Dataset Information

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

ABSTRACT: 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 X 10(6) (PG-25), 0.30 X 10(6) (PG-35) and 0.88 X 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.

SUBMITTER: Kapoor R

PROVIDER: S-EPMC1147452 | biostudies-other | 1986 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description: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.

Project description:1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs(2)SO(4) both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density rho=1.75g/ml whereas fraction PG-II banded sharply at rho=1.64g/ml. However, in Cs(2)SO(4), fraction PG-II banded at rho=1.51g/ml and fraction PG-I at rho=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs(2)SO(4) fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs(2)SO(4) gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88-42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs(2)SO(4) were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1x10(6)) than were those obtained previously for their ;monomeric' states (fraction PG-I 160000-410000, and fraction PG-II 70000-130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs(2)SO(4) (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs(2)SO(4). Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Project description:Cloned immortalized MC615 mouse chondrocytic cells were used to examine their capability to produce multiple types of matrix proteoglycans. Immunofluorescence staining indicated a uniform expression of aggrecan, biglycan and decorin by all cells. After culture with [35S]sulphate, proteo[35S]glycans secreted by the cells were found to elute in two peaks from a Sepharose CL-4B column. The first peak, at the void volume of the column, contained a large proteoglycan with an estimated average hydrodynamic mass of 10(3) kDa. The glycosaminoglycan chains of this proteoglycan had an average hydrodynamic size of 17 kDa, estimated by Sepharose CL-6B chromatography, indicating the presence of 30-70 glycosaminoglycan chains per core protein, which was consistent with the characteristics of aggrecan. Biglycan and decorin were immunoisolated from the second Sepharose CL-4B peak, and had average glycosaminoglycan hydrodynamic sizes of approx. 25 kDa and 32 kDa respectively. Glycosaminoglycan chains of the aggrecan, biglycan and decorin were treated with chondroitin ABC lyase, chondroitin AC lyase and chondroitin B lyase to determine the positions of sulphation and the degree of uronic acid epimerization. The aggrecan glycosaminoglycan chains were found to contain a 4-sulphate/6-sulphate ratio of 7:3, with no epimerization of glucuronic acid to iduronic acid. The biglycan glycosaminoglycan chains were found to contain a similar ratio of 4-sulphate/6-sulphate, but with approx. 40-45% of the glucuronic acid epimerized to iduronic acid. The decorin glycosaminoglycan chains were found to contain 4-sulphate but no detectable 6-sulphate, and approx. 30-35% epimerization of the glucuronic acid to iduronic acid. The results, using these cloned cells, indicated that a single MC615 cell is able to make all three proteoglycans with distinctive differences between the glycosaminoglycans of aggrecan, biglycan and decorin. These data indicate that a mechanism must exist for a single MC615 cell to regulate the sizes and fine structures of glycosaminoglycans on simultaneously produced, different proteoglycans in a core-protein-specific manner.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets