Glycosaminoglycan synthesis in the chick gastrula

Explanted definitive primitive streak to four somite chick embryos were labeled with [H³]glucosamine or S³⁵O₄ and the glycosaminoglycans were isolated and characterized. On the basis of susceptibility to Streptomyces hyaluronidase, which specifically degrades hyaluronic acid, hyaluronic acid is the major glycosaminoglycan produced by these embryos (at least 84%). On the basis of electrophoretic mobility, about 10% of the [H³]glucosamine-labeled glycoaminoglycan is sulfated. At least 55% of the sulfate-labeled glycosaminoglycan is sensitive to testicular hyaluronidase, and 36–39% is resistant to testicular hyaluronidase, but sensitive to nitrous acid treatment. About 94% of the labeled glycosaminoglycans can be accounted for in ratios of 22:1:5:1 as hyaluronic acid:chondroitin sulfate:heparan sulfate. No stage-related changes were observed. It is suggested that hyaluronic acid synthesis at this time might be related to the appearance of extensive cell-free spaces.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Change in the synthesis of glycosaminoglycans by fibrotic vitreous induced by erythrocytes

Vitreous fibrosis was induced in rabbit eyes by intravitreal injection of erythrocytes. The fibrotic vitreous removed from experimental animals were then incubated with [³H]glucosamine at 37°C for 24 h. The newly synthesized ³H-labeled glycosaminoglycans were isolated by 4 M guanidium hydrochloride extraction followed by pronase digestion. The ³H-labeled glycosaminoglycans were then characterized by gel filtration column chromatography and by specific enzymatic degradation, i.e., hyaluronidase, chondroitinase AC, and/or chondroitinase ABC. The disaccharides derived from chondroitinase ABC degradation were identified by thin-layer chromatography. We previously demonstrated that 91% of the total glycosaminoglycan synthesized by normal vitreous was hyaluronic acid. Our present results indicate that in the fibrotic vitreous, the synthesis of hyaluronic acid was decreased to 26%, whereas the synthesis of chondroitin sulfate increased to 59% of the total newly synthesized glycosaminoglycans. These results suggest that cells present in fibrotic vitreous resemble fibroblasts with respect to their activities in glycosaminoglycans synthesis.     

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.     

Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthesis.

The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in vitro. At levels of 10^?9 to 10^?8m retinoic acid, ³⁵SO₄ uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of ³⁵SO₄ or [¹⁴C]glucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased three- to fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin 6-sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of ³⁵SO₄ into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver

Nuclei isolated from rat liver were purified extensively and then subjected to extraction of glycosaminoglycans by the conventional method with a slight modification including the treatments with amylase nucleases, (DNAase and RNAase), and sialidase in addition to the pronase treatment. The nuclear glycosaminoglycan fraction thus prepared was subjected to chromatography on Dowex 1-X2 (Cl^?) and electrophoresis before or after digestion with specific enzymes such as Streptomyces hyaluronidase, chondroitinase ABC and AC. These results together with the results of chemical analyses have revealed that the purified nuclei from rat liver contain glycosaminoglycans equivalent to 0.2–0.3 μg hexuronic acid per mg DNA. A major component of the nuclear glycosaminoglycans has been identified as hyaluronic acid, while a minor component as chondroitin sulfate A (or C). Preliminary investigations have shown that most of the nuclear glycosaminoglycans are associated with the chromatin fraction.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

Active synthesis of glycosaminoglycans by liver parenchymal cells in primary culture

Rat liver parenchymal cells were evaluated after 2 days of primary culture for their ability to synthesize and accumulate heparan sulfate as the major component and low-sulfated chondroitin sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. The newly synthesized glycosaminoglycans secreted into the medium were different from those remaining with and/or on the cell layer. Low-sulfated chondroitin 4-sulfate, a major glycosaminoglycan in blood, was synthesized in the order of 320 μg/liver per day, more than 90% of which was secreted into the medium within 16 h and 40% of the glycan secreted was degraded during that time. On the other hand, heparan sulfate, the major glycosaminoglycan synthesized by the parenchymal cells, was mainly distributed in the cell layer. After 8 days of culture, the synthesis of glycosaminoglycans by the cells increased markedly, especially dermatan sulfate, chondroitin sulfate and hyaluronic acid.     

Changes in the organization and biosynthesis of cell surface acidic sugars during the phytohemagglutinin-induced blast formation of human T-lymphocytes

Use of cell electrophoresis combined with specific enzymes and varying ionic strength revealed a topological change of acidic sugars in lymphocyte membrane treated with a T-cell mitogen, phytohemagglutinin (PHA). The suggested alterations were an early translocation of hyaluronic acid to the cell periphery within 15 min of PHA addition and, 4 h later, the appearance of chondroitin sulphate in T-lymphocytes, but not in B-lymphocytes. As the contribution of chondroitin sulfate to the electrophoretic mobility increased with time up to 24 h, that of sialic acid decreased conversely. Several agents which block blast formation (2 mM ethylene glycol bis-β-aminoethylethyl-N,N,N′,N′-tetraacetic acid, 2 × 10⁻⁷ M ouabain, 0.1 μg/ml colchicine and 1 μg/ml cytochalasin B) also blocked the translocation of hyaluronic acid at the same concentrations. Chemical analysis of [¹⁴C]glycosaminoglycans by means of gel filtration followed by paper chromatography revealed a four-fold enhancement of the biosynthesis of chondroitin sulfate C after PHA stimulation. The presence of chondroitin sulfate in the cell periphery was also detected electrophoretically in T-cell type leukemia cells (MOLT-4B). These results suggest that the reorganization of glycosaminoglycans may be one of the membrane changes associated with blast formation of lymphocytes.     

The Influence of Hyaluronidase and Hyaluronidase Substrates on Penetration of Cultured Cells by Eimerian Sporozoites

SYNOPSIS Leighton tube cultures of bovine embryonic kidney cells were inoculated with Eimeria adenoeides sporozoites suspended in media containing either hyaluronidase, hyaluronidase substrates (chondroitin sulfate and hyaluronic acid) or Ficoll. After 1 hr at 41 C, coverslips were removed and cells were fixed and stained. Hyaluronidase (1 and 10 mg/ml) did not increase the number of intracellular sporozoites. Chondroitin sulfate (1 and 10 mg/ml) and hyaluronic acid (1 mg/ml) did not reduce the number of intracellular sporozoites. However, the number was reduced when the media contained either chondroitin sulfate (100 mg/ml) or hyaluronic acid (5 mg/ml), which were quite viscous.
Ficoll (117 mg/ml), which produced the same viscosity as 5 mg hyaluronic acid/ml, also reduced the number of intracellular sporozoites. This finding circumstantially indicates that sporozoites may be physically inhibited from entering cells by the high viscosity of the substrates.
Biochemical tests, which detected as little as 0.2 μg of known hyaluronidase, failed to detect hyaluronidase activity in excysted intact or fragmented E. adenoeides sporozoites or in sporozoites within E. tenella oocysts.     

Glycosaminoglycans in two mollusks, Aplysia californica and Helix aspersa, and in the leech, Nephelopsis obscura

The presence of glycosaminoglycans was examined in two mollusks (Pulmonates): the terrestrial garden snail, Helix aspersa, and the opishtobranchian sea slug, Aplysia californica and also in the leech (Hirudinea, Erpobdellidae, Nephelopsis obscura). Organs in the garden snail contained predominately chondroitin sulfate and heparan sulfate as a lesser component. The ctenidium of the sea slug contained mainly chondroitin sulfate and a compound which migrated on electrophoresis as heparin but additional data indicated that it could also represent a highly sulfated form of heparan sulfate. The foregut contained only the heparin-like polymer. No standard glycosaminoglycan could be identified in the leech although a polydispersed polysaccharide containing uronic acid, hexosamine and sulfate was shown to be present. A detailed analysis of the heparan sulfate isolated from the garden snail is also given.     

Cell cycle dependent rate of labelling of cellular and secreted glycosaminoglycans in mouse embryonic fibroblasts

Cultures of embryonic fibroblasts from Balb/c or CBA/J mice were given 12-h pulses of ¹⁴C-galactose, or were double-labelled with ³H-galactose and ³⁵H-sulfate. The time course of the rates of labelling of glycosaminoglycans – galactose label was found in the uronic acid moiety – was studied in synchronously and asynchronously growing cultures. Partial synchrony was achieved by trypsinising quiescent, confluent cells and subsequent transfer of cells to new cultures with fresh medium. Synchrony was monitored by measurement of thymidine uptake in parallel cultures. The distribution of label in the hyaluronic acid, chondroitin sulfate, and heparan sulfate fractions from cells and culture media was determined at each time point. Peaks of DNA synthesis were accompanied by or followed 12 h later by a maximal rate of labelling with galactose of secreted glycosaminoglycans, and – with the exception of hyaluronic acid – also of cellular glycosaminoglycans. The rate of labelling with galactose of glycosphingolipids in parallel cultures followed a different time course. In double-label experiments the rates of labelling of glycosaminoglycan sulfates with ³H-galactose and ³⁵S-sulfate did not go parallel. In older, quiescent cultures the labelling rate with galactose decreased while the sulfation rate increased. It is discussed that the labelling rate with galactose is indicative of the biosynthetic rate of the glycosaminoglycans. The conclusion is reached that glycosaminoglycans are preferentially synthesized and secreted after the S phase of the cell cycle.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Estimation of glycosaminoglycans by atomic absorption determination of copper in their alcian blue complexes

Glycosaminoglycans can be quantitated by determining the copper content of their alcian blue complexes. The use of this method is demonstrated with mixtures containing heparan sulfate, hyaluronic acid, and chondroitin sulfate after they have been resolved by cellulose acetate electrophoresis. Quantitation of alcian blue by atomic absorption is more sensitive than spectrophotometric techniques previously published. The method can be used to estimate the glycosaminoglycan composition in small amounts of tissue. This report demonstrates the use of this methodology in the quantitation of glycosaminoglycans in fetal and postnatal mouse brain and in the determination of the specific activities of glycosaminoglycans of fetal mouse brain labeled in vitro with [1-¹⁴C]glucosamine.     

Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy

Summary The ability of tannic acid to enhance binding of glycosaminoglycans to purified collagen was analysed in an in vitro system using amino sugar analysis on an amino acid analyser, transmission electron microscopy, and scanning electron microscopy. Collagen was purified by digestion with trypsin, papain, and hyaluronidase. Purified collagen was incubated with hyaluronic acid or with chondroitin sulphate glycosaminoglycan and then treated with tannic acid. Tannic acid was found to enhance retention during preparation for electron microscopy of either of the glycosaminoglycans onto collagen fibres. The ability of tannic acid to enhance binding of collagen and glycosaminoglycans might explain, at least in part, its structural reinforcement effect on resected synovial joint-apposing surfaces during preparation for scanning electron microscopy.     

Characterization of glycosaminoglycans in urine from patients with nephrotic syndrome and control subjects, and their effects on lipoprotein in lipase

Previously we found that the α₁-acid glycoprotein fraction from urine of patients with the nephrotic syndrome stimulated the lipoprotein lipase reaction in vivo and in vitro. The activator was separated from the α₁-acid glycoprotein and identified as a glycosaminoglycan. The studies reported here were undertaken to characterize and quantify the glycosaminoglycans contained in urine of patients with the nephrotic syndrome and to compare these to the glycosaminoglycans in urine of control subjects. We found that free low molecular weight glycosaminoglycans, heparan sulfate and chondroitin 4-sulfate, are excreted in both patients with the nephrotic syndrome and controls, however, patients with the nephrotic syndrome excreted much less of both glycosaminoglycans. The free form of heparan sulfate was found to be the activator which stimulated the lipoprotein lipase reaction in vitro in the presence of apolipoprotein C_II. In addition, the urine from patients with the nephrotic syndrome contained a protein-glycosaminoglycan complex which was absent in control urine. Glycosaminoglycans in the complex could be released by papain digestion or by trichloroacetic acid. Our evidence indicates that this glycosaminoglycan fraction is a low charge form of chondroitin sulfate.     

Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate

Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat.