Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin.

Rat skin heparin proteoglycans vary markedly in the proportions of their constituent polysaccharide chains that have high binding affinity for antithrombin. As the proportion of such chains in a proteoglycan rises, their degree of affinity for antithrombin also increases [Horner (1987) Biochem. J. 244, 693-698]. The antithrombin-binding-site densities of such chains have now been determined, by measuring heparin-induced enhancement of the intrinsic fluorescence of antithrombin and by chemical analysis for the disaccharide sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate), which is unique to this site in heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555]. Antithrombin-binding-site density ranged from one to five sites per chain.     

Heterogeneity of rat skin heparin chains with high affinity for antithrombin.

Subfractions of 35S-labelled rat skin heparin proteoglycans with various degrees of high affinity for antithrombin were obtained by gradient elution from a column of antithrombin-agarose. Heparin chains released from the proteoglycan preparations by beta-elimination with alkali were re-fractionated on the same column. Proportions of chains with high affinity for antithrombin (HA-chains) ranged from 17% to 76%. These separations also revealed three overlapping subfractions of HA-chains. Their proportions varied in a manner consistent with a stepwise increase in the degree of affinity of HA-chains for antithrombin, this presumably being due to the biosynthesis of increasing numbers of antithrombin-binding sites per chain. The anticoagulant activity, with respect to thrombin neutralization, ranged from 32 units/mg to 287 units/mg. It is suggested that HA-chains may have from one to five or six antithrombin-binding sites. Thus the asymmetric distribution of these sites in rat skin heparin proteoglycans is much more marked than was realized from the earlier work of Horner & Young [(1982) J. Biol. Chem. 257, 8749-8754].     

Location of antithrombin-binding regions in rat skin heparin proteoglycans.

Rat skin heparin proteoglycan labelled biosynthetically with 35S was fractionated on a column of antithrombin-Sepharose into fractions with varying degrees of affinity for antithrombin. These were treated with NaOH to release heparin chains (Mr 60,000-100,000), by beta-elimination or incubated with serum to produce fragments of the same order of size as commercial heparin (Mr 5000-30,000), by endoglycosidase cleavage. Chains and fragments were then fractionated on antithrombin-Sepharose. The various fractions were deaminated with HNO2 at pH 1.5 followed by reduction with NaB3H4. Approx 90% of the incorporated 3H was associated with disaccharides. These were fractionated by high-performance ion-exchange chromatography. A unique minor component corresponding to the sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate) in the polysaccharide was found only in fractions with high affinity for antithrombin. The glucosamine residue linked to C-4 of this glucuronosyl unit was predominantly (or exclusively) N-sulphated rather than N-acetylated, pointing to a structural difference between the antithrombin-binding region of rat heparin and that of pig mucosal heparin. Calculations based on the distribution of the glucosaminyl 3-O-sulphate group showed that approximately two-thirds of the total antithrombin-binding regions present in the unfractionated material were accommodated by only 20% of the proteoglycan molecules, and by 10% of the polysaccharide chains. While most of the proteoglycan molecules thus lacked such regions (and hence affinity for antithrombin) a minor proportion of the polysaccharide chains contained on the average three binding regions per molecule. These findings support by direct chemical analysis an earlier proposal, based on anticoagulant activities of similar rat skin heparin fractions, that the distribution of antithrombin-binding sites in intact heparin proteoglycans is markedly non-random.     

Rat heparins. A study of the relative sizes and antithrombin-binding characteristics of heparin proteoglycans, chains and depolymerization products from rat adipose tissue, heart, lungs, peritoneal cavity and skin.

35S-labelled heparins were recovered from adipose tissue, hearts, lungs, peritoneal cavities and skins of rats given H2(35)SO4. Their purification involved incubation with Pronase, precipitation with cetylpyridinium chloride in 1.0 M-NaCl, gradient elution from DEAE-Sephacel and incubation with chondroitinase ABC. Each product was divided into proteoglycan and "depolymerization products' fractions by gel filtration on Bio-Gel A-15m. Heparin chains were released from a portion of each proteoglycan fraction by beta-elimination with NaOH. Proteoglycans, chains and depolymerization products were separated by gradient elution from a column of antithrombin-agarose into fractions with no affinity, low affinity and high affinity for antithrombin. The relative sizes of the products were determined by gel filtration on columns of Bio-Gel A-50m, A-15m, A-1.5m and A-0.5m. Skin was the major source of heparin and contained the largest proteoglycans and the lowest proportion of depolymerization products. Lungs contained the smallest proteoglycans, the smallest depolymerization products and the highest proportion of depolymerization products. The highest proportions of proteoglycans, chains and depolymerization products with high affinity for antithrombin were found in adipose tissue. The lowest proportions of each of these fractions were found in the peritoneal cavity. The data suggest that there was relatively little biosynthesis of sites with high affinity for antithrombin in peritoneal-cavity mast cells and that heparin catabolism was most active in lungs. Each source of heparin was unique with respect to both biosynthesis and subsequent breakdown of its proteoglycans.     

[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages.]

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.     

Rat heparan sulphates. A study of the antithrombin-binding properties of heparan sulphate chains from rat adipose tissue, brain, carcase, heart, intestine, kidneys, liver, lungs, skin and spleen.

Adult male rats were given [35S]sulphate intraperitoneally. Heparan [35S]sulphate (HS) chains were recovered from adipose tissue, brain, carcase, heart, intestine, kidneys, liver, lungs, skin and spleen by digestion with Pronase, precipitation with cetylpyridinium chloride, digestion with chondroitin ABC lyase and DNAase and gradient elution from DEAE-Sephacel. Purity was confirmed by agarose-gel electrophoresis and degradation with HNO2. Fractionation by gradient elution from antithrombin-agarose indicated that the proportion of HS with high binding affinity for antithrombin (HA-HS) ranged from 4.7% (kidneys) to 21.5% (brain). On a mass basis the major sources of HA-HS were carcase, skin and intestine. HA-HS from intestine was arbitrarily divided into subfractions I-VI, with anticoagulant activities ranging from 1 to 60 units/mg [by amidolytic anti-(Factor IIa) assay] and from 4 to 98 units/mg [by amidolytic anti-(Factor Xa) assay], indicating that the antithrombin-binding-site densities of HA-HS chains covered a wide range, as shown previously for rat HA-heparin chains [Horner, Kusche, Lindahl & Peterson (1988) Biochem. J. 251, 141-145]. HA-HS subfractions II, IV and VI were mixed with samples of HA-[3H]heparin chains and rechromatographed on antithrombin-agarose. Affinity for matrix-bound antithrombin did not correlate with anticoagulant activity, e.g. HA-HS subfraction IV [38 anti-(Factor Xa) units/mg] was co-eluted with HA-heparin chains [127 anti-(Factor Xa) units/mg].     

Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor Xa

Oligosaccharides (10-20 monosaccharide units) with high affinity for antithrombin, as well as larger high-affinity heparin fractions (having relative molecular masses between 6,000 and 21,500), all markedly accelerated the inhibition of Factor Xa by antithrombin. Moreover, all high-affinity oligosaccharides and heparins enhanced, to a similar extent, the amount of free proteolytically modified antithrombin cleaved at the reactive bond by Factor Xa. In contrast, a minimum high-affinity heparin size of approximately 18 monosaccharide units was required to significantly accelerate the inactivation of thrombin by antithrombin and to enhance the production of modified antithrombin by this enzyme. All high-affinity fractions studied had similar affinities for antithrombin, as determined by fluorescence titrations. In competition experiments, binary complexes of antithrombin with octadecasaccharide or larger high-affinity heparins, but not with smaller oligosaccharides, displaced inactivated 125I-thrombin from matrix-linked low-affinity heparin. Moreover, similar binary complexes with 3H-labeled octadecasaccharide or larger chains, but not with smaller oligosaccharides, were capable of binding to matrix-linked inactivated thrombin. These results indicate that simultaneous binding of antithrombin and thrombin to high-affinity heparin is a prerequisite to the acceleration of the antithrombin-thrombin reaction and that the minimum heparin sequence capable of binding both proteins comprises approximately 18 monosaccharide units. Similar complex formation apparently is not required for the acceleration of the antithrombin-Factor Xa reaction.     

The molecular-weight-dependence of the anti-coagulant activity of heparin.

It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

Isolation and characterization of the heparan sulfate proteoglycans of brain. Use of affinity chromatography on lipoprotein lipase-agarose

Heparan sulfate proteoglycans were extracted from rat brain microsomal membranes or whole forebrain with deoxycholate and purified from accompanying chondroitin sulfate proteoglycans and membrane glycoproteins by ion-exchange chromatography, affinity chromatography on lipoprotein lipase-Sepharose, and gel filtration. The proteoglycan has a molecular size of approximately 220,000, containing glycosaminoglycan chains of Mr = 14,000-15,000. In [3H]glucosamine-labeled heparan sulfate proteoglycans, approximately 22% of the radioactivity is present in glycoprotein oligosaccharides, consisting predominantly of N-glycosidically linked tri- and tetraantennary complex oligosaccharides (60%, some of which are sulfated) and O-glycosidic oligosaccharides (33%). Small amounts of chondroitin sulfate (4-6% of the total glycosaminoglycans) copurified with the heparan sulfate proteoglycan through a variety of fractionation procedures. Incubation of [35S]sulfate-labeled microsomes with heparin or 2 M NaCl released approximately 21 and 13%, respectively, of the total heparan sulfate, as compared to the 8-9% released by buffered saline or chondroitin sulfate and the 82% which is extracted by 0.2% deoxycholate. It therefore appears that there are at least two distinct types of association of heparan sulfate proteoglycans with brain membranes.     

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.     

A study of the interaction in vitro between type I collagen and a small dermatan sulphate proteoglycan.

The interaction between a small dermatan sulphate proteoglycan isolated from human uterine cervix and collagen type I from human and rat skin was investigated by collagen-fibrillogenesis experiments. Collagen fibrillogenesis was initiated by elevation of temperature and pH after addition of proteoglycan, chondroitinase-digested proteoglycan or isolated side chains, and monitored by turbidimetry. Collagen-associated and unbound proteoglycan was determined by enzyme-linked immunosorbent assay after aggregation was complete. (1) The binding of proteoglycan to collagen could be explained by the presence of two mutually non-interacting binding sites, with Ka1 = 1.3 x 10(8) M-1 and Ka2 = 1.3 x 10(6) M-1. The number of binding sites per tropocollagen molecule was n1 = 0.11 and n2 = 1.1. The 0.1 high-affinity binding site per tropocollagen molecule indicates that the strong interaction between proteoglycan and collagen results from a concerted action of tropocollagen molecules in fibrils. Digestion of the proteoglycan with chondroitinase ABC did not affect these binding characteristics. (2) Proteoglycan did not affect the rate of fibrillogenesis, but increased the steady-state A400 by up to 90%. This increase was directly proportional to the saturation of the high-affinity type of binding sites. Neither isolated core protein nor isolated side chains induced a similar high increase in steady-state A400. (3) Electron micrographs showed that the fibril diameter was affected only to a minor extent, if at all, by the proteoglycan, whereas bundles of laterally aligned fibrils were common in the presence of proteoglycan. (4) Results obtained with human and rat collagen were similar.     

Occurrence of three distinct molecular species of chondroitin sulfate proteoglycan in the developing rat brain

More than 60% of brain chondroitin sulfate proteoglycans were extracted from 10-day-old rat brains by homogenization in ice-cold phosphate-buffered saline containing protease inhibitors. Although the soluble proteoglycan preparation was a mixture of chondroitin sulfate proteoglycans with a different hydrodynamic size as well as a different molecular density, each subfraction of the proteoglycans contained chondroitin sulfate side chains with virtually identical molecular weight (approximately 15,000) and chondroitin sulfate disaccharide composition (high content of 4-sulfate unit). Digestion of the purified proteoglycan preparation with protease-free chondroitinase ABC produced five core proteins with Mr = 250,000 (designated as 250K protein), 220,000 (220K), 150,000 (150K), 130,000 (130K), and 93,000 (93K). All these core proteins were obtained from chondroitin sulfate proteoglycan preparations extracted from various regions of the brain, but their composition varied among different brain regions. Analysis for amino acid composition of these core proteins and two-dimensional mapping of their proteolytic peptides revealed that three major core proteins (250K, 220K, and 150K proteins) were structurally different. These observations indicate that at least three distinct types of chondroitin sulfate proteoglycan occur in the developing rat brain.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Processing of macromolecular heparin by heparanase

Heparanase is an endo-glucuronidase expressed in a variety of tissues and cells that selectively cleaves extracellular and cell-surface heparan sulfate. Here we propose that this enzyme is involved also in the processing of serglycin heparin proteoglycan in mouse mast cells. In this process, newly synthesized heparin chains (60-100 kDa) are degraded to fragments (10-20 kDa) similar in size to commercially available heparin (Jacobsson, K. G., and Lindahl, U. (1987) Biochem. J. 246, 409-415). A fraction of these fragments contains the specific pentasaccharide sequence required for high affinity binding to antithrombin implicated with anticoagulant activity. Rat skin heparin, which escapes processing in vivo, was used as a substrate in reaction with recombinant human heparanase. An incubation product of commercial heparin size retained the specific pentasaccharide sequence, although oligosaccharides (3-4 kDa) containing this sequence could be degraded by the same enzyme. Commercial heparin was found to be a powerful inhibitor (I50 approximately 20 nM expressed as disaccharide unit, approximately 0.7 nM polysaccharide) of heparanase action toward antithrombin-binding oligosaccharides. Cells derived from a serglycin-processing mouse mastocytoma expressed a protein highly similar to other mammalian heparanases. These findings strongly suggest that the intracellular processing of the heparin proteoglycan polysaccharide chains is catalyzed by heparanase, which primarily cleaves target structures distinct from the antithrombin-binding sequence.     

Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan

Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.     

Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts.

1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmstr?m, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.     

Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.     

Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline