A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [³⁵S]sulphate (and in some cases [6-³H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [³H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.     

The stabilization of in vivo assembled collagen fibrils by proteoglycans/glycosaminoglycans

The effects of proteoglycans/glycosaminoglycans on the thermal stability of in vivo assembled collagen fibrils have been examined. The shrinkage temperature of tendon collagen was found to be linearly dependent on the concentration of chondroitin sulphate in the surrounding fluid. Enzymic pretreatment of articular cartilage, to reduce its glycosaminoglycan content, resulted in decreased stability of the collagen present. The stability of the collagen in hyaluronidase-treated cartilage was found to be higher when measured in a solution of chondroitin sulphate (30 g/dl) than in buffer alone. The results of this study demonstrate that the proteoglycans stabilize collagen fibrils in tissues such as articular cartilage.     

Reconstruction of glycosaminoglycan chains in decorin

The glycosaminoglycan chain of decorin from human spinal ligaments was digested using the hydrolysis of bovine testicular hyaluronidase. As a result, decorin with hexasaccharide, octasaccharide, and decasaccharide including the linkage region, GlcA-Gal-Gal-Xyl, was obtained. The obtained decorin as an acceptor and hyaluronic acid as a donor were incubated with bovine testicular hyaluronidase under the condition of transglycosylation reaction. The transglycosylation reaction product had hexasaccharide to triacontasaccharide. Judging from the analysis of glycosaminoglycan chain in the transglycosylation reaction product, it was confirmed that hyaluronic acid chain as a donor was transferred to the retained glycosaminoglycan chain of decorin as an acceptor. Similarly, it was possible to reconstruct the glycosaminoglycan chain in decorin to chondroitin, chondroitin 4-sulfate or chondroitin 6-sulfate. Therefore, we succeeded in synthesizing an artificial family of decorins.     

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.     

Expression of hyaluronan synthases and hyaluronidases in the MG63 osteoblast cell line.

The expression of hyaluronan synthases (1, 2 and 3) and hyaluronidases (1, 2, 3, 4 and PH20) was examined in the MG63 osteoblast cell line induced to mineralize in vitro and compared to the rate of glycosaminoglycan production. Real-time PCR analysis demonstrated a 13-fold decrease in hyaluronan synthase 3 expression in mineralising MG63 cells; no significant change in hyaluronan synthase 2 expression in mineralising cells and hyaluronan synthase 1 was not expressed. In mineralising MG63 cells a 62-fold increase in hyaluronidase 2, a 13-fold increase in hyaluronidase 3, and a 3-fold increase in hyaluronidase 4 expression were observed when compared to non-mineralising cells; hyaluronidase 1 and PH20 expression was not detected. After 5 weeks in mineralising culture conditions a 2-fold increase in total 3H-glucosamine incorporation was observed in cells when compared to 24 h or 5 week control cultures. This was made up of a 5-fold decrease in hyaluronan production, a 2-fold increase in chondroitin sulphate/dermatan sulphate and a 10-fold increase in 3H-glucosamine incorporation into the non-glycosaminoglycan fraction. A 3-fold increase in 35SO4 incorporation into chondroitin sulphate/dermatan sulphate was also observed. Thus there is co-ordinate expression of genes that control hyaluronan metabolism such that there is a general decrease in the expression of hyaluronan synthases, an increase in the expression of hyaluronidases and a corresponding decrease in hyaluronan production by mineralising MG63 cells.     

Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.     

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.     

Evidence for the existence of a multichain proteoglycan of heparan sulphate

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.     

The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.     

Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver.

Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.     

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.     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase

Explants of cartilage from tibiae of 11-12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.     

Sequential distribution of keratan sulphate and chondroitin sulphate epitopes during ameloblast differentiation

Proteoglycans are complex macromolecules containing one or more glycosaminoglycan chains and exhibiting a variety of biological functions in connective tissues. The aim of the present study was to immunolocalize the distribution of keratan sulphate and chondroitin sulphate epitopes during initial enamel formation in order to study temporo-spatial expression patterns of these macromolecules. Third molars of four-months-old pigs were used for immunolocalization of keratan sulphate and chondroitin sulphate epitopes in the developing enamel layer. Tooth organs were prepared for paraffin sections in order to perform indirect immunohistochemistry. The results demonstrated a mutually exclusive positioning between these two epitopes. Keratan sulphate epitopes were observed in pre-secretory pre-ameloblasts and adjacent stratum intermedium while chondroitin sulphate epitopes were demonstrated in secretory ameloblasts and adjacent stratum intermedium. Our findings suggest that proteoglycans containing glycosaminoglycan chains may play a regulatory role during enamel mineralization.     

Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta

Summary The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate.Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possibly also present at a high concentration in the endothelium. Staining of sections after treatment with 4m guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.     

Histochemical evidence of a functional heterogeneity in neonatal canine epiphyseal chondrocytes

Synopsis The chondrocytes of the neonatal proximal humeral chondroepiphyses of twelve purebred English pointer pups were investigated histochemically, using frozen serial sections, for chondroitin sulphate and for the following enzyme activities: lactate dehydrogenase, NAD and NADP transhydrogenases, glutamate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, ATPase, succinate dehydrogenase, and UDPgalactose 4-epimerase. By using Alcian Blue with and without a prior digestion in testicular hyaluronidase, and Alcian Blue in the presence of 0.9 M magnesium chloride, it was found that about half the chondrocytes stained as if they were producing significant amounts of chondroitin sulphate. Only one enzyme, UDPgalactose 4-epimerase (which is involved in the biosynthesis of chondroitin sulphate), was found to have a similar staining heterogeneity. Therefore, it was concluded that the chondrocytes studied possessed a functional heterogenicity with particular reference to chondroitin sulphate synthesis while appearing morphologically homogeneous.     

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.     

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.     

The molecular-weight distribution of glycosaminoglycans

1. A rapid and sensitive method for the accurate estimation of the molecular-weight distribution of keratan sulphate and chondroitin sulphate isolated from adult bovine nasal septum and intervertebral disc is described. The method utilizes gel chromatography of reductively labelled glycosaminoglycan and end-group estimation of number-average molecular weight for each fraction across the peak of eluted glycosaminoglycan. 2. Chain-length distribution data obtained by this procedure are used to evaluate mechanisms of chondroitin sulphate biosynthesis.