. In light-scattering experiments both proteoglycans I and II yielded high particle weights in 0.15 M-NaCl (3.1 X 10(6) and 3.4 X 10(6) daltons respectively). In the presence of 6 M-guanidine hydrochloride the molecular weights decreased to 410000 and 130000 respectively. The particle weights in 0.15 M-NaCl were not altered by the addition of hyaluronate or hyaluronate oligosaccharides. 5. The dermatan sulphate side chains of scleral proteoglycans (L-iduronate/D-glucuronate ratio 7:13) gave a particle weight of 100000 daltons in 0.15 M-NaCl. In 1.00 M-KCl/0.02M-EDTA the molecular weight was 24000. Addition of free scleral dermatan sulphate chains to a solution of proteoglycan II promoted further multimerization of the macromolecule.     

Extracellular matrix proteoglycans control the fate of bone marrow stromal cells

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-beta (TGF-beta)-binding proteoglycans, biglycan and decorin, prevents TGF-beta from proper sequestration within the extracellular matrix. The excess TGF-beta directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-beta signaling in bgn/dcn-deficient bone marrow stromal cells is a "switch in fate" from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass.     

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.     

Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage.

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 X 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 X 10(-8) cm2 X S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 X 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.     

Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera.

1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 x 10(-13) and 2.2 x 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.     

Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage.

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.     

Turnover of proteoglycans in articular-cartilage cultures. Characterization of proteoglycans released into the medium.

By using an e.l.i.s.a. method it was demonstrated that the majority of proteoglycans released into the medium of both control and retinoic acid-treated explant cultures of bovine articular cartilage did not contain a hyaluronate-binding region. This supports our previous findings [Campbell & Handley (1987) Arch. Biochem. Biophys. 258, 143-155] that proteoglycans released into the medium of both cultures were of smaller hydrodynamic size, more polydisperse and unable to form aggregates with hyaluronate. Analysis of 35S-labelled core proteins associated with proteoglycans released into the medium of both cultures by using SDS/polyacrylamide-gel electrophoresis and fluorography indicated the presence of a series of core-protein bands (Mr approx. 300,000, 230,000, 215,000, 200,000, 180,000, 140,000, 135,000, 105,000, 85,000 and 60,000) compared with three core proteins derived from the proteoglycans remaining in the matrix (Mr 300,000, 230,000 and 215,000). Further analysis of the core proteins released into the medium indicated that the larger core proteins associated with medium proteoglycans contain both chondroitin sulphate and keratan sulphate glycosaminoglycans whereas the smaller core proteins contain only chondroitin sulphate chains. These experiments provide definitive evidence that the loss of proteoglycans from the matrix involves proteolytic cleavage at various sites along the proteoglycan core protein.     

The hydration of proteoglycans of bovine cornea.

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

Characterization of the tissue-specific proteoglycans synthesized by chondrocytes from nanomelic chick embryos.

Cartilage from the avian mutant nanomelia has been reported to synthesize cartilage-specific proteoglycans, PGS(SC)-I, at 1-2% of normal values [McKeown & Goetinck (1979) Dev. Biol. 71, 203-215]. Proteoglycans were endogenously labelled with [35S]sulphate and extracted from cartilage in 4 M-guanidine hydrochloride and chromatographed on controlled-pore glass 1400. PGS(SC)-I was obtained from the void volume of these columns. Dissociative sucrose-density-gradient analysis revealed a greater than normal polydispersity in the nanomelic PGS(SC)-I. Fractions from both the controlled-pore glass 1400 void volume and sucrose gradients were tested for their ability to bind specific antibody against cartilage proteoglycan monomer. In all instances, binding of normal fractions was greater than 90%, whereas binding to nanomelic fractions ranged from 20 to 65%. Chromatography of PGS(SC)-I on controlled-pore glass 2500 resulted in 70% of the normal and 25% of the mutant proteoglycans eluting as aggregates. Chondroitin sulphate chains from mutant PGS(SC)-I appeared slightly larger than normal when chromatographed on controlled-pore glass 500. In addition, PGS(SC)-I from nanomelic cartilage is more susceptible to proteolysis in vitro than the PGS(SC)-I from normal cartilage. This evidence suggests that the small amount of cartilage-specific proteoglycan synthesized by nanomelic cartilage is not normal.