Mammalian Homologues of the Drosophila Slit Protein Are Ligands of the Heparan Sulfate Proteoglycan Glypican-1 in Brain

• Published in: The Journal of biological chemistry Vol. 274; no. 25; pp. 17885 - 17892
• Main Authors: Liang, Y, Annan, R S, Carr, S A, Popp, S, Mevissen, M, Margolis, R K, Margolis, R U
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 18-06-1999
• Subjects: Amino Acid Sequence; Animals; Brain - metabolism; Cloning, Molecular; Drosophila - metabolism; Drosophila Proteins; Heparan Sulfate Proteoglycans - metabolism; Humans; Ligands; Mice; Molecular Sequence Data; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Peptide Fragments - chemistry; Rats; Recombinant Fusion Proteins - genetics; RNA, Messenger; Sequence Alignment
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.25.17885

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.