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

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.     

Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin.

A minor low-sulphated dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass (Mr) ranging from 70 to 120 kDa is composed of about two dermatan sulphate chains (Mr 33 kDa) bound on a protein core of Mr 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the dermatan sulphate chain.     

The proteoglycans of the canine intervertebral disc

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.     

A role for disulphide bridges in the protein core in the interaction of proteodermatan sulphate and collagen

Proteodermatan sulphate from bovine skin retarded precipitation of fibrils from solutions of purified acid-soluble bovine skin collagen. The isolated protein core was as effective as the intact proteoglycan. Thermal denaturation leading to almost complete loss of the native secondary structure, (determined by circular dichroism spectroscopy to consist of about 60% beta structure) did not diminish the effect unless accompanied by reduction of disulphides, of which there were shown to be three per molecule. The reduced and alkylated protein core was totally ineffective. Electron-microscopy revealed a D-periodic arrangement of glycosaminoglycan on the surfaces of collagen fibrils precipitated in the presence of proteodermatan sulphate. Dermatan sulphate (with attached small peptide) prepared from the proteoglycan, had no effect on the rate of fibrillogenesis and was apparently not bound to the fibrils.     

Isolation and some structural analyses of a proteodermatan sulphate from calf skin.

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).     

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

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.     

Interaction of cartilage proteoglycans with collagen-substituted agarose gels.

1. Rat tail-tendon collagen was coupled to activated Sepharose 4B at 2.5 mg of collagen/ml of gel. Chromatographic columns of this gel were calibrated with T2 virus (Vo) and Dnp-alanine (Vt). 2. The chromatographic behaviour of cartilage proteoglycans on the collagen-substituted gel was studied under conditions of varying ionic strength. Proteoglycan subunit obtained from bovine nasal cartilage, the proteoglycan obtained after digestion with chondroitnase ABC and purified chondriotin sulphate were all retarded on the collagen gel by an interaction that abolished at I0.17. Purified keratan sulphate and hyaluronic acid were not retarded. 3. A strong ionic interaction between cartilage proteoglycan and collagen was demonstrated to depend on the structure of the protein core of the proteoglycan.     

Mineral-binding proteoglycans of fetal porcine calvarial bone

To provide a more definitive characterization of the hydroxylapatite-associated proteoglycans (HAPG) of bone, proteins were extracted from the mineralized matrix of fetal porcine calvaria with 0.5 M EDTA in the absence of guanidine HCl. The small proteoglycans obtained in the extract were fractionated by gel filtration on Sepharose CL-6B, purified by ion-exchange chromatography on Polyanion matrix (fast protein liquid chromatography), and then separated into three major populations of chondroitin sulfate proteoglycans by chromatography on hydroxylapatite, all in the presence of 7 M urea. Based on immunological and chemical properties, two classes of bone proteoglycan were resolved. In one class (HAPG1), the proteoglycan and specific CNBr-derived peptides cross-reacted with three monoclonal antibodies that recognize different epitopes of the protein core of bovine skin proteodermatan sulfate. The other class of proteoglycan included two species (HAPG2, HAPG3) which were not recognized by these antibodies. In addition, these proteoglycans did not stain with Coomassie Blue R-250 nor with silver stain nor did they bind to nitrocellulose membranes used in Western blots. However, the cationic dye Stains-all stained both HAPG2 and HAPG3; the protein cores of these proteoglycans were stained a characteristic turquoise blue, whereas the protein core of HAPG1 was stained pink. The average Mr values of the bone proteoglycans, from gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis were: HAPG1, 120,000, with a protein core (chondroitinase AC-digested) of 45,000; HAPG2 and HAPG3, 110,000, with protein cores of 37,000-38,000. On 15% polyacrylamide gel electrophoresis, the protein cores of HAPG2 and HAPG3 migrated with an Mr 30,000, while HAPG1 protein core was unchanged (Mr 45,000). Based on amino acid analysis, the protein chains of HAPG2 and HAPG3 appear to be identical, although minor differences in the relative amount of glucosamine were evident. In contrast, the composition of HAPG1 was quite different, with higher relative amounts of hydrophobic and aromatic residues and lower amounts of Asx and Glx. The presence of 360 residues/1,000 of Asx and Glx in HAPG2 and HAPG3 may in part explain the characteristic staining and immunotransfer properties of these proteoglycans. The unique amino-terminal sequence of HAPG2 (Asn-Pro-Val-Ala-Arg-Tyr-Gln), together with the immunological and chemical properties, would indicate that HAPG2 and HAPG3 are novel proteoglycans and, unlike HAPG1, could be unique to mineralized tissues.     

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.     

Isolation and characterization of dermatan sulfate proteoglycans synthesized by cultured bovine aortic endothelial cells

Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region.     

Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture.

35SO42(-)- and [3H]leucine-labelled proteoglycans were isolated from the medium and cell layer of human skin fibroblast cultures. Measures were taken to avoid proteolytic modifications during isolation by adding guanidinium chloride and proteolysis inhibitors immediately after harvest. The proteoglycans were purified and fractionated by density-gradient centrifugation, followed by gel and ion-exchange chromatography. Our procedure permitted the isolation of two major proteoglycan fractions from the medium, one large, containing glucuronic acid-rich dermatan sulphate chains, and one small, containing iduronic acid-rich ones. The protein core of the latter proteoglycan had an apparent molecular weight of 47000 as determined by polyacrylamide-gel electrophoresis, whereas the protein core of the former was considerably larger. The major dermatan sulphate proteoglycan of the cell layer was similar to the large proteoglycan of the medium. Only small amounts of the iduronic acid-rich dermatan sulphate proteoglycan could be isolated from the cell layer. Instead most of the iduronic acid-rich glycans appeared as free chains. The heparan sulphate proteoglycans found in the cell culture were largely confined to the cell layer. This proteoglycan was of rather low buoyant density and seemed to contain a high proportion of protein. The major part of the heparan sulphate proteoglycan from the medium had a higher buoyant density and contained a smaller amount of protein.     

Primary structure of a mouse mastocytoma proteoglycan core protein.

The complete nucleotide sequence of a mouse mastocytoma proteoglycan core protein mRNA was determined. The mRNA, estimated to contain 1.1 kb, encodes a protein with an Mr of 16715. A 21-amino acid-residue region of the protein is composed of alternating serine and glycine residues. Southern-blot analysis of mouse genomic DNA with cDNA containing sequences corresponding to the Ser-Gly repeat region revealed more than 15 gene fragments. Hybridization with a probe corresponding to the N-terminal portion of the core protein identified two fragments, and cDNA covering the C-terminal part of the core protein and the 3' untranslated part of the mRNA hybridized to a single fragment. Antibodies against the core protein, obtained after immunization of rabbits with a fusion protein, reacted with both chondroitin sulphate proteoglycans and heparin proteoglycans produced by the tumour. In immunoblotting of a microsomal fraction from the mastocytoma, the antiserum recognized a single protein (Mr 17,000), which probably represents the core protein before glycosylation.     

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.     

Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpyridium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractionated by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and a proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma.

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpirdium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractioned by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Proteoglycans of Wharton's jelly

Wharton's jelly (WJ) is a myxomatous substance surrounding the blood vessels of the umbilical cord. Proteoglycans (PGs) of Wharton's jelly have not been studied to date therefore it was decided to explore proteoglycan composition of this tissue. Proteoglycans were subjected to dissociative extraction with 4M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion-exchange chromatography and lyophilised. They were analysed by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after treatment with chondroitinase ABC. It was found that 1g of Wharton's jelly contains 2.43+/-0.63mg (n=10) of sulphated glycosaminoglycans (GAGs), reflecting the presence of proteoglycans. The proteoglycans were mainly substituted with chondroitin/dermatan sulphate (DS) chains. The predominant proteoglycan fraction included small proteoglycans with core proteins of 45 and 47kD, immunologically related to decorin (45 and 47kD) and biglycan (45kD). The expression of decorin core proteins was much higher than that of biglycan. Larger proteoglycans (core proteins of 90, 110, 220 and 260kD) were found in lower amounts. The most abundant of them (core protein of 260kD) was immunologically related to versican. Perlecan was not identified in Wharton's jelly. The study shows that Wharton's jelly contains mainly small chondroitin/dermatan sulphate proteoglycans, with decorin strongly predominating over biglycan. We suggest that an intensive expression of decorin is associated with very high content of its ligand, collagen.     

Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain.

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.     

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.