The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.     

Fluorescence method for the structural analysis of oligomannose-type sugar chains by partial acetolysis

Fluorescence labeling was used in the analysis of partial acetolysis products of oligomannose-type sugar chains with five to nine mannose residues. The principle of the method was the pyridylamination of fragments obtained by the partial acetolysis of pyridylamino sugar chains and the identification of the fragments with an HPLC apparatus equipped with a fluorescence spectrophotometer. The method was tested by analysis of eight oligomannose-type sugar chains with known chemical structures and was found to be effective for analysis of branching structures with samples of 0.5 nmol.     

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.     

Decorin inhibits cell migration through a process requiring its glycosaminoglycan side chain.

Several studies overwhelmingly support the notion that decorin (DCN) is involved in matrix assembly, and in the control of cell adhesion and proliferation. However, nothing is known about the role of DCN during cell migration. Cell migration is a tightly regulated process which requires both adhesion (at the leading edge of the cell) and de-adhesion (at the trailing edge of the cell) from the substratum. We have determined in this study the effect of DCN on MG-63 osteosarcoma cell migration and have analyzed whether its effect is mediated by the protein core and/or the glycosaminoglycan side chain. DCN impeded the migration-promoting effect of matrix molecules (fibronectin, collagen type I) known to interact with the proteoglycan. Conversely, DCN did not counteract the migration-promoting effect of fibrinogen lacking proteoglycan affinity. DCN bearing dermatan-sulfate chains (i.e., skin and cartilage DCN) was about 20-fold more effective in inhibiting cell migration than DCN bearing chondroitin-sulfate chains (i.e., bone DCN). In addition, chondroitinase AC-treatment of cartilage DCN (which specifically removes chondroitin-sulfate chains) did not attenuate the inhibitory effect of this proteoglycan, while cartilage DCN deprived of both chondroitin- and dermatan-sulfate chains failed to alter cell migration promoted by either fibronectin or its heparin- and cell-binding domains. These data assert that the dermatan-sulfate chains of DCN are responsible for a negative influence on cell migration. However, isolated glycosaminoglycans failed to alter cell migration promoted by fibronectin, indicating that strongly negatively charged glycosaminoglycans alone cannot account for the impaired cell motility seen with DCN. Overall, these results show that the inhibitory action of DCN is dependent of substratum binding, is differentially mediated by its glycosaminoglycan side chains (chondroitin-sulfate vs. dermatan-sulfate chains), and is independent of a steric hindrance effect exerted by its glycosaminoglycan side chains.     

Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages.

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536--544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.     

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

Effect of endothelial-cell-conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.     

Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes.

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.     

Nonlysosomal processing of cell-surface heparan sulfate proteoglycans. Studies of I-cells and NH4Cl-treated normal cells

The metabolism of cell-associated proteoglycans, labeled in the glycosaminoglycan portion with 35SO2-4, was studied in normal skin fibroblasts (SL66 cells), NH4Cl-treated SL66 fibroblasts, and in I-cells derived from patients with mucolipidosis II. Kinetic data from label-chase experiments and gel filtration analysis of the molecular weight distribution of the radiolabeled glycosaminoglycans indicated that I-cells and NH4Cl-treated normal fibroblasts (a) internalize cell surface proteoglycans, (b) remove glycosaminoglycan chains from proteoglycan core proteins, and (c) degrade heparan sulfate glycosaminoglycan chains via an endoglycosidic activity. These processes occur with rates comparable to those in normal fibroblasts. The data are consistent with the hypothesis that the glycosaminoglycan chains of cell-surface proteoglycans are separated from the protein cores in a nonlysosomal compartment prior to the transport of these chains to lysosomes for degradation. These observations also raise the possibility that this early step in separation of glycosaminoglycan chains from protein cores may serve to regulate the levels of glycosaminoglycan-free core protein observed in various cells.     

The influence of ovarian steroids on ovine endometrial glycosaminoglycans

The ovine endometrium is subjected to cyclic oscillations of estrogen and progesterone in preparation for implantation. One response to fluctuating hormonal levels is the degree of hydration of the tissue, suggesting cyclical alterations in glycosaminoglycan/proteoglycan content. The aim of the present study was to quantitate and characterize glycosaminoglycans in the ovine endometrium during estrogen and progesterone dominant stages. Endogenous endometrial glycosaminoglycan content was determined by chemical analysis and characterized by enzyme specific or chemical degradation. [(35)S]-sulphate and [(3)H]-glucosamine labeled proteoglycans/glycosaminoglycans were extracted by cell lysis or with 4M guanidine-HCl. Extracts were purified by anion exchange and gel chromatography and characterized as above. Estrogen and progesterone dominant endometrium contained 3.2 +/- 0.1 and 2.1 +/- 0.1 mg endogenous glycosaminoglycan/g dehydrated tissue, respectively. Characterization of endogenous glycosaminoglycan showed chondroitin sulphate and hyaluronan contributing over 80%. The major difference between hormonal dominant tissue was a higher estrogenic hyaluronan percentage and a higher progestational keratan sulphate percentage (p < 0.001). Estrogen dominant tissue incorporated 1.6-1.9 fold more radiolabeled proteoglycans/glycosaminoglycans (p < 0.001). Analysis of newly synthesized proteoglycans/glycosaminoglycans revealed a heparan/chondroitin sulphate ratio of 1:2.2-2.5. Keratan sulphate was not detected. Estrogenic hyaluronan was 1.6 fold greater in [(3)H]-labeled tissue. Analysis of labeled proteoglycans/glycosaminoglycans revealed two size classes with apparent molecular weights >2.0 x 10(6) and 0.8-1.1 x 10(5) and a charge class eluting between 0.1-0.5 M NaCl. The greater glycosaminoglycan content (particularly hyaluronan) and synthesis in estrogen dominant tissue supports a role for steroid hormones in endometrial glycosaminoglycan/proteoglycan regulation and consequent tissue hydration. It also suggests a role for these macromolecules in endometrial function and possibly the implantation process.     

Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.     

Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A

Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex. We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains. BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain. In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA. The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events. Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment.     

Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan

Perlecan, the predominant basement membrane proteoglycan, has previously been shown to contain glycosaminoglycans attached at serine residues, numbers 65, 71, and 76, in domain I. However, the C-terminal domains IV and V of this molecule may also be substituted with glycosaminoglycan chains, but the exact substitution sites were not identified. The amino acid sequence of mouse perlecan reveals many ser-gly sequences in these domains that are possible sites for glycosaminoglycan substitution. We expressed recombinant domain IV and/or V of mouse perlecan in COS-7 cells and analyzed glycosaminoglycan substitution. Both heparan sulfate and chondroitin sulfate chains could be detected on recombinant domain V. One site, ser-gly-glu (serine residue 3593), toward the C-terminal region of domain V is a substitution site for heparan sulfate. When this sequence was absent, chondroitin/dermatan sulfate substitution was deleted, and the likely site for this galactosaminoglycan substitution was ser-gly-ala-gly (serine residue 3250) on domain V.     

Synthesis of aberrant glycosaminoglycans during cartilage culture in 'sulfate free' medium

Incorporation of radiolabeled sulfate into glycosaminoglycans is a widely accepted assay to measure the rate of proteoglycan synthesis. Although glycosaminoglycan synthesis is dependent on the quantity of inorganic sulfate available to proteoglycan synthesizing cells, 'sulfate free' medium is regularly used in studies regarding proteoglycan synthesis. In this study murine patellar cartilage glycosaminoglycans synthesized under 'sulfate free' conditions were compared with those synthesized at physiological sulfate concentration. Under 'sulfate free' conditions synthesis was not only decreased but low sulfated glycosaminoglycans were made that were not synthesized during incubation at physiological sulfate concentration. The use of 'sulfate free' medium should be avoided in proteoglycan synthesis studies.     

Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages

Peritoneal macrophages from nude mice were found to be functionally similar to 'activated' macrophages from normal mice. The objective of the present study was to characterize the proteoglycans synthesized and secreted in vitro by peritoneal macrophages isolated from nude and normal Balb/c mice and to investigate the relationship between macrophage 'activation' and changes in the proteoglycan patterns. Macrophages obtained by peritoneal lavage were seeded in Petri dishes. After 2 h incubation at 37 degrees C, the adherent cells (macrophages) were exposed to [35S]sulphate for the biosynthetic labelling of proteoglycans. After incubation, the cell and medium fractions were collected and analysed for proteoglycans and glycosaminoglycans. The glycosaminoglycans were identified and characterized by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. It was shown that 3/4 of the total 35S-labelled glycosaminoglycans were in the extracellular compartment after 24-48 h. The macrophages synthesized dermatan sulphate (68%), chondroitin sulphate (7%) and heparan sulphate (25%). Both cell and medium fractions of normal and nude mouse macrophages contained glycosaminoglycans with the same ratios, although the nude mouse macrophages synthesized 2-fold less glycosaminoglycans than the normal mouse macrophages. Lower levels of 35S-proteoglycans were also obtained from in vitro 'activated' macrophages, but the ratios of dermatan sulphate:chondroitin sulphate: heparan sulphate were altered in these cells as compared to the control. Furthermore, all the 35S-macromolecules found in the extracellular compartment of nude and normal control cells were of proteoglycan nature, in contrast to the medium fractions of 'activated' macrophages, which contain both intact proteoglycans and 'free' glycosaminoglycan chains. These results indicate that, at least as regards the proteoglycans and glycosaminoglycans, the nude mouse macrophages are not identical to the 'activated' macrophages from normal mice.     

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.     

The spacing of S-domains on HS glycosaminoglycans determines whether the chain is a substrate for intracellular heparanases

Heparanases are mammalian endoglucuronidases that degrade heparan sulfate (HS) glycosaminoglycans to short 5-6 kDa pieces. In the Golgi, HS glycosaminoglycans are modified by a series of interdependent reactions which result in chains that have regions rich in N- and O-sulfate groups and iduronate residues (S-domains), separated by regions that are nearly devoid of sulfate. Structural analysis of the short HS chains produced by Chinese hamster ovary (CHO) cell heparanases indicate that the enzymes recognize differences in sulfate content between S-domains and unmodified sequences, and cleave the chain at junctions between these regions. To look more closely at whether the spacing of S-domains on the gly- cosaminoglycan influences its ability to be cleaved by heparanases, we examined the susceptibility of the HS chains synthesized by the proteoglycan synthesis mutant, pgsE-606. PGS:E-606 cells are deficient in the modification enzyme N-deacetylase/N-sulfotransferase I, and synthesize HS chains that have fewer N- and O-sulfate groups and iduronate residues compared to wild-type (Bame et al., (1991), J. Biol. Chem., 266, 10287). HS glycosaminoglycans were isolated from wild-type and pgsE-606 cells and separated into populations based on sulfate content. Compared to wild-type HS, which has 14 S-domains, pgsE-606 cells synthesize three HS species, 606-1, 606-2, and 606-3, with 1, 4, and 8 S-domains, respectively. The spacing of the S-domains on the pgsE-606 HS chains is similar to the spacing the modified sequences on wild-type HS, indicating that each mutant glycosaminoglycan is composed of wild-type-like sequences and sequences devoid of S-domains. When incubated with partially purified CHO heparanases, only the portion of the mutant HS chains that had S-domains were degraded. Structural analysis of the heparanase-products confirmed that both the number and the arrangement of S-domains on the HS glycosaminoglycan are important for heparanase susceptibility. The structure of the different pgsE-606 HS chains also suggests mechanisms for the placement of S-domains when the gly- cosaminoglycan is synthesized.     

Characterization and N-terminal sequence of a heparan sulphate proteoglycan synthesized by endothelial cells in culture.

We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains.     

Biochemical and functional characterization of proteoglycans isolated from basophils of patients with chronic myelogenous leukemia

Human basophils were obtained from three donors with myelogenous leukemia. Proteoglycans were labeled by using [35S]sulfate as precursor and were extracted in 1 M NaCl with protease inhibitors to preserve their native structure. [35S]proteoglycans filtered on Sepharose 4B with an average m.w. similar to that of a rat heparin proteoglycan that has an estimated m.w. of 750,000. The [35S]glycosaminoglycan side chains filtered with an average m.w. slightly smaller than a 60,000-m.w. glycosaminoglycan marker. The [35S]glycosaminoglycans were resistant to heparinase and susceptible to degradation by chondroitin AC lyase and chondroitin ABC lyase. The intact [35S]glycosaminoglycans chromatographed on DEAE Sepharose as a single peak eluting just before an internal heparin marker. These findings indicate that the [35S]glycosaminoglycans were made up only of chondroitin sulfates. No heparin was identified. The chondroitin sulfate disaccharides that resulted from the action of chondroitin ABC lyase on the basophil glycosaminoglycans consisted of 92% delta Di-4S, 6% delta Di-6S, and 2% disulfated disaccharides. The [35S]chondroitin sulfate proteoglycans were susceptible to cleavage with proteases and could be shown to be released intact from basophils during degranulation initiated by the calcium ionophore A23187. The basophil proteoglycans and glycosaminoglycans were capable of binding histamine in water, but not in phosphate-buffered saline, and had no anticoagulant activity.     

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.