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R K Margolis

Publications and source records attributed to R K Margolis.

At least 37 records · Page 2Linked to original sources
PubMed1994

Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.

We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase.

Alternative Splicing↗
PubMed1994

Nervous tissue proteoglycans.

The structure, biosynthesis, localization, and possible functional roles of nervous tissue glycosaminoglycans and proteoglycans were last reviewed several years ago. Since that time, there has been an exponential increase in publications on the neurobiology of proteoglycans. This review will therefore focus on reports which have appeared in the period after 1988, and especially on those concerning the properties of individual characterized nervous tissue proteoglycans. Related areas such as the regulation of glycosaminoglycan biosynthesis and the roles of cell surface proteoglycans in adhesion and growth control are covered in other contributions to this special topic issue.

Amyloid beta-Protein Precursor↗
PubMed1994

Disaccharide composition of heparan sulfates: brain, nervous tissue storage organelles, kidney, and lung.

We have characterized the structural properties of heparan sulfates from brain and other tissues after depolymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46-69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17-21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10-14%, together with smaller and approximately equal proportions (5-9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine- and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5-10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71%) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large dense cored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for approximately 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21% unsulfated disaccharides, 10% mono- and disulfated disaccharides, and 69% trisulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth.

We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.

Aggrecans↗
PubMed1994

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules.

Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.

Animals↗
PubMed1994

Immunocytochemical and in situ hybridization studies of the heparan sulfate proteoglycan, glypican, in nervous tissue.

Using immunocytochemistry and in situ hybridization histochemistry, we have investigated in embryonic and postnatal rat nervous tissue the localization and cellular sites of synthesis of glypican, a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan. Glypican immunoreactivity is present in the marginal layer (prospective white matter) and in the dorsal root entry zone of E13-16 spinal cord, as well as in the optic nerve and retina at this stage, but does not appear at significant levels in brain until approximately E19. The proteoglycan shows a wide distribution in grey matter and axonal projections of postnatal brain, including the hippocampal formation, the parallel fibers of cerebellar granule cells, and in the medulla and brainstem. Northern analysis demonstrated high levels of glypican mRNA in brain and skeletal muscle, and in rat PC12 pheochromocytoma cells. In situ hybridization histochemistry showed that glypican mRNA was especially prominent in cerebellar granule cells, large motor neurons in the brainstem, and CA3 pyramidal cells of the hippocampus. Our immunocytochemical and in situ hybridization results indicate that glypican is predominantly a neuronal membrane proteoglycan in the late embryonic and postnatal rat central nervous system.

Amino Acid Sequence↗
PubMed1993

Nervous tissue proteoglycans.

The structure, biosynthesis, localization, and possible functional roles of nervous tissue glycosaminoglycans and proteoglycans were last reviewed several years ago. Since that time, there has been an exponential increase in publications on the neurobiology of proteoglycans. This review will therefore focus on reports which have appeared in the period after 1988, and especially on those concerning the properties of individual characterized nervous tissue proteoglycans. Related areas such as the regulation of glycosaminoglycan biosynthesis and the roles of cell surface proteoglycans in adhesion and growth control are covered in other contributions to this special topic issue.

Amyloid beta-Peptides↗
PubMed1992

Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican.

We have obtained the complete coding sequence of a highly conserved heparan sulfate proteoglycan which we previously characterized biochemically after isolation from rat brain. An open reading frame of 558 amino acids encodes a protein with a molecular mass of 62 kDa containing three peptide sequences present in the isolated proteoglycan. The total sequence obtained is 3.5 kb long, including 1.6 kb of 3'-untranslated sequence and 0.2 kb of 5'-untranslated sequence. The deduced amino acid sequence and the 3'- and 5'-untranslated sequences have 89% and 66-80% identity, respectively, with those of a phosphatidylinositol-anchored human lung fibroblast heparan sulfate proteoglycan (glypican) for which mRNA is detectable in a large number of human cell lines. Our data therefore demonstrate that this major heparan sulfate proteoglycan of brain is the rat form of glypican.

Amino Acid Sequence↗
PubMed1992

Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain.

We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a 22-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the fibroblast and cartilage proteoglycans, versican and aggrecan, including two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Aggrecans↗
PubMed1991

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)

Animals↗
PubMed1991

Immunocytochemical localization of chondroitin and chondroitin 4- and 6-sulfates in developing rat cerebellum.

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Effects of beta-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum.

We have examined the effects of beta-xylosides, which act as exogenous acceptors for glycosaminoglycan chain initiation, on the morphology and proteoglycan biosynthesis of PC12 pheochromocytoma cells, and on monolayer, aggregate and explant cultures of early postnatal rat cerebellum. PC12 cells cultured for 13 days in the presence of nerve growth factor (NGF) and beta-xyloside, and labeled during days 11-13 with sodium [35S]sulfate, showed an 8- to 11-fold increase in [35S]sulfate-labeled macromolecules released into the culture medium. Most of the increase was accounted for by chondroitin sulfate, which was in the form of free glycosaminoglycan chains, which were not acid-precipitable. The presence of beta-xyloside also led to a 65-115% increase in [35S]sulfate incorporation into cell-associated glycosaminoglycans and glycoproteins of untreated and NGF-treated PC12 cells, respectively. beta-Xyloside treatment reduced the size of the chondroitin sulfate chains in both the cells and medium from approximately 34,000 to 10,000 Mr, but had much less effect on heparan sulfate, which decreased in size from 16,000 to 13,000-14,500 Mr (in the medium and cells, respectively). beta-Xyloside inhibition of proteoglycan biosynthesis was accompanied by significant morphological effects in NGF-treated PC12 cells, consisting of an increase in length and decrease in the branching, diameter and adhesion to the collagen substratum of the PC12 cell processes. p-Nitrophenyl- and 4-methylumbelliferyl-beta-D-xylosides produced similar effects, which were not seen with p-nitrophenyl-beta-D-galactoside. beta-Xylosides also produced distinct alterations in the adhesion and morphology of monolayer, aggregate, and explant cultures of early postnatal rat cerebellum, which occurred together with inhibition of chondroitin sulfate proteoglycan biosynthesis and a decrease in glycosaminoglycan chain size. These studies indicate that chondroitin sulfate (and probably also heparan sulfate) proteoglycans play a significant role in modulating cell-cell and cell-matrix interactions in nervous tissue development and differentiation.

Adrenal Gland Neoplasms↗
PubMed1990

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A.

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.

Adrenal Gland Neoplasms↗
PubMed1989

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.

Adrenal Gland Neoplasms↗
PubMed1989

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain.

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.

Animals↗
PubMed1989

Immunoelectron microscopic localization of hyaluronic acid-binding region and link protein epitopes in brain.

The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid.

Animals↗