Biochemical composition and heterogeneity of heparan sulfates isolated from AH-130 ascites hepatoma cells and fluid

The glycosaminoglycan composition of AH-130 ascites hepatoma cells and fluid were examined using enzymatic digestion, electrophoresis, and sequential partition fractionation. The cell-associated glycosaminoglycans were found to consist of 93% heparan sulfate, with the remainder consisting primarily of chondroitin sulfate. The glycosaminoglycans isolated from the ascitic fluid were found to consist of 58% heparan sulfate, 26% hyaluronic acid and 16% chondroitin sulfate. Dermatan sulfate was not detected in either cells or fluid. The heparan sulfate isolated from AH-130 cells is low-sulfate and highly heterogeneous with respect to biochemical composition. Fractions isolated by partition fractionation varied from 0.14 mol sulfate/mol uronic acid to 0.6 mol sulfate/mol uronic acid. Of the total sulfate 70–80% is N-sulfate in the former and 50% in the latter. Electrophoresis in 0.1 M HCl showed a highly heterogeneous material with mobility between that of hyaluronic acid and beef lung heparan sulfate. The heparan sulfate isolated from the fluid was similar to that isolated from the cells but was, however, somewhat more homogeneous with respect to charge.     

Age-related changes in the content and composition of glycosaminoglycans isolated from the mouse skeletal muscle: normal and dystrophic conditions

Glycosaminoglycans were isolated from the skeletal muscle of either normal or dystrophic mice aged from 3 to 18 weeks. The glycosaminoglycan content of the normal muscle, based on the tissue weight, decreased slightly during the period from 3 to 10 weeks, and remained almost unchanged after 10 weeks. The major glycosaminoglycan in normal muscle was hyaluronate, the relative amount of which increased slightly (from 70% to 80%) with age. Both dermatan sulfate and heparan sulfate were also obtained. The relative amounts of these sulfated glycosaminoglycans tended to decrease with age. On the other hand, the glycosaminoglycan content of the dystrophic muscle was higher than that of normal muscle even at 3 weeks. The proportion of hyaluronate was almost constant (about 65%) throughout the age range examined. The relative amount of dermatan sulfate increased from 20% to 30% with a compensatory decrease in the amount of heparan sulfate. Further, the incorporation of [35S]sulfate into glycosaminoglycans by the dystrophic muscle was reduced to about 60% of the normal. These differences in glycosaminoglycan composition and [35S]sulfate incorporation between the normal and the dystrophic muscles may be related to the progressive muscular dysfunction seen in this disease.     

The preparation of heparan sulfate from the mitral valve of the human heart

A study has been made of the glycosaminoglycan composition of the mitral valve of the normal human heart. Five glycosaminoglycans were isolated from tryptic digest of the material and were assayed by determining the carbohydrate content. Separation of these five polymers was achieved by Dowex 1 X 2 column chromatography. They were identified as hyaluronic acid, heparan sulfate, chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively. As far as the authors are aware, this is the first isolation of heparan sulfate from the preparation of the mitral valve of the normal human heart.     

Heparin and heparan sulfate delimit nephron formation in fetal metanephric kidneys

Formation of nephrons from primitive mesenchyme in fetal kidneys is induced by ureteric buds. Nephron induction is closely coordinated with branching morphogenesis of the ureteric bud. Having previously shown that branching of the primitive ureter is associated with de novo synthesis of chondroitin sulfate proteoglycan and release of free heparan sulfate glycosaminoglycan chains, we asked whether glycosaminoglycans influence nephron development. Fetal mouse kidneys were incubated in organ cultures containing heparan sulfate, heparin, chondroitin sulfate, or hyaluronate. After 48 hr the number of nephrons at each developmental stage was enumerated by light microscopic analysis of serial tissue sections. Kidneys incubated in heparin or in heparan sulfate contained up to 10-fold fewer nephrons than did kidneys incubated in control conditions or in chondroitin sulfate or hyaluronic acid. Maturation of nephrons, however, was unaffected. Inhibition of nephron development was associated with binding of labeled heparin to primitive mesenchyme and altered tissue distribution of fibronectin. Branching morphogenesis was impaired in kidneys exposed to heparin but not to heparan sulfate or to de-N-sulfated, N-acetylated heparin. The capacity of glycosaminoglycans to inhibit nephron formation depended on sugar composition and O-sulfation but not GAG chain size or charge density. Thus, heparan sulfate may have the capacity to specifically control formation of nephrons in fetal metanephric kidneys in vitro.     

Composition of glycosaminoglycans in human pancreatic cancer

Five glycosaminoglycans were isolated from tryptic digestion of both cancerous and normal tissues of the human pancreas and were assayed by determining the carbohydrate content of materials. Separation of these five polymers was achieved by Dowex 1-X2 column chromatography and fractionation with Benedict's solution. They were identified as hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate, respectively. The total amount of glycosaminoglycans in cancer tissue increased in comparison to the controls. The increase in tissue content of glycosaminoglycans was accompanied by increases in chondroitin-4-sulfate and chondroitin-6-sulfate levels.     

Ninhydrin Positive Compounds Produced by Heating of Poly-l-amino Acids,Tripeptides and an Amino Acid Mixture

Glycosaminoglycans were isolated from bovine and human milk fat globule membrane (MFGM) preparations and characterized. The amount of glycosaminoglycans in human MFGM protein fraction was 5 ~ 10 times higher than that in bovine MFGM protein fraction. On Dowex-1 column chromatography most of the glycosaminoglycans were eluted with 1 M and 3 M NaCl. Cellulose acetate electrophoresis, chondroitinase digestion and nitrous acid degradation indicated that the major glycosaminoglycan in both bovine and human MFGM was heparan sulfate. Bovine MFGM was shown to contain chondroitin sulfate A/C in addition to heparan sulfate. The glycosaminoglycan composition of bovine colostral MFGM was similar to that of bovine normal MFGM. The glycosaminoglycan fraction prepared from MFGM of human colostrum, however, did not contain heparan sulfate, suggesting some compositional changes in glycosaminoglycans during the lactation period.     

A transformation-dependent difference in the heparan sulfate associated with the cell surface.

Glycosaminoglycans from the surface of cultured mouse cells (3T3, SV40-3T3, 3T6) were released by trypsin digestion and separated by ion-exchange chromatography into hyaluronic acid, heparan sulfate and chondroitin sulfate. Using a double label technique, the glycosaminoglycans from 3T3 cells were compared with those from SV40-3T3 and 3T6 cells. No differences were apparent in either the hyaluronic acid or chondroitin sulfate fractions, however, the heparan sulfate from 3T3 cells was found to elute from DEAE-cellulose at a higher ionic strength than that from transformed cells. This altered behavior implies a structural difference in the cell surface heparan sulfate which appears to be dependent upon transformation.     

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.     

Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with ³H-glucosamine and Na₂³⁵SO₄ and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.     

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.     

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.     

Identification and turnover of glycosaminoglycans in rat kidneys.

The turnover of sulfate label in crude glycosaminoglycan fractions from rat kidney cortex, medulla, and papilla has been determined. Heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronate have been separated electrophoretically and their specific activities determined after injection of labeled sulfate or glucose. The half-lives of the sulfated glycosaminoglycans are within the ranges found for other organs and tissues, but hyaluornate has a somewhat faster turnover in the kidney than elsewhere.     

Alterations in human gingival glycosaminoglycan pattern in inflammation and in phenytoin induced overgrowth

Glycosaminoglycans were extracted from normal, inflamed and phenytoin induced overgrowth of human gingival tissue by proteolysis and alcohol precipitation. Extracts were run in a Dowex-1 column and the fractions were treated with mucopolysaccharidases. Cellulose acetate electrophoresis was carried out with or without enzyme digestion for identification of individual glycosaminoglycans. Glycosaminoglycans were found to be decreased in inflammation but were observed to increase in the overgrowth. Hyaluronic acid was found to be increased in both the pathological conditions. Dermatan sulphate, chondroitin sulphate and heparan sulphate were observed to be decreased in inflammation. In overgrowth, dermatan sulphate and chondroitin sulphate were found to increase while the presence of heparan sulphate was not significant. The changes in the pattern of individual glycosaminoglycan in the two varied conditions are discussed.Abbreviations GAG glycosaminoglycan - MPS mucopolysaccharide - DS dermatan sulphate - HS heparan sulphate - CS chondroitin sulphate - HA hyaluronic acid - KS keratan sulphate     

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.     

Hormonal effect on glycosaminoglycans and glycoproteins in uteri of ovariectomized rats

1. Glycosaminoglycans such as chondroitin sulfate A (or C), chondroitin sulfate B (dermatan sulfate), heparitin sulfate (heparan sulfate) and hyaluronic acid were identified as major glycosaminoglycan components in whole uteri as well as in uterine stroma of rats. Two types of sialoglycoproteins with different electrophoretic mobilities (fast- and slow-migrating) were detected in the glycosaminoglycan fraction from the luminal epithelia. 2. Treatment of ovariectomized rats with estradiol-17beta markedly increased the uterine contents of glycosaminoglycans. Chondroitin sulfate A (or C) was found to increase more than chondroitin sulfate B. Furthermore, it was found that the estrogen treatment specifically increases the fast-migrating sialoglycoprotein level in the luminal epithelia and results in the appearance of it in the uterine fluid. 3. Administration of progesterone to ovariectomized rats slightly increased the uterine glycosaminoglycan content without appreciable alteration of the uterine glycosaminoglycan pattern.     

Glycosaminoglycans in the vitreous body of patients with retinal detachment.

The variation and changes of glycosaminoglycans in human vitreous body from patients with retinal detachment were studied. The isolated glycosaminoglycans from normal vitreous were identified as hyaluronate, which is the main component (92%) and chondroitin sulphate (8%). In contrast, in pathologic samples up to 18% of total glycosaminoglycans were identified as chondroitin.sulphate. In addition, in pathologic vitreous two fractions of glycosaminoglycans about 10% were identified as undersulphated chondroitin and heparan sulphate. The hydrodynamic size of hyaluronate differs between normal and pathologic samples. In samples from the patients with detached retinas the hyaluronic acid was of small hydrodynamic size.     

Glycosaminoglycans of brain during development.

The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development.     

Postnatal development of rat lung. Changes in lung lectin, elastin, acetylcholinesterase and other enzymes

Non-pregnant and pregnant rats of known gestational age were killed at intervals and their uterine cervices were excised and digested with papain. Glycosaminoglycans thus extracted were separated by cellulose acetate electrophoresis and stained with Alcian Blue. Glycosaminoglycans were identified by comparison with standards and by serial degradation with chondroitin ABC lyase, butyl nitrite and leech hyaluronidase. Dermatan sulphate, hyaluronic acid and heparan sulphate were identified and quantitative determined by densitometry. The overall concentration of glycosaminoglycans changed little during pregnancy. A 3-fold total increase in uronic acid paralleled the increase in cervical weight. Hyaluronate content, however, increased 17-fold, and rose from 6% of total glycosaminoglycans in the non-pregnant state to 33% at term. Furthermore, the ratio of hyaluronate to hydroxyproline increased 10-fold. These changes are consistent with an accumulation of hyaluronate in the interstices between collagen fibres, resulting in the softening of this tissue that is seen in late pregnancy.     

Dermatan sulfate is the major metachromatic glycosaminoglycan in the integument of the anuran Bufo ictericus

Glycosaminoglycans from the ventral and dorsal integuments of the anuran Bufo ictericus were characterized based on biochemical and histochemical methods. Dermatan sulfate is the major metachromatic glycosaminoglycan found in these tissues, but small amount of heparan sulfate was also detected. The average molecular mass of the dermatan sulfate is approximately 20 kDa, similar to the glycosaminoglycan isolated from mammalian skin. In addition, the amphibian integument contains high amounts of hyaluronic acid, especially in the ventral area. We also observed that the glycosaminoglycans occur in the anuran integument as irregular deposits through the spongious dermis and in the mast cells, as revealed by histochemical analysis using Alcian blue, dimethylmethylene blue and toluidine blue stains. The concentration and composition of glycosaminoglycans found in the amphibian integument resemble those from mammalian skin except for the higher concentration of hyaluronic acid in the amphibian tissue. Possibly, this observation indicates that the function of the sulfated glycosaminoglycan in these tissues has been preserved during evolution, although the amphibian integument and the human skin have their own particular physiology.     

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.