Characterization of Slit Protein Interactions with Glypican-1

Abstract

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885-17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N-and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80 -110 nM range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80 -90% by heparin (2 g/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N-or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.In previous studies we biochemically characterized a major heparan sulfate proteoglycan of nervous tissue (1, 2) that we later cloned and identified as the rat homologue of glypican-1 (3), the initial member of a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that is currently composed of six vertebrate proteins. High levels of glypican-1 mRNA are present in brain and skeletal muscle, and in situ hybridization histochemistry demonstrated that glypican-1 mRNA is especially prominent in cerebellar granule cells, large motor neurons in the brain stem, and CA3 pyramidal cells of the hippocampus (4). These results and parallel immunocytochemical studies indicate that glypican-1 is predominantly a neuronal product in the late embryonic and postnatal rat nervous system. Based on genetic studies, it would appear that the glypicans play a significant role in cell growth and development. Dally, the Drosophila homologue of glypican-1, is required for the control of cell division in the developing visual system and for morphogenesis of other tissues (5), and the human homologue of glypican-3/OCI-5 (GPC3) is found to be mutated in pat...

PTPσ functions as a presynaptic receptor for the glypican-4/LRRTM4 complex and is essential for excitatory synaptic transmission

“…In vertebrates, GPCs are predominantly expressed during nervous system development, where they serve as guidance cues for axonal navigation and neuronal migration. Consistent with the role in axon guidance, GPCs also interact with the axon guidance molecule Slit (15,16). Moreover, they regulate several signaling pathways, including the Wnt, Hedgehog, fibroblast growth factor, and bone morphogenic protein pathways (17).…”

Robo1 and Robo2 Cooperate to Control the Guidance of Major Axonal Tracts in the Mammalian Forebrain

“…Thus, biochemical experiments have shown that Slits bind the heparan sulfate proteoglycan glypican-1 (Liang et al, 1999;Ronca et al, 2001). Heparan sulfate enhances the affinity of Slit2 for Robo1 receptors in vitro, and removal of cell surface heparan sulfate by heparinase III treatment or addition of saturating amounts of heparan sulfate abolishes the repulsive activity of Slit2 toward olfactory bulb axons in explant cultures (Hu, 2001).…”

Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

“…Several midline glial specializations such as the roof and floor plates and the chiasmatic glial palisade express slit (Erskine et al 2000 and that may occur in the presumptive derivatives of the midbrain homologues in culture. There is also convincing evidence that glypican-1 binds slit (Liang et al 1999, Ronca et al 2001 and there is no insurmountable obstacle to the formation of a complex of slit/ glypican and a (neuronal) robo receptor since glypican-1 is expressed by astrocytes (Bansal et al 1996) and is shed from the cell surAn Acad Bras Cienc (2002) 74 (4) face (Winkler et al 2002). Additionally, syndecans 2 and 4, that may also be shed, are heavily expressed in cultured astrocytes (Bansal et al 1996).…”

“…Three distinct slit genes (Slit1, Slit2 and Slit3) and three distinct robo genes (robo1, robo2 and rig-1) have been cloned in mammals. The mammalian homologues of slit are ligands of the heparan sulfate proteoglycan (HSPG) glypican-1 (Liang et al 1999, Ronca et al 2001.…”