Inhibition by heparin-modulated antithrombin III of amidolysis catalysed by m beta-acrosin.

Purified m beta-acrosin catalysed amidolysis of several p-nitroanilides with C-terminal arginine residues. Antithrombin III inhibited amidolysis catalysed by the enzyme. This effect of antithrombin III was potentiated by heparin, and to a modest extent by heparan sulphate, cellulose sulphate, dextran sulphate and xylan sulphate. De-N-sulphated heparin, de-N-sulphated N-acetylated heparin, heparin of low relative molecular mass, chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were ineffective.     

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycansubstrates was examined using viscosimetric measurements andgradient polyacrylamide gel electrophoresis (PAGE). Heparinlyase I (heparinase, EC 4.2.2.7 [EC] ) and heparin lyase II (no ECnumber) both acted on heparin in a random endolytic fashion.Heparin lyase II showed an ideal endolytic action pattern onheparan sulphate, while heparin lyase I decreased the molecularweight of heparan sulphate more slowly. Heparin lyase III (heparitinase,EC 4.2.2.8 [EC] ) acted endolytically only on heparan sulphate anddid not cleave heparin. Chondroitin ABC lyase (chondroitinaseABC, EC 4.2.2.4 [EC] ) from Proteus vulgaris acted endolytically onchondroitin-6-sulphate (chondroitin sulphate C) and dermatansulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate(chondroitin sulphate A) at a reduced rate, decreasing its molecularweight much more slowly. Two chondroitin AC lyases (chondroitinaseAC, both EC 4.2.2.5 [EC] ) were examined towards chondroitin-4- and-6-sulphates. The exolytic action of chondroitin AC lyase Afrom Arthrobacter aurescens on both chondroitin-4- and -6-sulphateswas demonstrated viscosimetrically and confirmed using bothgradient PAGE and gel permeation chromatography. ChondroitinAC lyase F from Flavobacterium heparinum (Cytophagia heparinia)acted endolytically on the same substrates. Chondroitin B lyase(chondroitinase B, no EC number) from F.heparinum acted endolyticallyon dermatan sulphate giving a nearly identical action patternas observed for chondroitin ABC lyase acting on dermatan sulphate. action pattern chondroitin lyase glycosaminoglycan heparin lyase.     

The effect of preganglionic stimulation on the incorporation of l-[U-14C]valine into the protein of the superior cervical ganglion of the guinea pig

The acid glycosaminoglycans were extracted from the skins of young rats less than 1 day post partum. The isolated products were fractionated by a cetylpyridinium chloride-cellulose column technique and identified by chemical analysis, electrophoretic mobility and susceptibility to testicular hyaluronidase digestion. Hyaluronic acid (56%) dermatan sulphate (15.6%) and chondroitin 6-sulphate (9.1%) were the major components, but chondroitin 4-sulphate, heparan sulphate and heparin were also present, together with two further fractions tentatively suggested to be a heparan sulphate-like fraction and a dermatan sulphate fraction, both of short chain length or low degree of sulphation.     

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels.The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [³H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [³H] acetate.Perfusion of lymph nodes with [³H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24[emsp4 ]h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19[emsp4 ]h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4–5×10⁵[emsp4 ]D; heparan sulfate proteoglycan was 2.8×10⁴ to 9.4×10⁵[emsp4 ]D; heparin 7.9×10⁴[emsp4 ]D and chondroitin sulfate 1.3×10⁴[emsp4 ]D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation.It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.     

Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils.

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48--61% chondroitin 4-sulphate, 20--30% dermatan sulphate, 10--15% heparin and 7--9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Selective binding of zinc ions to heparin rather than to other glycosaminoglycans.

The relative binding affinity of Zn2+ to several glycosaminoglycans was determined by gel-filtration chromatography. Binding was observed only between Zn2+ and heparin. No binding was observed between Zn2+ and chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate of hyaluronic acid. All of the glycosaminoglycans contained carboxy groups, but only heparin bound Zn2+. This observation suggests that, contrary to a previously proposed hypothesis, simple electrostatic interactions between the negatively charged carboxy groups of the glycosaminoglycans and the positively charged Zn2+ cannot explain the observed binding.     

The influence of the type of sulphate bond and degree of sulphation of glycosaminoglycans on their interaction with lysosomal enzymes.

Significant differences occur between the interaction of several sulphated glycosaminoglycans with a particular lysosomal protein, leading to inhibition in the case of lysosomal enzymes. The order of strength of inhibition at pH4 was: heparin greater than chondroitin 4-sulphate = chondroitin 6-sulphate greater than dermatan sulphate.     

Optical properties of Cu(II) complexes with heparin and related glycosaminoglycans.

Absorption and circular dichroism measurements have been carried out to obtain information regarding the stability and the nature of complexes between Cu(II) and heparin, and between Cu(II) and related glycosaminoglycans. In the presence of Cu(II), all glycosaminoglycans, except keratan sulfate, show a characteristic absorption band near 237 nm, which we assign to charge-transfer bands involving ligands to metal ion. From the absorbance values, the formation constants of Cu(II)-heparin and Cu(II)-(itN)-desulfated heparin have been determined to be approximately 1 × 10⁴ and 2 × 10² mol^?1, respectively. The large difference in the stability constant values is attributed to the difference in the charge density of the polymers, and to involvement of more than one ligand in the case of heparin. The CD characteristics of the Cu(II)-heparin complex suggest that both carboxyl and sulfamino groups are involved as ligands. The appearance of extrinsic CD bands in heparin, heparan sulfate, dermatan sulfate, and N-desulfated heparin at pH > 5 is ascribed to asymmetry of chelate rings. Absence of CD change in chondroitin sulfate and N-desulfated heparin (pH < 5) in the presence of Cu(II) suggests that only the carboxyl group is involved in those complexes. The differences either in iduronic acid conformation (C-1 vs. 1-C) or in intersaccharide linkages between dermatan sulfate and heparin (or heparan sulfate) are revealed in the difference CD spectra between the complexes and the polymers. The change in the intrinsic Cotton effect on complex formation is accounted for as a change in spatial orientation of the ligand groups rather than as a major conformational change of the polymers.     

Heparin binds to Leishmania donovani promastigotes and inhibits protein phosphorylation.

We show that promastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar), possess heparin receptors on their surface. From a linear Scatchard plot of the binding data obtained using [3H]heparin and viable promastigotes, one derives a binding constant of 4.7 x 10(-7) M and an estimate of 860,000 receptors per parasite. The [3H]heparin bound to parasites could not be displaced by hyaluronic acid or by three other glycosaminoglycans (dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate). It was demonstrated that exponential phase promastigotes growing in medium 199 supplemented with fetal bovine serum incorporate 35SO4 into a cell-associated macromolecule that has the properties of heparin proteoglycan. Heparin inhibits the activity of the cell-surface histone-protein kinase; incubation of viable promastigotes with [gamma-32P]ATP and MgCl2 (10 mM) in the absence and presence of heparin (0.01-0.5 mg/ml) for 10 min, followed by analysis by SDS/polyacrylamide-gel electrophoresis and autoradiography, revealed that the phosphorylation of 12 or 13 parasite proteins was inhibited by the glycosaminoglycan. These data suggest that heparin may play a role in the host-parasite relationship.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

X-ray diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate (Short Communication)

Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating β-d-(1→4) and α-d-(1→4).     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

X-ray-diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate

Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating beta-d-(1-->4) and alpha-d-(1-->4).     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

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

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

Low-density lipoprotein binding affinity of arterial wall proteoglycans: characteristics of a chondroitin sulfate proteoglycan subfraction

The characteristics of an arterial wall chondroitin sulfate proteoglycan (CS-PG) subfraction that binds avidly to low-density lipoproteins (LDL) was studied. A large CS-PG was extracted from bovine aorta intima-media under dissociative conditions, purified by density-gradient centrifugation and gel filtration chromatography, and further subfractionated by affinity chromatography on LDL-agarose. A proteoglycan subfraction, representing 25% of the CS-PG, showed an elution profile (with dissociation from LDL-agarose occurring between 0.5 and 1.0 M NaCl) corresponding to that of heparin, heretofore considered to be the most strongly binding glycosaminoglycan with LDL. The proteoglycan subfraction which migrated as a single band on composite agarose-polyacrylamide gel electrophoresis contained chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in a proportion of 70:22:8. The core protein of the proteoglycan had an apparent molecular weight of 245,000, and contained approx. 33 glycosaminoglycan chains with an average molecular weight of 32,000. The CS-PG subfraction, like heparin, formed insoluble complexes in the presence of 30 mM Ca2+. Complexing of LDL with proteoglycan resulted in two classes of interactions with 0.1 and 0.3 proteoglycan monomer bound per LDL particle characterized by an apparent Kd of 4 and 21 nM, respectively. This indicates that multiple LDL particles bind to single proteoglycan monomers even at saturation. In contrast, LDL-heparin interactions showed a major component characterized by an apparent Kd of 151 nM and a Bmax of 9 heparin molecules per LDL particle. The occurrence of a potent LDL-binding proteoglycan subfraction within the family of arterial CS-PG may be of importance in terms of lipid accumulation in atherogenesis.     

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of proteoglycans. The development of proteoglycan glycotechnology to exploit the functionality of glycosaminoglycan chains is an extremely important aspect of glycobiology. Here we describe an efficient and widely applicable method for chemoenzymatic synthesis of conjugate compounds comprising intact long chondroitin sulfate (ChS) chains. An alkyne containing ChS was prepared by an enzymatic transfer reaction and linked with a chemically synthesized core compound containing an azido group using click chemistry. This method enabled highly efficient introduction of ChS into target materials. Furthermore, the ChS-introduced compounds had marked stability against proteolysis, and the chemically linked ChS chain contributed to the stability of these core compounds. We believe the present method will contribute to the development of proteoglycan glycobiology and technology.     

Analysis of plasma proteins that bind to glycosaminoglycans

Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-alpha-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties.     

Endothelial heparan sulphate: Compositional analysis and comparison of chains from different proteoglycan populations

From cultures of human umbilical vein endothelial cells incubated with³H-glucosamine or³⁵S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (>1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW>50,000) of low overall sulphation (32–35%N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.Abbreviations HSPG heparan sulphate proteoglycan - DSPG dermatan sulphate proteoglycan - GlcNAc(6S) N-acetylglucosamine 6-sulphate - GlcNAc6R glucosamine with either-OH or-OSO₃ at C-6 - GlcNR glucosamine with either-SO₃ or-COCH₃ as N-substituent - GlcNSO₃ N-sulphated glucosamine - GlcNSO₃(3S) N-sulphated glucosamine 3-sulphate - GlcA d-glucuronic acid - IdoA l-iduronic acid - IdoA(2S) iduronic acid 2-sulphate - HexA hexuronic acid - DHexA hexuronic acid with a 4,5-double bond - Xyl xylose - SAX strong anion exchange - d.p. degree of polymerization (a disaccharide has d.p.=1 etc) - AUFS absorbance units full scale The codes used for proteoglycans denote in turn: C 2, low-density (1.35–1.28 g/ml) HSPG from the cell extract; M 1a, high density (>1.4 g/ml) HSPG fraction from the spent medium; M 2a, low-density (1.31 g/ml) HSPG from the spent medium [6].