Comparative acute toxicity and primary irritancy of the ethylidene and vinyl isomers of norbornene

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.     

Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.     

The core protein of the chondroitin sulfate proteoglycan phosphacan is a high-affinity ligand of fibroblast growth factor-2 and potentiates its mitogenic activity

Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.     

High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth-associated molecule

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.     

A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans

The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.     

Multiple heparan sulfate chains are required for optimal syndecan-1 function

Syndecans have three highly conserved sites available for heparan sulfate attachment. To determine if all three sites are required for normal function, a series of mutated syndecans having two, one, or no heparan sulfate chains were expressed in ARH-77 cells. Previously, we demonstrated that expression of wild-type syndecan-1 on these myeloma cells mediates cell-matrix and cell-cell adhesion and inhibits cell invasion into collagen gels. Here we show that to optimally mediate each of these activities, all three sites of heparan sulfate attachment are required. Generally, an increasing loss of syndecan-1 function occurs as the number of heparan sulfate attachment sites decreases. This loss of function is not the result of a decrease in either the total amount of cell surface heparan sulfate or syndecan-1 core protein. In regard to cell invasion, cells expressing syndecan-1 bearing a single heparan sulfate attachment site exhibit a hierarchy of function based upon the position of the site within the core protein; the presence of an available attachment site at serine 47 confers the greatest level of activity, while serine 37 contributes little to syndecan-1 function. However, when all three heparan sulfate chains are present, significantly greater biological activity is observed than is predicted by the sum of the activities occurring when the chains act individually. This synergy provides a functional basis for the evolutionary conservation of the three heparan sulfate attachment sites on syndecans and supports the idea that molecular heterogeneity, which is characteristic of proteoglycans, contributes to their functional diversity.     

Binding of human phospholipase A2 type II to proteoglycans. Differential effect of glycosaminoglycans on enzyme activity

Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 microM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 microM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.     

A sensitive enzymatic-isotopic method for the analysis of tyramine in brain and other tissues

The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 mumol hexosamine/100 mg protein, comprising 57% chondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 mug/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.     

Foam cell conversion of macrophages alters the biosynthesis of heparan sulfate

Heparan sulfate is thought to regulate the biological activities of several proteins implicated in the pathogenesis of atherosclerosis. While the interactions of heparan sulfate with lipoprotein lipase and various growth factors have been actively studied, little is known of the cellular regulation of heparan sulfate biosynthesis in response to lipid accumulation. We have investigated heparan sulfate biosynthesis during conversion of murine J774 macrophages into lipid-laden foam cells. Such conversion is shown to accelerate the rate of glycosaminoglycan synthesis and the transport of newly synthesized proteoglycans into the medium. Moreover, the structure of heparan sulfate is specifically altered due to an approximately 30% increase in the 6-O-sulfation of glucosamine residues within the N-sulfated heparan sulfate domains, whereas the sulfation of chondroitin sulfate remains unaffected. These results suggest a selective effect of foam cell conversion on the biosynthesis of heparan sulfate.     

Prospective study of the significance of contact sensitization caused by metal implants

Cell nuclei of mouse hepatoma contain various proteoglycans (PG) which include heparan sulfate proteoglycan (HS-PG), dermatan sulfate proteoglycan (DS-PG), and chondroitin sulfate proteoglycan (CS AC-PG). The latter is not found in cell nuclei of normal mouse liver. Heparan sulfate (HS) and dermatan sulfate (DS) are the main constituents of carbohydrate chains of nuclear proteoglycans of tumor and normal cells, respectively. Changes in the composition of nuclear PG during malignant transformation are discussed considering the concept of their possible involvement in the regulation of cell mitotic activity.     

Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transfromation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamine- and galactosamine-containing GAG were labeled equivalently by [3H=A1-glucose regardless of culture type, allowing incorporation into the various GAG to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized GAG and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media , which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into GAG relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular GAG due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more GAG label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density-dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAG's in the culture behavior of normal and transformed cells may occur only at high cell density. The density-induced GAG alterations most likely involved are increased condroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface.     

Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.     

Differential release of proteoglycans during human B lymphocyte maturation

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.     

Isolation and characterization of chondroitin sulfate proteoglycans from embryonic quail that influence neural crest cell behavior

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 microg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.     

The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.     

Using forensic autopsies to teach advanced pathophysiology

Cell-surface proteoglycans participate in cell adhesion, growth-factor signalling, lipase activity and anticoagulation. Until recently, only the roles of the glycosaminoglycan chains were investigated. Now, with molecular characterization of several core proteins, the roles of each individual proteoglycan species in cellular signalling pathways are being determined. This review describes some of the recent advances in our understanding of the major transmembrane group of heparan sulfate proteoglycans, the syndecans, including evidence that they play an important role as accessory signalling molecules modulating integrin-based adhesion.     

Regulation of anti-HIV-1 activity of RANTES by heparan sulfate proteoglycans

The role of cell surface proteoglycans in CC chemokine-mediated anti-HIV-1 activity in T cells and macrophages was investigated. Enzyme digestion of heparan sulfate (HS), but not chondroitin sulfate, from the surface of PM1(CD26H) cells (a human T cell line selected for high CD26 expression) rendered them resistant to the antiviral effects of RANTES and macrophage-inflammatory protein-1beta at otherwise inhibitory chemokine concentrations. HIV-1 infection of macrophages, however, was inhibited only partially, even at high concentrations of RANTES, and this inhibition was not prevented by HS removal. Flow cytometry revealed that digestion of cell surface proteoglycans, including HS, prevented the binding of RANTES at 10 to 100 nM concentrations to PM1(CD26H) cells. However, the binding of RANTES to activated macrophages occurred only at higher concentrations (100-300 nM) and was mostly chondroitin sulfate, and not HS, dependent. These results support a role for HS in facilitating the interaction of CC chemokines with the cell surface and the consequent inhibition of HIV-1 infection. The absence of HS-dependent binding of RANTES at lower concentrations to macrophages is consistent with the resistance of these cells to the antiviral effects of chemokines.     

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.     

Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction

Wingless, the Drosophila homologue of the proto-oncogene Wnt-1, encodes a secreted glycoprotein that regulates differentiation and proliferation of nearby cells. Here we report on the biochemical mechanism(s) by which the wingless signal is transmitted from cell to cell. When expressed in S2 cells, the majority (approximately 83%) of secreted wingless protein (WG) is bound to the cell surface and extracellular matrix through specific, noncovalent interactions. The tethered WG can be released by addition of exogenous heparan sulfate and chondroitin sulfate glycosaminoglycans. WG also binds directly to heparin agarose beads with high affinity. These data suggest that WG can bind to the cell surface via naturally occurring sulfated proteoglycans. Two lines of evidence indicate that extracellular glycosaminoglycans on the receiving cells also play a functional role in WG signaling. First, treatment of WG-responsive cells with glycosaminoglycan lyases reduced WG activity by 50%. Second, when WG-responsive cells were preincubated with 1 mM chlorate, which blocks sulfation, WG activity was inhibited to near-basal levels. Addition of exogenous heparin to the chlorate-treated cells was able to restore WG activity. Based on these results, we propose that WG belongs to the group of growth factor ligands whose actions are mediated by extracellular proteoglycan molecules.     

Eukaryotic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity

Biglycan is a small chondroitin sulfate proteoglycan found in many tissues and is structurally related to decorin, fibromodulin, and lumican. The biological function of biglycan is poorly understood, although several studies have indicated interaction with other extracellular matrix components. We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vaccinia virus/T7 bacteriophage expression system. A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistidine fusion protein under control of the T7 phage promoter was expressed in HT-1080 cells and UMR106 cells. The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules labeled in the presence of [35S]sulfate, [3H]glucosamine, and [35S]methionine. Glycoforms of biglycan were separated by imidazole gradient elution, under non-denaturing conditions, and comprised: a large proteoglycan form substituted with two chondroitin sulfate chains of molecular mass approximately 34 kDa (HT-1080 cells) or approximately 40 kDa (UMR106 cells); a small proteoglycan form substituted with two chondroitin sulfate chains with a median molecular mass approximately 28 kDa; and a core protein form secreted devoid of glycosaminoglycan chains. All the glycoforms were substituted with two N-linked oligosaccharides, and the disaccharide composition of the two glycosaminoglycan populations were identical. Approximately 70% of the recombinant biglycan secreted by HT-1080 cells was substituted with chondroitin sulfate chains, whereas about 50% of the biglycan expressed by UMR106 cells was substituted with chondroitin sulfate chains. Infection with vBGN4 in both HT-1080 and UMR106 cells resulted in the production of approximately 10 mg of biglycan/10(9) cells per 24 h. The native recombinant biglycan was shown to bind to collagen type V and the complement protein, C1q. However, when the secondary structure of recombinant biglycan was disrupted by exposure to 4 M guanidine hydrochloride, the affinity for collagen type V was dramatically reduced. These data demonstrate the importance of secondary structure to the function of this small proteoglycan.