Characterization of monoclonal antibody 155.8 and partial characterization of its proteoglycan antigen on human melanoma cells

Immunization of mice with a plasma membrane-enriched fraction from human malignant melanoma cells and subsequent generation of hybridomas resulted in the isolation of an IgG1 monoclonal antibody, 155.8, that recognizes chondroitin sulfate proteoglycans. By cell binding analysis, 155.8 was shown to react with seven of eight cultured melanoma cell lines, but not with a variety of lymphoblastoid cell lines or cultured tumor cells derived from other solid tumor types. Indirect immunoprecipitation of the 155.8 antigen from intrinsically labeled melanoma cells revealed a glycoprotein of Mr = 250,000 and a sulfated molecule of Mr greater than 400,000. The antigen was identified as a chondroitin sulfate type A/C proteoglycan synthesized by melanoma cells on the basis of its sensitivity to chondroitinase ABC digestion and the identification of sulfated glycosaminoglycans released from the antigen immunoprecipitated by 155.8. The determinants recognized by antibodies 155.8 and 9.2.27, another anti-chondroitin sulfate proteoglycan, immunoprecipitate only a proteoglycan from high density cesium chloride gradient fractions, (1.487 g/liter); however, they immunoprecipitate a free glycoprotein of Mr = 250,000 from low density fractions (1.317 g/liter). This demonstrated that the 155.8 and 9.2.27 determinants, both of which reside on the glycoprotein of Mr = 250,000, are also present in the proteoglycan, suggesting that this glycoprotein is the proteoglycan core protein. Monoclonal antibody 155.8 reacts with a determinant on the core protein distinct from that recognized by 9.2.27. Proteoglycans bearing 155.8 determinants are distributed on the surface of cultured melanoma cells in a punctated fashion that apparently resolves to short, filamentous structures at high magnification. Immunohistochemical analysis demonstrated that 155.8-defined proteoglycans are found in freshly biopsied melanoma tissue, suggesting that these antigens are also synthesized in vivo by melanoma cells.     

N-linked glycan of a sperm CD52 glycoform associated with human infertility.

In a benchmark study, Isojima and colleagues established H6-3C4, the first successful heterohybridoma immortalized from the peripheral blood lymphocytes of an infertile woman who exhibited high sperm-immobilizing antibody titers. The present report demonstrates the identity between the glycoprotein antigens recognized by the human H6-3C4 monoclonal antibody (mAb) and the murine S19 mAb, generated in our laboratory to sperm agglutination antigen-1 (SAGA-1). Both mAb's recognize N-linked carbohydrate epitopes on the 15-25 kDa, polymorphic SAGA-1 glycoprotein that is localized to all domains of the human sperm surface. Treatment with phosphatidylinositol-specific phospholipase C demonstrated that SAGA-1 is anchored in the sperm plasmalemma via a GPI-lipid linkage. Immunoaffinity purification and microsequencing indicated that the core peptide of the SAGA-1 glycoprotein is identical to the sequence of CD52, a GPI-anchored lymphocyte differentiation marker implicated in signal transduction. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that the sperm form of CD52 exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6-3C4 mAb, which are not detected on lymphocyte CD52. Thus, the two populations of the CD52 glycoprotein on lymphocytes and spermatozoa represent glycoforms, glycoprotein isoforms with the same core amino acid sequence but different carbohydrate structures. Furthermore, mAb's to the unique carbohydrate epitopes on sperm CD52 have multiple inhibitory effects on sperm function, including a cytotoxic effect on spermatozoa in the presence of complement. These results are the first to implicate unique carbohydrate moieties of a sperm CD52 glycoform as target epitopes in the anti-sperm immune response of an infertile woman. Furthermore, localization of CD52 on all domains of the sperm surface coupled with the multiple sperm-inhibitory effects of antibodies to its unique carbohydrate moieties suggest opportunities for immunocontraceptive development.     

Distinct in vivo roles of colony-stimulating factor-1 isoforms in renal inflammation

CSF-1, the major regulator of macrophage (Mphi) development, has three biologically active isoforms: a membrane-spanning, cell surface glycoprotein, a secreted glycoprotein, and a secreted proteoglycan. We hypothesized that there are shared and unique roles of individual CSF-1 isoforms during renal inflammation. To test this, we evaluated transgenic mice only expressing the cell surface or precursors of the secreted CSF-1 isoforms for Mphi accumulation, activation, and Mphi-mediated tubular epithelial cell (TEC) apoptosis during unilateral ureteral obstruction. The only difference between secreted proteoglycan and secreted glycoprotein CSF-1 isoforms is the presence (proteoglycan) or absence (glycoprotein) of an 18-kDa chondroitin sulfate glycosaminoglycan. We report that 1) cell surface CSF-1 isoform is sufficient to restore Mphi accumulation, activation, and TEC apoptosis to wild-type levels and is substantially more effective than the secreted CSF-1 isoforms; 2) the chondroitin sulfate glycosaminoglycan facilitates Mphi accumulation, activation, and TEC apoptosis; 3) increasing the level of secreted proteoglycan CSF-1 in serum amplifies renal inflammation; and 4) cell-cell contact is required for Mphi to up-regulate CSF-1-dependent expression of IFN-gamma. Taken together, we have identified central roles for the cell surface CSF-1 and the chondroitin sulfate chain on secreted proteoglycan CSF-1 during renal inflammation.     

Chondroitin sulfate at the plasma membranes of cultured fibroblasts

We have previously shown that in confluent human fibroblast cultures chondroitin sulfate proteoglycan is a component of the fibronectin-containing pericellular matrix fibers. In the present work the distribution of chondroitin sulfate was studied in subconfluent cell cultures using antibodies that bind to a chemically defined carbohydrate fragment of chondroitinase ABC-modified chondroitin sulfate proteoglycan. Using immunofluorescence microscopy, we observed, in addition to the fibrillar matrix staining, chondroitin sulfate diffusely distributed at the cell surface. In indirect immunoferritin electron microscopy this staining corresponded to patchy binding of ferritin close (24 nm) to the outer aspect of the plasma membrane. The patchy organization appeared uniform in all cell surfaces. The cell surface chondroitin sulfate could not be removed from the plasma membrane by agents that dissociate electrostatic interactions. These data show that in fibroblasts chondroitin sulfate is a component of the outer aspect of the plasma membrane, and raise the possibility of an integral plasma membrane chondroitin sulfate proteoglycan.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

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.     

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.     

Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin.

The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615-1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH-terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH-terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin.     

Heparan sulfate modification of the transmembrane receptor CD47 is necessary for inhibition of T cell receptor signaling by thrombospondin-1

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M(r) > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M(r) 230,000) and CD47 (apparent M(r) > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser(64) and Ser(79). Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser(64).     

Epitope-specific changes in chondroitin sulfate/dermatan sulfate proteoglycans as markers in the lymphopoietic and granulopoietic compartments of developing bursae of Fabricius

The types and distributions of chondroitin sulfate proteoglycans within developing chick bursae of Fabricius were determined by indirect immunocytochemical analyses using mAb specific for chondroitin/dermatan sulfate epitopes. Analyses obtained from the use of well characterized mAb known to specifically identify chondroitin 4- and dermatan sulfates (antibody 2B6) and chondroitin 6-sulfate (antibody 3B3) were compared with those obtained from two additional mAb raised against chick chondroitin sulfates proteoglycans derived from hemopoietic tissue. The results indicate that chondroitin sulfate compositions of the adjacent lymphopoietic and granulopoietic compartments differ. Chondroitin 6-sulfate, notably absent from lymphopoietic regions, is a major chondroitin sulfate species in granulopoietic regions of day 13 bursae. Moreover, chondroitin 6-sulfate disappears from the granulopoietic compartment in a time course that corresponds to the decline in granulopoietic activity. Simultaneously, there is an apparent increase in chondroitin sulfates associated with developing medullary regions of lymphoid follicles. The content of chondroitin 4-/dermatan sulfates and, most significantly, of chondroitin/dermatan sulfates identified by antibodies raised against chick proteoglycans, increases within developing follicles. As a consequence, by day 18 of incubation, immunostained follicles become clearly demarcated from the connective tissue of the tunica propria. This study provides evidence that chondroitin sulfates are constituents of both lymphopoietic and granulopoietic microenvironments and that subtle changes occur within these proteoglycan structures during bursal development. These developmental changes in chondroitin sulfate compositions are consistent with these molecules playing a functional role in hemopoiesis.     

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts.     

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.     

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.     

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.     

Characterization of an activity-dependent, neuronal surface proteoglycan identified with monoclonal antibody Cat-301

Monoclonal antibody Cat-301 was previously shown to recognize a surface-associated antigen on subsets of mammalian CNS neurons whose expression is regulated by neuronal activity early in an animal's postnatal life. We now present the partial purification and characterization of the Cat-301 antigen and demonstrate that it is a chondroitin sulfate proteoglycan. Extracellular localization of the Cat-301 epitope is demonstrated by staining live, intact neurons in situ. Extraction of the antigen from membranes in the absence of detergent indicates that it is either a peripheral membrane protein or a component of an extracellular matrix. The Cat-301 antigen migrates on Western blots of SDS gels with a molecular weight of integral of 680,000 dalton and is purified by DEAE chromatography and Sepharose gel filtration in 8 M urea (pH 4.9) buffer. The antigen is sensitive to chondroitinase ABC, indicating that it is a chondroitin sulfate proteoglycan. Furthermore, we provide strong evidence that the biochemically characterized antigen is indeed the histologically detected species by using a second antibody, Cat-304, that produces immunohistological staining patterns identical to those of Cat-301 and reacts with the purified antigen, but at a distinct epitope. Our earlier developmental findings and the present localization and biochemical results suggest that the antigen may play a role in the maturation of functional connections between neurons, perhaps through stabilization of axosomatic and axodendritic synapses.     

The high molecular weight Cat-301 chondroitin sulfate proteoglycan from brain is related to the large aggregating proteoglycan from cartilage, aggrecan.

Monoclonal antibodies Cat-301 and Cat-304 recognize a neuronal cell surface-associated chondroitin sulfate proteoglycan (CSPG), which is expressed during critical periods of postnatal development in the mammalian central nervous system (CNS). In the present study we show that the CNS CSPG identified by Cat-301/304 is similar to aggrecan, the high molecular weight CSPG from cartilage. By Western blot analysis, cartilaginous tissues, which are rich sources of aggrecan, have a high concentration of a high molecular weight CSPG which is immunoreactive with Cat-301 and 304. The Cat-301 and 304 epitopes, however, are partially masked by chondroitin sulfate glycosamino-glycan and are unmasked by digestion of the antigen with chondroitinase ABC. Although the antigen from both cartilage and CNS can be purified by CsCl buoyant density gradient centrifugation, a standard technique for purifying aggrecan, most of the antigen from the CNS has a lower buoyant density than that of cartilage. This may be due, in part, to the paucity of keratan sulfate substitution on the CNS antigen compared with that of the cartilage antigen. Both the CNS and cartilage antigens bind to hyaluronic acid, a feature characteristic of aggrecan. The physiochemical, biochemical, and functional properties of the Cat-301/304 antigen from cartilage are identical to aggrecan. The CNS antigen is similar, but not identical, to the cartilage antigen, and may thus represent another member of the family of high molecular weight CSPGs which bind to and aggregate with hyaluronic acid.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

Occurrence of three distinct molecular species of chondroitin sulfate proteoglycan in the developing rat brain

More than 60% of brain chondroitin sulfate proteoglycans were extracted from 10-day-old rat brains by homogenization in ice-cold phosphate-buffered saline containing protease inhibitors. Although the soluble proteoglycan preparation was a mixture of chondroitin sulfate proteoglycans with a different hydrodynamic size as well as a different molecular density, each subfraction of the proteoglycans contained chondroitin sulfate side chains with virtually identical molecular weight (approximately 15,000) and chondroitin sulfate disaccharide composition (high content of 4-sulfate unit). Digestion of the purified proteoglycan preparation with protease-free chondroitinase ABC produced five core proteins with Mr = 250,000 (designated as 250K protein), 220,000 (220K), 150,000 (150K), 130,000 (130K), and 93,000 (93K). All these core proteins were obtained from chondroitin sulfate proteoglycan preparations extracted from various regions of the brain, but their composition varied among different brain regions. Analysis for amino acid composition of these core proteins and two-dimensional mapping of their proteolytic peptides revealed that three major core proteins (250K, 220K, and 150K proteins) were structurally different. These observations indicate that at least three distinct types of chondroitin sulfate proteoglycan occur in the developing rat brain.     

Isolation of a cell-surface receptor for chick neural retina adherons

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).