Renée K. Margolis

Mammalian homologues of the Drosophila slit protein are ligands of the heparan sulfate proteoglycan glypican-1 in brain.

Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, we have isolated from rat brain at least two proteins that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled us to isolate from a rat brain cDNA library a 4.1-kilobase clone that encoded two of our peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that our peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523–1534 amino acids, and our peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using our affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8.6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where we have previously also demonstrated glypican-1 mRNA and immunoreactivity. Our results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis.

The Core Protein of the Chondroitin Sulfate Proteoglycan Phosphacan Is a High-affinity Ligand of Fibroblast Growth Factor-2 and Potentiates Its Mitogenic Activity

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

The fibrinogen-like globe of tenascin-C mediates its interactions with neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta.

Two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan (the extracellular domain of protein-tyrosine phosphatase-ζ/β), are high-affinity ligands of tenascin-C. Using portions of tenascin-C expressed as recombinant proteins in human fibrosarcoma cells, we have demonstrated both by direct radioligand binding assays and inhibition studies that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen-like globe and that phosphacan binds to this single domain with nearly the same affinity (K d ∼12 nm) as to native or recombinant tenascin-C. However, maximum binding of neurocan requires both the fibrinogen globe and some of the adjacent fibronectin type III repeats. Binding of phosphacan and neurocan to intact tenascin-C, and of phosphacan to the fibrinogen globe, is significantly increased in the presence of calcium. Chondroitinase treatment of the proteoglycans did not affect their binding to either native tenascin-C or to any of the recombinant proteins, demonstrating that these interactions are mediated by the proteoglycan core proteins rather than through the glycosaminoglycan chains. These results are also consistent with rotary shadowing electron micrographs that show phosphacan as a rod terminated at one end by a globular domain that is frequently seen apposed to the fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma cells adhere to and spread on deletion variants of tenascin-C containing only the epidermal growth factor-like domains or the fibronectin type III repeats and the fibrinogen globe. In both cases cell adhesion was inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C.

Ion dependence of amino acid uptake in brain slices.

1. n1. The concentrative uptake of amino acids by mouse brain slices was measured after 5 and 90 min of incubation in the presence of varying concentrations of Na+ and K+. Lowering the Na+ concentration in the medium from 148 mM to 10–30 mM decreased the tissue concentration of both Na+ and K+, with the Na+ levels approximating those of the medium. Media containing 148 mM Na+ and devoid of K+ led to elevated Na+ levels in the tissue. Decreasing only Na+ in the medium led to a greater loss of tissue R+ than the omission of K+ from the medium. n n2. n2. The Na+ requirement for transport of individual amino acids was not uniform. Lowering the Na+ concentration of the medium strongly inhibited the uptake of α-aminoisobutyric acid, l-α,γ-diaminobutyric acid, d-glutamic acid and lysine, whereas the uptake of d- and l-aspartic acids, l-glutamic acid and leucine was inhibited to a lesser degree. Even though the two isomers of glutamate were accumulated to approximately the same extent under control conditions, differences in uptake were observed in Na+-deficient media. n n3. n3. The absence of K+ from the incubation medium led to a marked inhibition of amino acid uptake (ranging from 64 to 82 %), and the degree of inhibition depended upon the Na+ concentration as well as on the amino acid studied. n n4. n4. There was no correlation between tissue levels of ATP and the degree of inhibition of amino acid uptake in ion-deficient media. n n5. n5. Although the data clearly show that accumulation of amino acids by brain slices is ion dependent, the presence of a physiological Na+ gradient between tissue and medium is not necessary.

Glycosaminoglycans and glycoproteins associated with rat brain nuclei

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

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.

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.

Isolation and properties of a soluble chondroitin sulfate proteoglycan from brain.

A proteoglycan in which the glycosaminoglycans are predominantly chondroitin sulfate has been isolated from the soluble fraction of rat brain by ion exchange chromatography and gel filtration. Glycoprotein oligosaccharides are also present, and result in adsorption of the proteoglycan by Concanavalin A-Sepharose. The proteoglycan-glycoprotein complex eluted from the affinity column by alpha-methylglucoside floats near the top of a cesium chloride density gradient run under dissociative conditions (4 M guanidine), but after beta-elimination of the chondroitin sulfate polysaccharide chains from their low buoyant density glycoprotein complex they sediment to the bottom of the gradient. These results suggest that relatively few polysaccharide chains are covalently linked to a large protein core in the dissociated chondroitin sulfate proteoglycan subunit from brain, and that the proteoglycans are closely associated with soluble glycoproteins.

The carbohydrate composition of brain microtubule protein

Abstract Brain microtubule protein was found to contain 1.3% carbohydrate, consisting of glucosamine, galactosamine, galactose, mannose, fucose and sialic acid. Alkaline borohydride treatment destroyed 40% of the galactosamine and led to the formation of an equimolar amount of galactosaminitol, indicating the presence of alkali-labile O -glycosidic linkages of N -acetyl-galactosamine to serine and/or threonine residues. Since a portion of the carbohydrate is attached to protein by alkali-stable linkages, at least two types of oligosaccharide are present, and can be calculated to contain seven monosaccharide residues per mole of microtubule protein dimer.

Effects of nerve growth factor-induced differentiation on the heparan sulfate of PC12 pheochromocytoma cells and comparison with developing brain

We have examined the size, charge, and sulfation pattern of heparan sulfate in the cell-soluble fraction, membranes, and culture medium of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF) and compared the structural features of PC12 cell heparan sulfate to that of rat brain at several stages of early postnatal development. Nitrous acid degradation studies revealed significant differences in the distribution of N-sulfate and N-acetyl groups in heparan sulfate present in the PC12 cell-soluble fraction, membranes, and medium and demonstrated that NGF treatment led to an increased proportion of N-sulfated segments in the cell-associated heparan sulfate, although no such change was seen in that released into the culture medium. There was very little change in the N-sulfation of brain heparan sulfate during the first 30 days after birth. In brain, most of the heparan sulfate glucosamine residues are N-sulfated and yield predominantly di- and tetrasaccharide nitrous acid degradation products, whereas PC12 cell heparan sulfate contains large blocks of N-acetylglucosamine residues. There was very little difference in the overall charge or size (approximately 15,000 Da) of heparan sulfate chains between the different PC12 cell fractions or brain, although NGF treatment led to a decrease in the proportion of less-charged chains in the PC12 cell membranes and a small increase in molecular size. Our studies therefore demonstrate the presence in PC12 cells of several pools of heparan sulfate having different structural properties, and that significant alterations in the charge, size, and sulfation pattern of PC12 cell heparan sulfate accompany NGF-induced differentiation and neurite outgrowth.