Free Glycosaminoglycan Chains Research Articles

Aortic Smooth Muscle Cells from ApoE(-/-) Mice Secrete Biglycan with Hyperelongated Glycosaminoglycan Chains

Atherosclerosis is the major underlying process of cardiovascular disease. Atherosclerosis commences with an initial pre-inflammatory phase of the trapping and accumulation of lipids in the vessel wall and this is followed by an inflammatory response. The trapping of lipids occurs via binding to proteoglycans, specifically biglycan, with hyperelongated glycosaminoglycan (GAG) chains. Models have been developed to study the aetiology as well as the effectiveness of medical and experimental interventions in preventing atherosclerosis. ApoE is an apolipoprotein associated with removal of lipids from peripheral tissues. Disruption of the ApoE gene in C57BL/6 mice produces mice which are ApoE-/- (ApoE deficient) and show elevated plasma lipids and accelerated development of atherosclerosis which is exacerbated on a high fat diet. These mice are widely used in studies of atherosclerosis. We investigated the possibility that changes in the size of biglycan might participate in the development of atherosclerosis in ApoE -/mice. We prepared Aortic Smooth Muscle Cell (ASMC) cultures by the digestion technique from ApoE-/- and ApoE+/+ mice and studied the size of biglycan secreted by both cell types. The biglycan secreted by ASMCs for ApoE -/mice was larger than that from ApoE+/+ ASMCs. The difference was not eliminated by treatment of cells with a high concentration of Platelet Derived Growth Factor (PDGF) or by treatment with the PDGF antagonist, imatinib. The size difference was however observed in the small free GAG chains (xyloside GAGs) secreted in cells supplemented with exogenous xyloside as a cellular assay of GAG synthesizing capacity. This result suggests that there is a hyperactivity of the fundamental GAG synthesizing capacity in the ASMCs from ApoE -/mice. These data suggest that hyperelongated biglycan might be contributing to lipid accumulation in ApoE-/- mice used in studies of atherosclerosis and that some of the medical interventions might have an action to reverse this hyperelongation response.

Invasive Candidiasis: New Insights Presaging New Therapeutic Approaches?

The expanding universe of amyloid has revealed an intriguing new galaxy, invasive candidiasis, with the publication by Gilchrist et al in this issue of the Journal. Invasive infection of the gut by Candida albicans is a serious, intractable condition that occurs most commonly but not exclusively in neutropenic patients and for which there is very poorly effective treatment. Gilchrist et al now report that amyloid fibrils are produced on the surface of the invading fungal cells in vivo, just as they are in vitro, and that human serum amyloid P component binds to the fibrils both in vitro and in vivo. What does this mean? Amyloid, used in the strict sense, which is essential for correct diagnosis and management of sick patients, is a pathological extracellular deposit composed predominantly of characteristic protein fibrils [1]. About 30 different, unrelated human proteins are now known to form amyloid fibrils in different clinical conditions. Despite widely different structures among their precursor molecules, all amyloid fibrils share the same cross–β core structure and all bind alkaline alcoholic Congo red to produce unique red-green birefringence when observed in strong cross-polarized light. This pathognomonic tinctorial property is the gold standard for histopathological diagnosis of amyloid. The fibrillar cross–β aggregate is a very stable protein assembly, and almost any polypeptide can form amyloid-like fibrils in vitro if subjected to suitable denaturing conditions that enable refolding. In vivo most types of amyloid fibrils are formed by misfolding of native globular proteins, often associated with limited proteolytic cleavage, and some are derived from natively unfolded precursors, but it is not known why only approximately 30 among all human proteins form amyloid in vivo. Regardless of their different protein compositions, all amyloid fibrils are rigid, non–branching, of indeterminate length, about 10 nm in diameter, and composed of 2–6 twisted protofibrils. In addition to the fibrils, amyloid deposits in vivo also always contain abundant heparan and dermatan sulfate proteoglycan and free glycosaminoglycan chains, some of which are tightly associated with the fibrils. Amyloid deposits also always contain serum amyloid P component (SAP), a normal plasma protein of the pentraxin family that shows avid specific calcium dependent binding to amyloid fibrils of all types [2]. Indeed,

Glycosaminoglycans in Human and Bovine Serum: Detection of Twenty-Four Heparan Sulfate and Chondroitin Sulfate Motifs Including a Novel Sialic Acid-Modified Chondroitin Sulfate Linkage Hexasaccharide

Heterogeneous heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) polysaccharides are important components of blood circulation. Changes in GAG quantity and structure in blood have been indicated in cancers and other human diseases. However, GAG quantities and structures have not been fully characterized due to lack of robust and sensitive analytical tools. To develop such tools, we isolated GAGs from serum and plasma. We employed liquid chromatography (LC) for GAG quantification and LC/mass spectrometry (MS) for GAG structural analysis. Twenty-four heparan and chondroitin sulfate motifs were identified, including linkage hexasaccharides, repeating disaccharide compositions, reducing, and non-reducing end mono-, di-, tri-, and tetrasaccharide structures. Disaccharides were detectable at picomolar level without radiolabeling or derivitization, so only a few ml of human and fetal bovine serum was required for this study. The detection of different reducing end structures distinct from GAG linkage hexasaccharides revealed that free GAG chains generated by GAG degradation enzymes co-existed with proteoglycans in serum. In addition, a novel sialic acid-modified linkage hexasaccharide was found conjugated to bikunin, the most abundant serum proteoglycan.

Biosynthesis of proteoglycan in bone and cartilage of parathyroid hormone-related protein knockout mice

Proteoglycans are suggested to regulate cell adhesion, differentiation and mineralization of hard tissues. In vitro studies have shown that many humoral and local factors regulate proteoglycan synthesis. Among them, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have potent stimulating effects on proteoglycan synthesis. However, the exact role of PTHrP on the biosynthesis and metabolism of proteoglycans during skeletal development is not clear. To clarify this point, we examined bony and cartilaginous explants of newborn mice with disrupted PTHrP alleles. Ribs of homozygous PTHrP-knockout mice and wildtype littermates were dissected into bony and cartilaginous regions and metabolically labeled with [35S]sulfate in culture. Radiolabeled proteoglycans were analyzed by column chromatography. The elution profiles of [35S]-labeled proteoglycan from cartilaginous explants did not differ between homozygous PTHrP-knockout mice and wild-type littermates. However, the amount of labeled proteoglycan in homozygous PTHrP-knockout mice was only 4%-5% that of wild-type littermates. In contrast with cartilaginous explants, the amount of labeled proteoglycans in bony explants did not differ between the two genotypes. Interestingly, besides the common major peak (Kd = 0.10-0.16) observed in the bony explants of both genotypes, a minor peak (Kd = 0.42) was specifically present in homozygous PTHrP-knockout mice. This minor peak was earlier than that of free glycosaminoglycan (GAG) chains, suggesting that the core protein, but not GAG chain, was cleaved in the bony explants of homozygous PTHrP. These findings demonstrate a crucial nonredundant role of PTHrP in the regulation of proteoglycan synthesis and metabolism during skeletal development.

Design of Peptides with High Affinities for Heparin and Endothelial Cell Proteoglycans

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.

Differential inhibition of retrovirus transduction by proteoglycans and free glycosaminoglycans.

We have previously shown that the efficiency of retrovirus-mediated gene transfer is limited in part due to the presence of chondroitin sulfate proteoglycans in virus stocks. In this study, we have used a model recombinant retrovirus encoding the Escherichia coli lacZ gene, bovine aorta chondroitin sulfate proteoglycan (CSPG), various free glycosaminoglycan chains (GAGs), and quantitative assays for retrovirus transduction to explore the mechanism by which proteoglycans and glycosaminoglycans inhibit retroviruses. We found that CSPG and GAGs block an early step in virus-cell interactions but do not act by inactivating viruses or by reducing the growth rate of the target cells. CSPG and most of the GAGs tested (chondroitin sulfate A, chondroitin sulfate B, heparin, heparan sulfate, and hyaluronic acid) inhibited transduction, but with widely varying degrees of activity. The chemical structure of GAGs was found to be an important determinant of their inhibitory activity, which suggests that GAGs do not inhibit transduction simply because they are highly negatively charged polymers. When GAGs were used in combination with a cationic polymer (Polybrene), however, their inhibitory activity was neutralized, and interestingly, at optimal doses of GAG and Polybrene, transduction efficiency was actually enhanced by as much as 72%. In contrast, the inhibitory activity of CSPG, due to the influence of its core protein, was not substantially reduced by Polybrene. The importance of these findings to our understanding of retrovirus-cell interactions and to the development of more efficient retrovirus gene transfer protocols is discussed.

Proteoglycans in macrophages: characterization and possible role in the cellular uptake of lipoproteins.

The murine macrophage cell line J774 was incubated with [35S]sulphate. The cell-associated 35S-labelled macromolecules were shown to be proteoglycans and glycosaminoglycans in similar amounts. The possible presence of cell-surface proteoglycans was investigated by incubating [35S]sulphate-labelled cells with trypsin for 15 min. The released material contained approx. 70% free glycosaminoglycan chains and 30% proteoglycans. The latter component was demonstrated by HNO2 treatment to contain heparan sulphate. In the total cell fraction not treated with trypsin a small but significant portion was shown to be chondroitin sulphate proteoglycan. The cell-associated glycosaminoglycans contained both chondroitin sulphate and heparan sulphate. To investigate possible biological functions of cell-surface proteoglycans in macrophages, cells were incubated with NaClO3 to inhibit sulphation of proteoglycans and beta-d-xyloside to abrogate proteoglycan expression. The uptake of oxidized 125I-tyraminylcellobiose-labelled low-density lipoprotein (125I-TC-LDL) was typically two to three times higher than that of native 125I-TC-LDL in untreated J774 cells. The cellular uptake at 37 degreesC of native 125I-TC-LDL was decreased 25% after both NaClO3 and xyloside treatment, whereas the uptake of oxidized 125I-TC-LDL was decreased 35% after both types of treatment. The mRNA levels for the scavenger receptor A-II and the LDL receptor were not affected by NaClO3 or xyloside treatment. Furthermore, fluid-phase endocytosis, measured as uptake of horseradish peroxidase, and receptor-mediated endocytosis, measured as uptake of 125I-TC-ovalbumin, were not affected by NaClO3 treatment of J774 cells. Removal of cell-surface chondroitin sulphate with chondroitinase ABC decreased only the binding of native 125I-TC-LDL, whereas removal of heparan sulphate with heparitinase decreased the binding of both oxidized and native 125I-TC-LDL. Addition of lipoprotein lipase increased the uptake of oxidized 125I-TC-LDL 1.7 times and the uptake of native 125I-TC-LDL 2.1 times. The binding of the former was more sensitive to NaClO3 treatment than the latter. The results presented support the notion that some of the uptake pathways for lipoproteins in the foam-cell-forming macrophages depend on the presence of cell-surface heparan sulphate and chondroitin sulphate.

Neurite Outgrowth in Brain Neurons Induced by Heparin-binding Growth-associated Molecule (HB-GAM) Depends on the Specific Interaction of HB-GAM with Heparan Sulfate at the Cell Surface

Heparin-binding growth-associated molecule (HB-GAM) is a cell-surface- and extracellular matrix-associated protein that lines developing axons in vivo and promotes neurite outgrowth in vitro. Because N-syndecan (syndecan-3) was found to function as a receptor in HB-GAM-induced neurite outgrowth, we have now studied whether the heparan sulfate side chains of N-syndecan play a role in HB-GAM-neuron interactions. N-Syndecan from postnatal rat brain was found to inhibit HB-GAM-induced but not laminin-induced neurite outgrowth when added to the assay media. The inhibitory activity was abolished by treating N-syndecan with heparitinase, but it was retained in N-syndecan-derived free glycosaminoglycan chains, suggesting that N-syndecan heparan sulfate at the cell surface is involved in HB-GAM-induced neurite outgrowth. Binding to HB-GAM and inhibition of neurite outgrowth was observed with heparin-related polysaccharides only; galactosaminoglycans were inactive. Significant inhibition of neurite outgrowth was induced by heparin and by N-syndecan heparan sulfate but not by heparan sulfates from other sources. A minimum of 10 monosaccharide residues were required for HB-GAM-induced neurite outgrowth. Experiments with selectively desulfated heparins indicated that 2-O-sulfated iduronic acid units, in particular, are of importance to the interaction with HB-GAM, were implicated to a lesser extent. Structural analysis of N-syndecan from 6-day-old rat brain indicated that the heparan sulfate chains contain sequences of contiguous, N-sulfated disaccharide units with an unusually high proportion (82%) of 2-O-sulfated iduronic acid residues. We suggest that this property of N-syndecan heparan sulfate is essential for HB-GAM binding and induction of neurite outgrowth.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Proteoglycan-mediated Inhibition of Aβ Proteolysis: A POTENTIAL CAUSE OF SENILE PLAQUE ACCUMULATION

Senile plaques of Alzheimer's disease brain contain, in addition to beta amyloid peptide (A beta), multiple proteoglycans. Systemic amyloidotic deposits also routinely contain proteoglycan, suggesting that these glycoconjugates are generally involved in amyloid plaque formation and/or persistence. We demonstrate that heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) inhibit the proteolytic degradation of fibrillar, but not non-fibrillar, A beta at physiological pH. In accordance with the proteolysis studies, high affinity binding of proteoglycans to fibrillar A beta(1-40) and A beta(1-42) is observed from pH 4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar peptide is only seen at pH < 6. This differing pH dependence of binding suggests that a lysine residue is involved in proteoglycan association with fibrillar A beta, whereas a protonated histidine appears to be needed for binding of the glycoconjugates to non-fibrillar peptide. Scatchard analysis of fibrillar A beta association with proteoglycans indicates a single affinity interaction, and the binding of both HSPG and CSPG to fibrillar A beta is completely inhibited by free glycosaminoglycan chains. This implies that these sulfated carbohydrate moieties are primarily responsible for proteoglycan.A beta interaction. The ability of proteoglycans to bind fibrillar A beta and inhibit its proteolytic degradation suggests a possible mechanism of senile plaque accumulation and persistence in Alzheimer's disease.

β‐Elimination for Release of O‐Linked Glycosaminoglycans from Proteoglycans

O-linked glycosaminoglycan (GAG) chains in proteoglycans are readily released from their core proteins by treatment with alkali at room temperature. This beta-elimination is the same type of reaction as that for releasing O-linked oligosaccharides from their core proteins. Under the reaction conditions described here, N-linked oligosaccharides remain attached to the core protein, but any O-linked oligosaccharides will be released along with the GAG chains. The procedure can be used to isolate the free GAG chains, the free O-linked oligosaccharides, and the core protein (which will still have any N-linked oligosaccharides that were originally present).

The Effect of the β-D-Xyloside Naroparcil on Circulating Plasma Glycosaminoglycans: AN EXPLANATION FOR ITS KNOWN ANTITHROMBOTIC ACTIVITY IN THE RABBIT

Beta-D-Xylosides are known to initiate or prime free glycosaminoglycan (GAG) chain synthesis in cell and tissue culture. As such, the effect of the venous antithrombotic beta-D-xyloside, naroparcil, was investigated on the plasma GAG profile in the rabbit after oral administration. Using dose-response experiments, we showed that antithrombin activity via antithrombin III and heparin cofactor II was increased in parallel with GAG plasma levels compared to control. A more detailed qualitative examination of plasma GAGs by cellulose acetate electrophoresis and ion-exchange chromatography, following oral administration of naroparcil at 400 mg/kg, revealed the presence of higher density charged molecules compared to control. The extracted GAGs were found to activate inhibition of thrombin by heparin cofactor II and contained approximately 25% of a dermatan sulfate-like compound (undetectable in control), which could be responsible for the antithrombotic effect. Using radiolabeled naroparcil, we found radiolabeled GAG fractions and the fact that naroparcil was a substrate for galactosyltransferase I, the second enzyme responsible for GAG chain polymerization, suggested that the compound could initiate in vivo the biosynthesis of antithrombotic free GAG chains. This is, to our knowledge, the first description of the in vivo effect of a beta-D-xyloside on GAG biosynthesis; furthermore, this is correlated with an antithrombotic action.

Growth modulation and proteoglycan turnover in cultured mesangial cells.

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with 35SO4(2-) by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (Kd of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (Kd = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with Kd values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (Kd = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular 35S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 microgram/ml) decreased by 45% the incorporation of [3H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G1/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 microgram/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity.

Proteoglycan metabolism in normal and inflammatory human macrophages

To study proteoglycan metabolism in inflammatory macrophages, primary cultures of human macrophages were cultured in the absence and presence of bacterial lipopolysaccharide (LPS). When exposed to [35S]sulfate, the cells incorporated the label almost exclusively into chondroitin sulfate proteoglycan (CSPG), which was recovered from the culture medium and the cell layer. Cells stimulated with LPS secreted approximately three times more [35]CSPG into the culture medium than control cells. Furthermore, cell adhesion was also found to promote proteoglycan secretion; when nonadherent monocytic cells were induced to adhere, the release of proteoglycan increased two times. The increased secretion seen in LPS-stimulated macrophages was partly due to increased biosynthesis, but was mostly due to increased sorting of CSPG to the secretory pathway. Only about 20% of the CSPG synthesized in unstimulated cells was secreted, whereas the corresponding figure in LPS-treated cells was 35%. In both cell types, the remaining [35S]CSPG was degraded, probably in the lysosomes. The degradation was a two-step process. First, the [35S]CSPG was rapidly cleaved to yield free glycosaminoglycan (GAG) chains (t1/2 = 15 to 30 minutes). Secondly, the GAG chains were completely depolymerized (t1/2 = 2 to 3 hours). Neither resting nor LPS-stimulated cells sorted CSPG to intracellular storage, as is evident in many hematopoietic cells. The LPS-treated cells synthesized [35S]CSPG of smaller molecular size than did control cells, with GAG chains of approximate molecular mass of 12 kD versus 16 kD in control cells. No difference was seen in the disaccharide composition of the GAG chains; both LPS-stimulated and unstimulated cells expressed a mixture of 80% to 90% chondroitin 4-sulfate and 10% to 20% chondroitin 4,6-disulfate. N-terminal sequence and Northern blot analysis indicate that the core protein of the CSPG secreted by human macrophages is serglycin.

Proteoglycan Metabolism in Normal and Inflammatory Human Macrophages

AbstractTo study proteoglycan metabolism in inflammatory macrophages, primary cultures of human macrophages were cultured in the absence and presence of bacterial lipopoly-saccaride (LPS). When exposed to [35S] sulfate, the cells incorporated the label almost exclusively into chondroitin sulfate proteoglycan (CSPG), which was recovered from the culture medium and the cell layer. Cells stimulated with LPS secreted approximately three times more [35S]CSPG into the culture medium than control cells. Furthermore, cell adhesion was also found to promote proteoglycan secretion; when nonadherent monocytic cells were induced to adhere, the release of proteoglycan increased two times. The increased secretion seen in LPS-stimulated macrophages was partly due to increased biosynthesis, but was mostly due to increased sorting of CSPG to the secretory pathway. Only about 20% of the CSPG synthesized in unstimulated cells was secreted, whereas the corresponding figure in LPS-treated cells was 35%. In both cell types, the remaining [35S]CSPG was degraded, probably in the lysosomes. The degradation was a two-step process. First, the [35S]CSPG was rapidly cleaved to yield free glycosaminoglycan (GAG) chains (t1/2 = 15 to 30 minutes). Secondly, the GAG chains were completely depoly-merized(t1/2 = 2 to 3 hours). Neither resting nor LPS-stimulated cells sorted CSPG to intracellular storage, as is evident in many hematopoietic cells. The LPS-treated cells synthesized [35S]CSPG of smaller molecular size than did control cells, with GAG chains of approximate molecular mass of 12 kD versus 16 kD in control cells. No difference was seen in the disaccharide composition of the GAG chains; both LPS-stimulated and unstimulated cells expressed a mixture of 80% to 90% chondroitin 4-sulfate and 10% to 20% chondroitin 4,6-disulfate. N-terminal sequence and Northern blot analysis indicate that the core protein of the CSPG secreted by human macrophages is serglycin.

Size-Dependent Separation of Proteoglycans by Electrophoresis in Gels of Pure Agarose

An improved system for electrophoresis of proteoglycans is presented, using a discontinuous buffer system that allows stacking of the sample. The molecular sieving in pure agarose was studied using cartilage proteoglycans separated into fractions of different size (Kav value) by gel chromatography. These fractions were used to establish the relationship between size and mobility in agarose gels of different concentrations. With low agarose concentration, the separation was largely dependent on charge density. With increasing agarose concentration, the separation became increasingly dependent on size. Electrophoresis provides a suitably sensitive tool for analysis of the proteoglycan and glycosaminoglycan fractions prepared by the Alcian blue precipitation described in the preceding paper. Alcian blue precipitated cartilage proteoglycans appeared identical in size to molecules prepared by conventional procedures when analyzed by electrophoresis. Proteoglycans of various sizes were prepared by precipitation with Alcian blue of 4 M guanidine-HCl extracts of bovine nasal cartilage, human articular cartilage, human skin, and bovine sclera. Proteoglycans were also precipitated from human synovial fluid and plasma. The samples were electrophoresed on 1.2% agarose gels, which was the highest agarose concentration suitable for large aggregating proteoglycans but still separating the smallest proteoglycans with one or two glycosaminoglycan chains from each other. Several sizes of proteoglycans were found in each sample except blood plasma, which only contained one small proteoglycan/glycosaminoglycan. Electrophoresis in agarose is also suitable for separating proteoglycan aggregates and monomers and allows rapid analysis of several samples at a time. The proteoglycans prepared by Alcian blue precipitation retained their ability to form aggregates with hyaluronic acid. In addition to separation according to size, the electrophoresis method is also useful for separating free glycosaminoglycan chains according to charge. Electrophoresis in 0.6% agarose was used to demonstrate that both chondroitin sulfate and keratan sulfate were precipitated with Alcian blue at 0.4 M guanidine-HCl. No keratan sulfate, either alone or together with chondroitin sulfate, was precipitated at 1.0 M guanidine-HCl concentration.

Topography of Proteoglycan and Glycosaminoglycan Free Chain Expression in 3T3 Fibroblasts and Human Keratinocytes

Synthesis of heparan sulfate-free chains by human keratinocytes is upregulated during terminal differentiation. The cellular location of this product class and the significance of the differentiation effect are unknown. Differential plasma membrane shearing with cationized colloidal silica was used to evaluate the compartmentalization of the heparan and chondroitin sulfate free chains and their respective proteoglycans in 3T3 fibroblasts and human keratinocytes. The method exploits the topologic segregation of plasma membranes of adherent cells into ventral, dorsal, and intracellular domains and the selective binding of the silica to the dorsal membranes, which by shearing can be separated from ventral membranes adherent to the substratum. Analysis of membrane preparations from sheared cells that had been prelabeled with [35S]-sulfate revealed the proteoglycans to be predominantly ventral, at which location a matrix binding function could be accommodated. Proteoglycans were also recovered from dorsal and intracellular membranes, suggesting active trafficking between intra- and extra-cellular sites. In contrast, the major fraction of heparan and chondroitin sulfate free chains was either cytosolic or associated with intracellular membranes, with the remaining approximately 20% segregated to dorsal and ventral membranes. These results suggest different cellular functions for the proteoglycans and glycosaminoglycan free chains. The partial localization of the free chains to peripheral membranes is compatible with our prior hypothesis that they arise by processing of precursor proteoglycans on cell surfaces. Following this origin, the free glycosaminoglycan polymers could be available to bind ligands such as cytokines prior to transport to intracellular sites of action.

Interleukin-1-induced alterations in glomerular proteoglycans: biochemical and tissue autoradiographic aspects.

The effect of interleukin-1 alpha (IL-1) on the synthesis of glomerular basement membrane heparan sulfate-proteoglycan (HS-PG) was investigated. An ex vivo recirculating organ perfusion system was used. Kidneys were perfused with a medium containing (35S) sulfate and IL-1 (0.625 to 6.25 ng/mL). After radiolabeling was performed, a small cortical piece was saved for tissue autoradiography; the remaining kidney and perfusion medium were used for biochemical studies. Renal cortices were dissected out, and glomeruli were isolated; the PG were extracted and characterized. With exposure to IL-1 (5 ng/mL), an approximately twofold increase of the radioactivity in the glomerular fraction was noted. The increase was unaffected by indomethacin treatment. By Sepharose CL-6B chromatography, a single peak of radioactivity with Kav = 0.25 (macromolecular form of PG) was observed in the control group. The IL-treated group had two peaks of radioactivity with Kav = 0.25 and 0.45: the first peak contained PG, and the second peak consisted of free glycosaminoglycan (GAG) chains. Elution profiles of hydrolyzed tissue GAG chains were similar. No change in the ratio of chondroitin to heparan sulfate was observed. By DEAE-Sephacel chromatography, the glomerular PG/GAG of the IL-treated group eluted at a relatively lower salt concentration, suggesting a change in the charge density characteristics. No differences in the elution profiles of media PG/GAG were observed. (35S)methionine labeling of proteins showed no significant increase of the total radioincorporation in the IL-treated group. Immunoprecipitation studies revealed an approximately 88% increase in the de novo synthesis of PG. Tissue autoradiography revealed an approximately twofold increase of (35S) sulfate radioactivity over the glomerular mesangium, epithelium, and basement membranes. These results indicate that IL-1 enhances the synthesis of the macromolecular form of PG and the generation of free chains. Conceivably, such alterations may lead to defective macromolecular interactions among various components of the glomerular basement membrane and compromise the integrity of the ultrafiltration unit of the glomerulus.

Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from β- d-xyloside inhibition of proliferation

β- d-Xylosides have been used to perturb proteoglycan (PG) synthesis to elucidate the function of PGs in a number of cellular processes, including proliferation, migration, and differentiation. This study was designed to examine whether specific xylosides affect the proliferation of several different cell types and, if so, whether this effect is dependent on altered PG synthesis via the false acceptor pathway. Both methylumbelliferyl β- d-xylopyranoside and p-nitrophenyl β- d-xylopyranoside (PNP β-xyloside) inhibit cell proliferation and modulate PG synthesis; however, the α form of PNP xyloside which does not perturb PG synthesis inhibits the proliferation of cultured cells on a molar basis equally as well as the β form. Conversely, β-methyl xylopyranoside stimulates the synthesis of free glycosaminoglycan chains equally as well as PNP β-xyloside and yet has no measurable effect on cell proliferation at comparable doses, indicating that cells can grow normally while experiencing disruption of their proteoglycan metabolism. At doses ranging from 0.5 to 5 m m, PNP β-xyloside arrests cells in the G1 phase of the cell cycle at the same time point as serum starvation. It also delays the exit of cycling cells from the S phase. This treatment is not cytotoxic and is rapidly reversed by the replacement of PNP β-xyloside containing medium with control medium. Dimethyl sulfoxide, the most commonly used solvent for β-xyloside in proteoglycan studies, potentiates the inhibitory effect of PNP β-xyloside on cell proliferation. These results indicate that the perturbation of PG synthesis via the false acceptor pathway can be uncoupled from control of cell proliferation.

Proteoglycan and glycosaminoglycan free chain expression in keratinocytes, endothelium, and mesenchymal cells

Cultured fibroblasts, bovine aortic endothelial cells, and human keratinocytes synthesize both proteoglycans and glycosaminoglycan free chains, the proportions varying between cell types. The major metabolic labeling is in proteoglycans, except for keratinocytes with approximately 60% of product as free chains. The proteoglycans range from approximately 50- greater than 1000 kDa, and the glycosaminoglycan side chains derived by alkaline elimination are approximately 30- greater than 100 kDa. The glycosaminoglycan free chains, in contrast, are smaller, from approximately 7-40 kDa in mass. The proteoglycans are both medium and cell layer constituents, whereas the glycosaminoglycan free chains are essentially confined to cells. The cellular proteoglycans and a portion of the free chains are accessible to in situ digestion by Flavobacterial glycosaminoglycan lyases, presumably reflecting localization to the cell surface. Collectively, the data show the free chains to be a common feature of all cells studied and to be partly expressed on cell surfaces. We hypothesize that the processing that creates these free chains occurs on cell surfaces, in which location they could serve ligand receptor functions.