Connective Tissue Polysaccharides Research Articles

Albert Dorfman and the Biosynthesis of Hyaluronic Acid

Albert Dorfman and the Biosynthesis of Hyaluronic Acid

Regulation of proximal tubular epithelial cell CD44-mediated binding and internalisation of hyaluronan

Background: Increased expression of the connective tissue polysaccharide hyaluronan (HA) in the renal corticointerstitium is associated with progressive renal fibrosis. Numerous studies have demonstrated involvement proximal tubular epithelial cells in the fibrotic process and in the current study we have characterised their expression of the HA receptor, CD44, and examined changes in CD44 expression and function in response to either IL-1β or glucose. Methods: Characterisation of CD44 splice variant expression was carried out in primary cultures of human proximal tubular cells (PTC) and HK2 cells. Binding and internalisation HA was examined by addition of exogenous of fluorescein-HA (fl-HA), and expression of CD44 examined by immunoblot analysis and flow cytometry. Alteration in “functional” CD44 was determined by immunoprecipitation of CD44 following stimulation in the presence of fl-HA. Results: PTC, both primary culture and the PTC cell line, HK2, express at least 5 CD44 splice variants, the expression of which are not altered by addition of either IL-1β or 25 mM d-glucose. Addition of either stimulus increased cell surface binding and internalisation of fl-HA and increased expression of functionally active CD44. Increased binding and internalisation of fl-HA, was blocked by anti-CD44 antibody, and by the inhibition of O-glycosylation. Conclusions: The data demonstrate that stimuli inducing PTC HA synthesis also regulate PTC–HA interactions. Furthermore increased HA binding and internalisation is the result of post-translational modification of CD44 by O-glycosylation, rather than by alteration in expression of CD44 at the cell surface, or by alternate use of CD44 splice variants.

Regulation of renal proximal tubular epithelial cell hyaluronan generation: Implications for diabetic nephropathy

Proximal tubular cells (PTCs) contribute to pathological changes in the renal interstitium by the generation of cytokines and alterations in the composition of the extracellular matrix. Hyaluronan (HA) is a ubiquitous connective tissue polysaccharide that regulates cell function and tissue remodeling. In the current study, we investigated the regulation of HA generation by PTCs. Primary cultures of human PTCs were grown to confluence and stimulated under serum-free conditions with either interleukin-1 (IL-1) or 25 mmol/L D-glucose. Alterations in HA generation were detected by enzyme-linked immunosorbent assay, and alterations in HA synthase gene expression were examined by reverse transcription-polymerase chain reaction. Subsequently, the mechanisms of IL-1 beta and glucose-induced alterations in HA were examined utilizing HK-2 cells. Stimulation of human PTCs (HPTCs) with either IL-1 beta or 25 mmol/L D-glucose led to a significant increase in the HA concentration in the culture supernatant. In contrast, stimulation of HPTCs with transforming growth factor-beta1, basic fibroblast growth factor, or platelet-derived growth factor-AB did not stimulate HA production. The addition of IL-1 beta or 25 mmol/L D-glucose also increased HA generation in HK-2 cells and was associated with the induction of HAS2 mRNA. HAS3 mRNA was constitutively expressed and was not influenced by the addition of either stimulus. HAS1 mRNA expression was not detected in either unstimulated or stimulated cells. Inhibition of gene transcription or protein synthesis abolished HA production in response to either IL-1 beta or glucose. Inhibition of nuclear factor-kappa B (NF-kappa B) activation either by sulindac or by the proteosome inhibitor (PSI) abrogated both IL-1 beta and glucose-mediated alteration in HA synthesis. This study demonstrates, to our knowledge for the first time, that increased HA synthesis in response to either IL-1 beta or elevated 25 mmol/L D-glucose is associated with NF-kappa B-activated transcription of HAS2.

Role of endocardial endothelial cells in the turnover of hyaluronan in Atlantic cod (Gadus morhua).

The fate of the major connective tissue polysaccharide hyaluronan, as it appears after release from the matrix, was studied in the Atlantic cod by use of subcutaneous administration of hyaluronan conjugated with fluorescein isothiocyanate (FITC) and labelled with 125I. After administration, the ligand was transported to the heart, which contained 58% of the recovered label after 22 h, whereas 36% remained at the injection site. Uptake in other organs was low. Results from intravenous co-injection studies showed that 125I-FITC-hyaluronan and native hyaluronan were in competition for uptake by the same receptor in cod heart. Fluorescence microscopy revealed that FITC-hyaluronan accumulated in spherical structures and discrete vesicles in endocardial endothelial cells lining the muscular trabeculae of both heart chambers. Immunoelectron microscopy showed that, in these cells, the ligand lined the limiting membrane of endosomes and filled the lumen of late endosomes or lysosomes. We conclude that, in the cod, heart endothelial cells are essential for the turnover of hyaluronan. Atrial endothelial cells were also able to ingest 2-microm latex beads, although these were far more effectively phagocytosed by head kidney macrophages. The present results strengthen the notion that the cod endocardium consists of specialized scavenger endothelial cells, resembling sinusoidal endothelial cells of salmonid kidney and mammalian liver. These cells should therefore be regarded as an important part of the cod reticulo-endothelial system.

Serum hyaluronan as a disease marker.

Hyaluronan is a connective tissue polysaccharide which has also been found in blood serum in concentrations < 100 micrograms/L (average 30-40 micrograms/L in middle-aged persons). The serum level is regulated by the influx of the polysaccharide from the tissues via lymph and its receptor-mediated clearance by liver endothelial cells. Markedly high serum levels are noted in certain liver diseases, especially in patients with cirrhosis, when the clearance is impaired. In these cases serum hyaluronan can be used to follow the development of the disease. Serum hyaluronan is also a sensitive marker for impending rejection of liver transplants. Patients with rheumatoid arthritis constitute another major group with increased serum hyaluronan, but in this case the level varies markedly during the day corresponding to physical activity. There are good indications that in these subjects the excess hyaluronan comes from the joints. Under stringent sampling conditions of serum it should be possible to extract interesting information on the inflammatory joint process. Increased hyaluronan levels are also seen in other inflammatory diseases and it is of special interest that high hyaluronan levels in patients with septic conditions is a sign of poor prognosis. Certain tumours, notably Wilms' tumour and mesothelioma, produce factors which activate synthesis of hyaluronan and increase its serum level. Rare hereditary diseases with disturbances of hyaluronan metabolism and elevated blood levels have also been discovered, e.g. Werner's syndrome and cutaneous hyaluronanosis. Information accumulated during the last decade regarding the metabolism of hyaluronan has made this polysaccharide an interesting clinical marker for a number of pathological conditions.

An early look at macromolecular crowding

Interest in the interaction between proteins and polysaccharides in semidilute systems developed in the late 1950's and early 1960's both in the field of matrix biology and in the construction of new separation techniques. The author gives an account of how his work in the 50's on the characterization of a connective tissue polysaccharide, hyaluronan, led him into studies on polysaccharide networks, sieve effects, molecular exclusion, a theory of gel filtration, solubility of proteins and chemical equilibria in polymer solutions, water and protein homeostatis in vivo, and ordered convectional flow in concentrated polymer systems.

Extracellular matrix interactions: Sulfation of connective tissue polysaccharides creates macroion binding templates and conditions for dissipative structure formation

Evidence is now accumulating that the post-polymer modification process of sulfation of connective tissue polysaccharides is primarily to provide an interactive macroion for enthalpic interactions rather than influence thermodynamic non-ideality which primarily affects water distribution in biological systems. Metabolic energy considerations also distinguish these physicochemical classifications. Thermodynamic non-ideality is embodied in the carboxyl group and polysaccharide chain which are energetically favoured in biosynthesis, whereas considerable energy input is required for sulfation. The sulfation process gives rise to macroions, with a wide variety of negative charge patterns, that may participate in heterotypic macromolecular interactions. This partial informational specificity is discussed in terms of evolutionary flexibility of the extracellular matrix as rationalized on the qualitative aspects of dissipative structure formation. The concept of multiple binding interactions of varying specificity associated with connective tissue polysaccharides raises the awareness of a more random, less highly ordered, extracellular matrix as compared to the tight machine-like organization generally found for processes in the cell. This is discussed in terms of the physiological adaptation and development of multicellular-tissue systems.

Hydrodynamic properties of connective-tissue polysaccharides

The major hydrodynamic properties of the connective-tissue polysaccharides are those that describe polysaccharide-water interaction as embodied in their osmotic-pressure and hydraulic-conductivity properties. This study shows that, for polysaccharides such as chondroitin sulphate, hyaluronate and the heparin-like polysaccharides, their hydrodynamic properties depend primarily on the presence of the uronic residue and the nature of the glycosidic linkage. Other parameters such as the degree of N-acetylation and sulphation were found not to influence these properties to any great extent. These studies particularly delineate structural-functional aspects of the connective-tissue polysaccharides in terms of their primary structure.

Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-glucose

Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.

Silk—A new substrate for UDP- d-xylose: Proteoglycan core protein β- d-xylosyltransferase

The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP- d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [ 14C]xylose transferred from UDP- d-[ 14C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the V max for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. K m values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [ 14C]xylose transfer to silk was alkali labile, and [ 14C]xylitol was formed when [ 14C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [ 14C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [ 14C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [ 14C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had V max values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a V max value close to 20% of that of intact fibroin.

Dermatan sulfate: Molecular conformations and interactions in the condensed state

The molecular conformations and manner of aggregation has been determined for three allomorphs of the connective tissue polysaccharide dermatan sulfate by analysis of X-ray diffraction from oriented, polycrystalline fibers of sodium salts. One allomorph is unique among glycosaminoglycans in having right-handed (8(3)) helical chains. Two such chains pack antiparallel in a tetragonal unit cell (a = b = 1.267 nm, c = 7.353 nm) with P4(3)2(1)2 space group symmetry. The 3(2) chains of the second allomorph are organized in a trigonal unit cell (a = b = 1.460 nm, c = 2.823 nm, space group symmetry P3(2)21) containing two left-handed antiparallel polysaccharide molecules. (The chirality of this allomorph has been assumed to be the same as in other 3-fold glycosaminoglycan helices, since discrimination between 3(1) and 3(2) symmetries was found not to be possible.) The archiral 2(1) helices of the third allomorph, pack probably in an orthorhombic unit cell (a = 1.151 nm, b = 1.065 nm, c = 1.878 nm, space group symmetry P2(1)2(1)2(1)) that contains again two antiparallel polymer molecules. Each dermatan sulfate chain is stabilized intramolecularly by O3-O5 hydrogen bonds across the beta (1 leads to 4) linkage. There are two intermolecular hydrogen bonds per tetrasaccharide repeat in the tetragonal structure and two per disaccharide in the trigonal structure. Fourier difference syntheses indicated equivalents of four sodium ions per tetrasaccharide and two sodium ions per disaccharide in the tetragonal and trigonal structures, respectively. The cations are either partially or fully hydrated and link dermatan sulfate chains either intra- or intermolecularly by involving besides other polyanion oxygen atoms, carboxylate and sulfate oxygen atoms. The probable mode of packing in the orthorhombic structure indicates a pair of hydrogen bonds between adjacent antiparallel polysaccharide chains and suggests plausible cationic sites in the unit cell.

Connective tissue polysaccharides: cations as determinants of conformations

Connective tissue polysaccharides: cations as determinants of conformations

Physiological function of connective tissue polysaccharides.

Physiological function of connective tissue polysaccharides.

Electric potentials generated by connective tissue polysaccharides in glass capillaries

Attention is drawn to a possible source of artifact in measurements of capillaary and displacement potentials of potassium hyaluronate solutions in glass capillaries. The experimental evidence demonstrates that there exist no electrochemical activity gradients of the polysaccharide in these systems.

Relationship of the ultrastructure and mechanical properties in tendinous collagen, a highly ordered macromolecular composite

A relationship has been found between the stress and deformation in tendon and, from an analysis of the data obtained, it was established that there is a major stress-bearing fiber, the diameter of which increases with increasing age of the animal. The relationship of the results obtained with the structure and properties of tendon as well as with the aging process is stressed. Models of the ultrastructure of collagen fibers in rat tail tendon or in other soft connective tissue were prepared by sealing rigid fibers in a considerably softer elastic matrix. Loss of fiber resistance was achieved by contraction of this matrix. These synthetic composite systems exactly reflect a number of the features of the collagen ultra-structure. Models were also used for studying stresses in the matrix about the fibers as this necessary information could not be obtained by a direct study of the biological tissues. Examination of the reaction between tropocollagen and the connective tissue polysaccharides in the process of collagen fiber formation shows that a mechanism exists through which the fibers may lose their resistance also in vivo.

X-ray diffraction studies on the connective tissue polysaccharides: Two-dimensiodnal packing schemes for threefold hyaluronate chains

An extended conformation of sodium and calcium hyaluronate with helical parameters n (number of residues per turn) = 3 and h (axial rise per disaccharide) = 0.95 ± 0.01 nm has been found to pack in a number of two-dimensional arrays. Each of the structures exhibits a variant of a motif consisting of six chains surrounding a “vacant” site. The distribution of chains around this site, together with certain aspects of the periodicity, suggests that there might be a special relationship between this conformation of hyaluronic acid and water.

X-ray diffraction studies on the connective tissue polysaccharides. Molecular conformations of dermatan sulphate

Three distinct molecular conformations of the connective tissue polysaccharide dermatan sulphate have been observed by X-ray diffraction. These comprise an 8-fold type helix, a 3-fold helix and a 2-fold helix with disaccharide repeats projected onto the helix axis of 0.93 nm, 0.96 nm and 0.97 nm, respectively. The relative merits of the various 8-fold helices are discussed. The evidence favours the l-iduronic acid moiety to be in the Cl chair form for all three structures.

Molecular conformations of chondroitin-4-sulphate.

CHONDROITIN-4-SULPHATE is a member of the connective tissue group of polysaccharides and occurs mainly in cartilage. The molecular chains are covalently linked to a protein core and act as a flexible connecting matrix between the tough protein filaments1–3. In common with many of the connective tissue polysaccharides, the isolated carbohydrate chains are based on a disaccharide repeat of the type (-A-B-)n, where A represents N-acetylgalactosamine-4-sulphate and B represents glucuronic acid. The glycosidic linkages and disposition of side groups are shown schematically in Fig. 1.

Metabolic turnover of sulphated heteroglycans in the gastrointestinal tract, cartilage and skin of the rat

Turnover rates of sulphated saccharides in the gastrointestinal tract of the rat were estimated with the aid of [ 35S]sulphate. Two long (16 and 33 days) and three short (3–3.5 days) experiments were made. Entities containing carbohydrate were isolated from the glandular stomach, upper, middle and lower parts of the small intestine and the colon. For comparison, glycosaminoglycans from costal cartilage (chondroitin 4(6)-sulphates) and from skin (dermatan sulphate) were isolated in two of the short experiments. It was noted that, in the gastrointestinal tract, there were large amounts of saccharides which originally belonged to the group of epithelial glycoproteins. These saccharides were composed principally of D-glucosamine, D-galactosamine, L-fucose, D-galactose, sialic acid and sulphate and some also contained D-mannose, D-glucose, L-arabinose and D-xylose, but no uronic acids were detected. The sulphated forms of saccharides were separated from neutral forms by anion-exchange chromatography, and their turnover rates were estimated. The biological half-lives were very short, approximately 14–24 h. The apparent reason for this is that they were secreted into the gut, and in this case the half-life merely reflects the secretion rate of a polysaccharide. In the gastrointestinal tract were also found typical connective tissue polysaccharides, which contain uronic acid, such as hyaluronic acid, heparan sulphate, chondroitin 4-sulphate and dermatan sulphate. The results of the long experiments indicated that the loss of sulphate from the polysaccharides was not constant. The biological half-lives varied considerably, depending on the period of time after sulphate injection during which the estimations were performed. On the other hand, the results of the short experiments indicated that sulphation of polysaccharides begins immediately after the injection of sulphate, and maximal labelling was usually achieved 6–8 h after sulphate injection. Thereafter there were periods of fast decrease in the radioactivity of polysaccharides and periods of slower decrease or even increase in radioactivity. The turnover rates of chondroitin 4-sulphate in costal cartilage and of dermatan sulphate in the skin were also estimated, and similar phenomena were observed. The results were similar in all three experiments. It is difficult to explain these findings. There may be diurnal variations in the rate of biosynthesis of polysaccharides. There may be metabolic heterogenity due to the occurrence of different pools of subjected glycosaminoglycans. Finally the sulphate moieties may metabolise independently of the complete macromolecules to which they are attached.

The rotational diffusion of albumin in solutions of connective-tissue polysaccharides.

The rotational behaviour of dansyl‐labelled human serum albumin in solutions of connective tissue polysaccharides (a hyaluronic acid and a sulphated proteoglycan) has been studied by fluorescence depolarization measurements. Over a wide range of pH, including the physiological conditions, the presence of the polymers up to concentrations of 10 mg/ml has no effect on the relaxation time of the protein. At higher concentrations of the sulphated proteoglycan (20–30 mg/ml) a moderate suppression of the rotational diffusion is observed. At pH values below the isoionic point of the protein, a marked effect on the polarization occurs which is interpreted as an extensive associative interaction between the protein and the polysaccharide materials. A quantitative interpretation of the effect is attempted.