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

an affinity matrix in which a recombinant glypican-Fc fusion protein expres sed 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 ge l electrophoresis as a single 200-kDa silver-stained band, from which 16 pa rtial peptide sequences were obtained by nano-electrospray tandem mass spec trometry. Mouse expressed sequence tags containing two of these peptides we re employed for oligonucleotide design and synthesis of probes by polymeras e 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 represente d the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also s howed 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-l ike motifs, and in Drosophila Slit is necessary for the development of midl ine glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and our peptide sequences cor respond 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 hepa ran sulfate chains, but the interaction appears to be relatively specific f or glypican-Fc insofar as no other identified heparin-binding proteins were isolated using our affinity matrix. Northern analysis demonstrated the pre sence 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 histochemist ry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where we have pr eviously also demonstrated glypican-1 mRNA and immunoreactivity, Our result s therefore indicate that Slit family proteins are functional ligands of gl ypican-1 in nervous tissue and suggest that their interactions may be criti cal for certain stages of central nervous system histogenesis.