The interaction of the retina cell surface N- acetylgalactosaminylphosphotransferase with an endogenous proteoglycan ligand results in inhibition of cadherin-mediated adhesion

We have previously shown that the binding to cells of a monoclonal antibody directed against the chick neural retina N- acetylgalactosaminylphosphotransferase (GalNAcPTase) results in inhibition of cadherin-mediated adhesion and neurite outgrowth. We hypothesized that the antibody mimics the action of an endogenous ligand. Chondroitin sulfate proteoglycans (CSPGs) are potential ligands because they inhibit adhesion and neurite outgrowth and are present in situ at barriers to neuronal growth. We therefore assayed purified CSPGs for their ability to inhibit homophilic cadherin-mediated adhesion and neurite outgrowth, as well as their ability to bind directly to the GalNAcPTase. A proteoglycan with a 250-kD core protein following removal of chondroitin sulfate chains (250-kD PG) inhibits cadherin-mediated adhesion and neurite outgrowth whether presented as the core protein or as a proteoglycan monomer bearing chondroitin sulfate. A proteoglycan with a 400-kD core protein is not inhibitory in either core protein or monomer form. Treatment of cells with phosphatidylinositol-specific phospholipase C, which removes cell surface GalNAcPTase, abolishes this inhibitory effect. Binding of the 250-kD core protein to cells is competed by the anti-GalNAcPTase antibody 1B11, suggesting that 1B11 and the 250-kD core protein bind to the same site or in close proximity. Moreover, soluble GalNAcPTase binds to the immobilized 250-kD core protein but not to the immobilized 400-kD core protein. Concomitant with inhibition of cadherin mediated adhesion, binding of the 250-kD core protein to the GalNAcPTase on cells results in the enhanced tyrosine phosphorylation of beta-catenin and the uncoupling of N-cadherin from its association with the cytoskeleton. Moreover, the 250-kD PG is present in embryonic chick retina and brain and is associated with the GalNAcPTase in situ. We conclude that the 250-kD PG is an endogenous ligand for the GalNAcPTase. Binding of the 250-kD PG to the GalNAcPTase initiates a signal cascade, involving the tyrosine phosphorylation of beta-catenin, which alters the association of cadherin with the actin-containing cytoskeleton and thereby inhibits adhesion and neurite outgrowth. Regulation of the temporal and spatial expression patterns of each member of the GalNacPTase/250-kD PG interactive pair may create opportunities for interaction that influence the course of development through effects on cadherin-based morphogenetic processes.     

Molecular interactions of neural chondroitin sulfate proteoglycans in the brain development

Aggrecan family proteoglycans, phosphacan/RPTPzeta/beta, and neuroglycan C (NGC) are the major classes of chondroitin sulfate proteoglycan in the developing mammalian brain. A multidomain is a common structural feature of these proteoglycans which can interact with various molecules including growth factors, cell adhesion molecules, and extracellular matrix molecules. Individual proteoglycans are distributed in the developing brain in a distinct temporal and spatial pattern, suggesting that they are involved in distinct phases of the brain development through multiple molecular interactions. This review mainly summarizes recent studies on the involvement of these three classes of proteoglycan in cell-cell and cell-substratum interactions during the brain development. Their expressions and proposed functional roles in injured brains are also mentioned. In addition, this review briefly covers potential functions of other neural chondroitin sulfate proteoglycans such as decorin, testican, NG2 proteoglycan, and amyloid precursor protein (APP) in developing and injured brains.     

Distribution of testican expression in human brain

Testican is a putative extracellular heparan/ chondroitin sulfate proteoglycan of unknown function that is expressed in a variety of human tissues at widely different levels but is most abundant in the brain. In mice, testican mRNA has been detected only in brain and it is therefore likely to have an important function in the central nervous system. RNA blot analysis reveals the relative intensity of testican in various regions of the human brain. Levels of testican message are most pronounced in the thalamus, hippocampus, occipital lobe, nucleus accumbens, temporal lobe, and caudate nucleus, with somewhat lower levels in the cerebral cortex, medulla oblongata, frontal lobe, amygdala, putamen, spinal cord, substantia nigra, and cerebellum. In situ hybridization reveals the cellular distribution of the mRNA within these areas to be highest in neurons and in choroid plexus epithelium, and moderately lower in ependymal cells lining the ventricles and in vascular endothelial cells. Testican mRNA is not detected in oligodendrocytes or in most astrocytes. However, astrocytes in regions of reactive gliosis do express testican mRNA. These findings, along with a cysteine-rich pattern similarity to neurocan, brevican, versican, and other proteoglycans found in brain, suggest that testican may be a part of the specialized extracellular matrix of the brain.     

Testican in human blood

Testican is a highly conserved, differentially expressed gene product of unknown function. Since testican is expressed by human endothelial cells and includes a signal sequence, it was our hypothesis that testican protein would be present in blood. We have developed chicken antibodies specific for testican sequence near the N-terminal and identified a 130-kDa form of testican in human plasma. This is much larger than the calculated molecular weight of the encoded polypeptide, suggesting glycosylation of this plasma protein, and large forms of recombinant testican produced in culture were found to include chondroitin sulfate. The 130-kDa form of testican is unstable in plasma. It is converted to smaller stable forms by separable plasma factors that can be blocked by certain serine protease inhibitors. Testican size conversion may be important in its functional activation or decay. One testican domain has strong homology to thyropin-type cysteine protease-inhibitors. Thus, testican may have a function related to protease inhibition in the blood.     

Chondronectin interactions with proteoglycan

We have investigated whether proteoglycans are involved in the attachment of embryonic chick chondrocytes to type II collagen. Chondroitin sulfate proteoglycan, when added exogenously, promotes the binding of chondronectin, the chondrocyte attachment factor, to type II collagen substrates and thereby stimulates chondrocyte adhesion. Blockage of endogenous proteoglycan synthesis with beta-xylosides prevents chondronectin-mediated chondrocyte attachment, confirming that proteoglycan is required. The intact proteoglycan must be present since chondroitin sulfate glycosaminoglycans did not promote chondronectin-mediated cell attachment but, rather, inhibited it in a dose-dependent manner. This inhibition, however, could be overcome with excess exogenous proteoglycan. Consequently, chondronectin interacts with proteoglycan and then the complex interacts with the collagen substrate and with the cell surface to promote cell adhesion. Further evidence for a direct interaction of chondronectin with the glycosaminoglycan portion of the proteoglycan is the selective binding of chondronectin to dextran-Sepharose, dextran having been shown to inhibit attachment to an extent similar to that of chondroitin sulfate.     

Digestion of proteoglycan by Bacteroides thetaiotaomicron

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.     

Cell-substratum adhesion in chick neural retina depends upon protein- heparan sulfate interactions

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.     

Testican‐3: a brain‐specific proteoglycan member of the BM‐40/SPARC/osteonectin family

The testicans are a three‐member family of secreted proteoglycans structurally related to the BM‐40/secreted protein acidic and rich in cystein (SPARC) osteonectin family of extracellular calcium‐binding proteins. In vitro studies have indicated that testicans are involved in the regulation of extracellular protease cascades and in neuronal function. Here, we describe the biochemical characterization and tissue distribution of mouse testican‐3 as well as the inactivation of the corresponding gene. The expression of testican‐3 in adult mice is restricted to the brain, where it is located diffusely within the extracellular matrix, as well as associated with cells. Brain‐derived testican‐3 is a heparan sulphate proteoglycan. In cell culture, the core protein is detected in the supernatant and the extracellular matrix, whereas the proteoglycan form is restricted to the supernatant. This indicates possible interactions of the testican‐3 core protein with components of the extracellular matrix which are blocked by addition of the glycosaminoglycan chains. Mice deficient in testican‐3 are viable and fertile and do not show an obvious phenotype. This points to a functional redundancy among the different members of the testican family or between testican‐3 and other brain heparan sulphate proteoglycans.     

Structure and synthesis of intracellular proteoglycan in HL-60 human leukemic promyelocytes

The structure, biosynthesis, and metabolism of proteoglycans in the HL-60 human promyelocytes were studied by metabolic labeling in culture with [35S]sulfate, [3H]glucosamine, [3H]serine, and [3H]leucine. These cells synthesize a single predominant species of intracellular proteoglycan with an approximate molecular weight of 100,000. The cells contain about 1 microgram of proteoglycan/million cells. The proteoglycan is turned over within the cells in two apparent pools with half-lives of about 0.6 and 27 h, respectively. The fast pool represents secretion into medium in an apparently intact form, whereas the slow pool represents intracellular degradation to free chondroitin sulfate chains and smaller fragments. The proteoglycan contains a protein core with an apparent Mr on gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of about 20,000-30,000. To the core protein are attached an average of six or seven chondroitin sulfate chains, each with an Mr of about 10,000. The chondroitin sulfate chains contain approximately 85% 4-sulfated and approximately 15% nonsulfated disaccharides. The chondroitin sulfate attachment region of the core protein is essentially resistant to trypsin and elastase, whereas the remainder of the protein core is readily degraded by proteases. The size of the chondroitin sulfate attachment region peptide generated by trypsin was estimated to be approximately 5 kDa. Based on the molecular size, distribution of amino acids, protease susceptibility, and the extent of O-glycosylation, we propose that the intracellular proteoglycan characterized in this study is the translation product of a proteoglycan gene reported to be present in these cells (Stevens, R.L., Avraham, S., Gartner, M.C., Bruns, G.A., Austen, K.E., and Weis, J.H. (1988) J. Biol. Chem. 263, 7287-7291).     

Modification of proteoglycans during maturation of fibroblast substratum adhesion sites

Newly formed adhesion sites, left bound to the tissue culture substratum after [ethylenebis(oxyethylenenitrilo)] tetraacetic acid mediated detachment of simian virus 40 transformed Balb/c 3T3 cells, have been extracted with 0.5 M guanidine hydrochloride or Zwittergent (3-12), extractions which identify different subfractions of proteoglycans in these sites. The compositions of these extracts were then compared to similar extracts of "maturing" adhesion sites in an effort to identify structural and metabolic changes which may occur with time and which may play a role in altering adhesion during cell movement. Guanidine hydrochloride (0.5 M) extracts both hyaluronate and chondroitin sulfate proteoglycan from newly formed sites (but which are not complexed in an aggregate similar to that found in cartilage) but only hyaluronate from fully matured sites, indicating that the chondroitin sulfate proteoglycans somehow become resistant to extraction with time. Both high and low molecular weight forms of hyaluronate also accumulate in sites with time. Zwittergent 3-12 solubilizes free chains of heparan sulfate but not heparan sulfate proteoglycan from either class of sites. Most of the heparan sulfate in newly formed sites occurs as a large proteoglycan excludable from Sepharose CL-6B columns under stringent dissociative conditions; however, as adhesion sites "mature", a portion of this proteoglycan appears to be converted by some unknown mechanism to free heparan sulfate chains. This process may very well weaken the close adhesive contacts between the cell and substratum mediated by fibronectin's binding to the highly multivalent heparan sulfate proteoglycans. These studies further indicate that there is considerable metabolism and changing intermolecular associations of proteoglycans within these sites during movement of fibroblasts over this model extracellular matrix.     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

Affinity Purification of Proteoglycans that Bind to the Amyloid Protein Precursor of Alzheimer's Disease

Abstract: The binding of the amyloid protein precursor (APP) to heparan sulfate proteoglycans has been shown to stimulate the neurite-promoting activity of APP. In this study, proteoglycans that bind with high affinity to APP were characterized. Conditioned medium from cultures of postnatal day 3 mouse brain cells was applied to an affinity column containing a peptide homologous to a heparin-binding domain of APP. A fraction 17-fold enriched in proteoglycans was recovered by elution with a salt gradient. APP bound saturably and with high affinity to the affinity-purified proteoglycan fraction. Scatchard analysis of the binding showed that APP bound to high- and low-affinity sites with equilibrium dissociation constants of 1.4 × 10⁻¹¹ and 6.5 × 10⁻¹⁰ M , respectively. APP, in conjunction with the affinity-purified proteoglycan fraction, promoted neurite outgrowth. The affinity-purified proteoglycan fraction contained a heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan. Digestion of the affinity-purified fraction with heparitinase I revealed a core protein of 63–69-kDa molecular mass, whereas digestion with chondroitinase ABC revealed a core protein of 100–110 kDa. The results suggest that expression of specific APP-binding proteoglycans may be an important step in the regulation of the neurite outgrowth-promoting activity of APP.     

Cell surface proteoglycan of mouse mammary epithelial cells is shed by cleavage of its matrix-binding ectodomain from its membrane-associated domain

The cell surface proteoglycan on normal murine mammary gland (NMuMG) epithelial cells consists of a lipophilic domain, presumably intercalated into the plasma membrane, and an ectodomain that binds via its glycosaminoglycan chains to matrix components, is released intact by proteases and is detected by monoclonal antibody 281-2. The antibody 281-2 also detects a proteoglycan in the culture medium conditioned by NMuMG cells. This immunoactive proteoglycan was purified to homogeneity using DEAE-cellulose chromatography, isopycnic centrifugation, and 281- 2 affinity chromatography. Comparison of the immunoreactive medium proteoglycan with the trypsin-released ectodomain revealed that these proteoglycans are indistinguishable by several criteria as both: (a) contain heparan sulfate and chondroitin sulfate chains; and (b) are similar in hydrodynamic size and buoyant density; (c) have the same size core protein (Mr approximately 53 kD); (d) are nonlipophilic as studied by liposomal intercalation and transfer to silicone-treated paper. Kinetic studies of the release of proteoglycan from the surface of suspended NMuMG cells are interpreted to indicate that the immunoreactive medium proteoglycan is derived directly from the cell surface proteoglycan. Suspension of the cells both augments the release and inhibits the replacement of cell surface proteoglycan. These results indicate that the cell surface proteoglycan of NMuMG cells can be shed by cleavage of its matrix-binding ectodomain from its membrane- associated domain, providing a mechanism by which the epithelial cells can loosen their proteoglycan-mediated attachment to the matrix.     

Regulation of cell substrate adhesion: effects of small galactosaminoglycan-containing proteoglycans.

Cell adhesion is a process which is initiated by the attachment of cells to specific sites in adhesive matrix proteins via cell surface receptors of the integrin family. This is followed by a reorganization of cytoskeletal elements which results in cell spreading and the formation of focal adhesion plaques. We have examined the effects of a class of small galactosaminoglycan-containing proteoglycans on the various stages of cell adhesion to fibronectin-coated substrates. Our results indicate that dermatan sulfate proteoglycans (DSPGs) derived from cartilage, as well as other related small proteoglycans, inhibit the initial attachment of CHO cells and rat embryo fibroblasts to substrates composed of the 105-kD cell-binding fibronectin fragment, but do not affect cell attachment to intact fibronectin. Although this effect involves binding of DSPGs to the substrate via the protein core, the intact proteoglycan is necessary for the observed activity. Isolated core proteins are inactive. The structural composition of the galactosaminoglycan chain does not appear to be functionally significant since both chondroitin sulfate and various dermatan sulfate proteoglycans of this family inhibit cell attachment to the fibronectin fragment. Neither the percentage of cells spread nor the mean area of spread cells adhering to substrates of intact fibronectin was significantly affected by the DSPGs. However, significantly fewer cells formed focal adhesions in the presence of DSPGs as compared with untreated control cells. These results suggest that the binding of small galactosaminoglycan-containing proteoglycans to a fibronectin substrate may affect several stages in the cell adhesion process.     

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.     

Multiple domains of the large fibroblast proteoglycan, versican.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.     

Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.     

Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using beta-D-xyloside

In 7-day chick embryo dorsal root ganglia and epidermis cocultures, nerve fibers avoid the epidermis. Previous studies have indicated that glycoproteic factors, secreted by epidermis, could be involved in this phenomenon. Treatment of epidermis by beta-D-xyloside, a specific proteoglycan synthesis inhibitor, abolishes the avoidance reaction. The same result is obtained when anti-chondroitin sulfate antibodies are added to the culture medium. Using HPLC and 35SO4 labeling combined with chondroitinase and hyaluronidase treatment, it has been demonstrated that chondroitin sulfate is present in the epidermal conditioned medium. This suggests that a chondroitin sulfate proteoglycan secreted by the epidermis is implicated in the neurite avoidance reaction and that epidermis could therefore control its own "noninnervation". In vivo, inhibitory influences by local extracellular components may control the guidance of growth cones during nerve pattern formation.