Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells

Chondroitin sulfate-rich proteoglycans secreted by arterial intima smooth muscle cells appear involved in low density lipoprotein entrapment and modification. Hypothetically, such a process may contribute to atherogenesis. We compared composition and size of those proteoglycans synthesized by proliferating and resting human arterial smooth muscle cells for which low density lipoprotein had affinity. Lipoprotein-binding proteoglycans secreted by proliferating cells were larger than those of resting cells (M(r) = 1.1 x 10(6) versus 0.8 x 10(6). This was primarily caused by increased M(r) of the chondroitin sulfate chains (6 x 10(4) versus 3.5 x 10(4)). The glycosaminoglycan chains of the proteoglycans from both cells were made of more than 90% chondroitin 6-sulfate and chondroitin 4-sulfate in a 6:4 ratio. Affinity chromatography indicated that low density lipoprotein had a higher affinity with the proteoglycans synthesized by proliferating cells than those from resting cells. Measured with gel mobility shift assay, the apparent affinity constant of low density lipoproteins for proteoglycans from proliferating cells was 3-fold higher than that for proteoglycans from resting cells. This increased affinity appeared related to the higher relative proportion of proteoglycans with longer glycosaminoglycan chains secreted by the proliferating cells than those secreted by the resting cells.     

Activation of protein kinase C pathway by phorbol ester results in a proteoglycan synthesis increase in peritubular cells from immature rat testis

In cultured peritubular cells (PT) from rat testis, protein kinase C (PKC) was activated by phorbol 12-myristate 13-acetate (PMA). PMA enhanced the synthesis of proteoglycans (PG) and to a lesser extent their catabolism; the stimulation of the synthesis appeared to be due to an increase in PG protein moiety production and, at the same time, to an increase in the glycanation process as revealed by the use of an exogenous acceptor, p-nitrophenyl-beta-d-xyloside. In the presence of PMA, the molecular weight of neosynthesized PG and the length of their constitutive glycosaminoglycan chains were not modified. Moreover, the distribution of proteochondroitin sulfate and proteoheparan sulfate in medium and in cell layer remained unchanged. However, PMA reduced the sulfation level of chondroitin sulfate and heparan sulfate chains, suggesting that PKC activation resulted in an independent modulation of the sugar chain formation and of the sulfate residue transfer. PMA effect on the synthesis of hyaluronan was also determined: PMA dramatically enhanced its production by PT cells.     

Widespread expression of chondroitin sulfate-type serglycins with CD44 binding ability in hematopoietic cells

Serglycin is a family of small proteoglycans with Ser-Gly dipeptide repeats and is modified with various types of glycosaminoglycan side chains. We previously demonstrated that chondroitin sulfate-modified serglycin is a novel ligand for CD44 involved in the adherence and activation of lymphoid cells. In this study, we investigated the production and distribution of CD44 binding serglycins in various hematopoietic cells and characterized their carbohydrate side chains. Immunoprecipitation analysis using CD44-IgG and polyclonal antibody against the serglycin core peptide demonstrated that various serglycin species capable of binding CD44 are produced by a variety of hematopoietic cells including lymphoid cells, myeloid cells, and a few tumor cell lines. Glycosaminoglycans on these serglycins, which are essential for CD44 binding, are composed of chondroitin 4-sulfate or a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate, but no heparin or heparan sulfate side chain was detected. The serglycins are also secreted by normal splenocytes, lymph node lymphocytes, and bone marrow cells, whereas they are secreted in very small amounts by normal thymocytes. Secretion of serglycins is greatly enhanced by mitogenic stimulation with concanavalin A or lipopolysaccharide. Our results showed that serglycin, unlike hyaluronate, is produced and secreted in a functional (CD44 binding) form by many members of the hematopoietic system including various lymphocyte subsets. Our data suggest that serglycin may serve as a major ligand for CD44 in various events in the lymphohematopoietic system.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system.     

Acute osteo-articular infection of the limbs of children. Physiopathology, diagnosis, progression, prognosis, treatment

Chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitinase ABC, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S) were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of delta Di-OS/delta Di-6S/delta Di-4S was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.     

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.     

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.     

Glycosaminoglycan and collagen distribution in the developing human vitreous

BACKGROUND: We determined the distribution of glycosaminoglycans and collagens in the developing human vitreous. METHODS: Eighty human eyes from 5 gestational weeks to 2 postnatal years of age were used. Glycosaminoglycan components were determined by enzyme digestion with hyaluronidase or chondroitinase AC and ABC and immunohistochemistry for chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate. Collagen distribution was determined by immunohistochemistry for types I, II, and III collagens. RESULTS: Enzyme digestion showed that throughout development hyaluronic acid is the main glycosaminoglycan in the vitreous and in the extraocular space at 5-7 gestational weeks. Both areas were filled with mesenchymal cells. Immunohistochemistry showed chondroitin-6-sulfate in the vitreous between 6 and 40 gestational weeks, and chondroitin-4-sulfate between 12 and 40 gestational weeks. Hyaluronic acid and chondroitin sulfate appeared in the retina and around the hyaloid vessels at 12-40 weeks. Immunohistochemistry showed type III collagen in the vitreous and around the mesenchymal cells at 5-7 weeks that was replaced by type II collagen after 8 weeks. CONCLUSIONS: Hyaluronic acid is the major glycosaminoglycan in the vitreous throughout development, except for the transient appearance of chondroitin sulfate at 6-40 gestational weeks. Type III is the main collagen in the early developing vitreous that converts to type II collagen at 8 weeks. The primary and secondary vitreous has the same components as these macromolecules. These vitreous glycosamino-glycans and collagens seem to be produced by mesenchymal cells at an early stage and by the retina and hyaloid vessels during middle and late development.     

Bikunin present in human peritoneal fluid is in part derived from the interaction of serum with peritoneal mesothelial cells

We recently reported that peritoneal fluid mainly contains two proteoglycans; one is the interstitial proteoglycan referred to as decorin, and the other an uncharacterized small chondroitin sulfate proteoglycan. In the present study, we have used a two-step process to isolate the small chondroitin sulfate proteoglycan free of decorin. The purified molecule ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular mass 50 kd made up of a chondroitin-4-sulfate glycosaminoglycan chain and a 30-kd core protein. NH2-terminal analysis of the core protein showed significant sequence homology with bikunin, a component of the human inter-alpha-trypsin inhibitor (IalphaI) family. A Western blot analysis using anti-human inter-alpha-trypsin inhibitor confirmed the identity of the small chondroitin sulfate proteoglycan as bikunin, and a trypsin inhibitor counterstain assay confirmed its anti-trypsin activity. Examination of serum from patients receiving continuous peritoneal dialysis suggests that free bikunin in peritoneal fluid may be the result of leakage of serum proteins into the peritoneum. Our findings further show that the interaction of serum with peritoneal mesothelial cells offers a new and novel explanation for the presence of bikunin in peritoneal fluid.     

Age-related changes in populations of aortic glycosaminoglycans: species with low affinity for plasma low-density lipoproteins, and not species with high affinity, are preferentially affected

Glycosaminoglycans were extracted from the intima and media layers of normal human thoracic aortas from donors of different ages. The arterial segments were devoid of macroscopically visible lesions obtained from patients who had no clinically evident cardiovascular disease. Total glycosaminoglycan content increases during the first 40 years of life. Changes in the content of hyaluronic acid and heparan sulfate are less noticeable. The content of chondroitin sulfate (mainly the 6-isomer) increases, whereas dermatan sulfate remains constant. Plasma LDL-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions allowed the separation of LDL high- and low-affinity glycosaminoglycan species. Remarkably, only glycosaminoglycan species with low affinity for plasma LDL increase with age in the disease-free areas of human thoracic aortas studied. These results suggest that age-related changes in glycosaminoglycan composition of the arterial wall do not contribute to increased deposition of plasma LDL. However, the alternative explanation that individuals with arterial glycosaminoglycans that avidly bind LDL would develop early and severe cardiovascular disease and would thus be excluded from our analysis cannot be ruled out.     

Two-year prospective, randomized trial comparing an innovative twice-a-week progestin regimen with a continuous combined regimen as postmenopausal hormone therapy

OBJECTIVE: To determine the relationship between matrix metalloproteinases (MMPs), their inhibitors, and the turnover of matrix molecules in articular cartilage from patients with osteoarthritis (OA). METHODS: Synovial fluid samples were collected from the knees of 54 patients with OA. Radiographic evaluations and magnetic resonance imaging were performed on the knees of 34 OA patients to classify the stage of the disease. Biochemical analyses and immunoassays were used to measure the concentrations of MMP-1, MMP-3, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, the disaccharide of hyaluronic acid, the proteoglycan glycosaminoglycan disaccharides of chondroitin 4-sulfate (delta di-CS4) and chondroitin 6-sulfate (delta di-CS6), the 846 epitope on chondroitin sulfate of cartilage proteoglycan aggrecan (putative biosynthetic marker), the keratan sulfate (KS) epitope of aggrecan (putative degradation marker), and the C-propeptide of cartilage type II procollagen (CPII) (biosynthetic marker). RESULTS: The concentration of TIMP-1 was directly correlated with the levels of MMP-1 and MMP-3 (both were also correlated with each other), confirming earlier results. There was an inverse correlation between the delta di-CS6:delta di-CS4 ratio and the concentration of MMP-3. The level of delta di-CS6 was correlated with that of the KS epitope, and to a lesser degree, with that of the 846 epitope (the latter was also correlated with the level of delta di-CS4). The concentration of TIMP-1 correlated with that of the 846 epitope, whereas TIMP-2 levels correlated with those of CPII. There were significantly lower concentrations of delta di-CS6, delta di-CS4, the 846 epitope, and CPII in synovial fluid from patients with late-stage OA. CONCLUSION: These observations suggest a link between proteolysis and inhibitor concentrations in OA cartilage. Production of TIMPs appears to be individually linked to the synthesis of specific cartilage molecules. The reduction in the amount of cartilage-matrix structural components suggests that there is a measurable loss of cartilage in the late stages of the disease, as suggested previously. The resultant composition of the cartilage suggests that the loss may primarily involve "resident" molecules originally present in healthy cartilage.     

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.     

Human follicular fluid proteoglycans in relation to in vitro fertilization

OBJECTIVE: To determine whether the concentrations of proteoglycans and hyaluronan in human follicular fluid (FF) are associated with follicular volume, oocyte fertilization, and ET during IVF. DESIGN: The FF from individual follicles were collected. Enzyme-linked immunosorbent assay methods for quantification of a larger chondroitin sulfate proteoglycan and a smaller composite heparan-chondroitin sulfate proteoglycan were established. Hyaluronan and E2 were measured by RIA techniques. PATIENT(S): Sixteen infertile women participating in the IVF program. MAIN OUTCOME MEASURE(S): Concentrations of the proteoglycans, follicular volume, fertilization, and ET rates. RESULT(S): The follicles contained high concentrations of proteoglycans with an average of 0.8 mg/mL of FF, and approximately 70% consisted of the larger chondroitin sulfate proteoglycan, and 30% of the heparan-chondroitin sulfate proteoglycan. A negative correlation was found between the follicular volume, the chondroitin sulfate proteoglycan (r = -0.43), and hyaluronan (r = -0.56). The percentage of embryos developed in culture was significantly higher in follicles larger than 2 mL. A significant and 35% lower concentration of the chondroitin sulfate proteoglycan was found in larger follicles from which subsequent ET was observed. THe heparan-chondroitin sulfate proteoglycan and hyaluronan were both unrelated to fertilization and ET in vitro. CONCLUSION(S): Lower concentrations of chondroitin sulfate proteoglycan were associated with higher follicular volumes and greater fertilization and ET rates. These associations could merely reflect the maturation of the follicle or a role of the chondroitin sulfate proteoglycan in the fertilization process.     

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.     

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.     

Human monocyte-derived macrophages secrete two forms of proteoglycan-macrophage colony-stimulating factor that differ in their ability to bind low density lipoproteins

This study evaluated whether human monocyte-derived macrophages synthesize specific types of proteoglycans with lipoprotein-binding capability that could contribute to lipid retention in the arterial wall. After labeling with either [35S]SO4 or [35S]methionine, macrophages secreted a high molecular mass proteoglycan, with glycosaminoglycan chains of approximately 18 kDa and core protein bands of approximately 100 and 55 kDa. Both core protein bands were recognized by an antibody to PG-100, an antibody that recognizes the proteoglycan form of macrophage colony-stimulating factor (PG-100/PG-MCSF). The interaction between PG-100/PG-MCSF and low density lipoproteins (LDL) was examined by gel mobility shift. In this system, PG-100/PG-MCSF was resolved further into two forms. The two forms had the same core proteins but differed in their overall size and glycosaminoglycan content. The larger form contained glycosaminoglycan chains that were entirely chondroitin ABC lyase-sensitive, whereas the smaller form contained chains that were sensitive to both chondroitin ABC lyase and heparinase. Both forms bound native LDL with high affinity, but the larger form bound LDL with higher affinity than the smaller form. The glycosaminoglycan chains of PG-100/PG-MCSF, but not the core proteins, were responsible for binding to native LDL. Mildly oxidized LDL and methyl-LDL, which have an electrophoretic charge similar to that of native LDL, also bound PG-100/PG-MCSF. In contrast, extensively oxidized LDL and acetyl-LDL, which are more electronegative than native LDL, did not bind to either form of PG-100/PG-MCSF. The demonstration of two forms of human monocyte-derived macrophage PG-100/PG-MCSF which bind LDL may represent an additional role for macrophages in the extracellular trapping of lipoproteins in atherosclerosis.     

SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.     

Reduction of GABAA receptor binding of [3H]muscimol in the barrel field of mice after peripheral denervation: transient and long-lasting effects

Proteoglycans play a role in regulating proliferation and adhesion of cells to each other and to the basal lamina. Synthesis of proteoglycans is disrupted by beta-xylosides, which serve as alternate substrate sites for glycosaminoglycan chain attachment and therefore prevent glycosylation of the core protein. We have investigated the effects of p-nitrophenyl-beta-D-xylopyranoside (PNP-xyloside) on cultured human keratinocytes. Stratified cultures were incubated for 7 days with PNP-xyloside (0.05-2.0 mM). Concentrations as low as 0.05 mM increased the secretion of free chondroitin sulfate by 10-15-fold over untreated cultures. Cell-associated proteoglycan decreased as PNP-xyloside concentration increased. At 2 mM PNP-xyloside, heparan sulfate as well as chondroitin sulfate addition to core proteins was disrupted: the core protein of epican, a heparan sulfate form of CD44 found on keratinocytes, was detected immunologically but lacked heparan sulfate. 2.0 mM PNP-xyloside reduced the number of attached cells by 20-25% after 7 days, but had little effect on morphology or protein synthesis. These results indicate that intact proteoglycans are not critical for maintaining epidermal keratinocyte stratification, cell-cell adhesion, or growth.     

Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.