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INTRODUCTION |
Glypican-1, whose primary structure was first reported based on
its cloning from human lung fibroblasts (1), was the initial member of
a rapidly expanding family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that is currently composed of four other
vertebrate proteins, cerebroglycan (glypican-2, Ref. 2), OCI-5
(glypican-3, Ref. 3), K-glypican (glypican-4, Ref. 4), and glypican-5
(5). We previously described a major heparan sulfate proteoglycan of
nervous tissue (6, 7) that we later cloned and identified as the rat
homologue of glypican-1 (8). Northern analysis demonstrated high levels
of glypican-1 mRNA in brain and skeletal muscle, and in
situ hybridization histochemistry showed 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
(9). From this work and parallel immunocytochemical studies (9) we
concluded that glypican-1 is predominantly a neuronal product in the
late embryonic and postnatal rat nervous system. Glypican-1 was also
found to be a dual modulator capable of enhancing the mitogenic
response of fibroblast growth factor-1 but inhibiting the effects of
fibroblast growth factor-7 in keratinocytes (10), and it can inhibit
neurite outgrowth induced by amyloid precursor protein in
vitro (11).
Genetic studies provide additional support for a role of glypicans 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 (12), and the human homologue of
glypican-3/OCI-5 (GPC3) was found to be mutated in patients with the
Simpson-Golabi-Behmel overgrowth syndrome (13). We have also recently
demonstrated a novel nuclear localization of glypican-1 in nervous
tissue (14), suggesting that it may be involved in the regulation of
cell division and survival by direct participation in nuclear processes.
Because the functional roles of glypican-1 in nervous tissue remain
unknown, we have begun studies aimed at identifying ligands that may
aid in understanding how it is involved in developmental and other
neurobiological processes. By affinity chromatography of brain extracts
on a matrix in which a recombinant glypican-Fc fusion protein was
coupled to protein A-Sepharose, we isolated and partially cloned
proteins whose sequences allowed us to identify mammalian homologues of
the Drosophila Slit protein as ligands of glypican-1. Slit,
which was initially identified by cross-hybridization using the
sequence coding for tandem epidermal growth factor repeats of Notch, a
gene involved in Drosophila neurogenesis (15), is necessary
for development of midline glia and commissural axon pathways in
Drosophila (16). Although information has only very recently
become available concerning the functions of mammalian Slit proteins,
our results suggest that interactions of these presumably extracellular
proteins with cell surface glypican-1 may be important in axonal
pathfinding and nervous tissue histogenesis.
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EXPERIMENTAL PROCEDURES |
Preparation of Glypican-Fc Fusion Protein Affinity
Matrix--
Human embryonic kidney 293 cells were transfected with a
glypican-Fc fusion protein construct (14) using LipofectAMINE (Life Technologies, Inc.) and grown in serum-free Dulbecco's modified Eagle's medium containing 1% ITS+ (Collaborative
Biomedical Products, Bedford, MA). The conditioned medium was
continuously collected and frozen after centrifugation for 30 min at
27,000 × g and addition of sodium azide to a
concentration of 0.02%. The amount of the glypican-1 fusion protein in
aliquots of the conditioned medium was estimated by Coomassie Blue
staining following SDS-PAGE1
in comparison with bovine serum albumin standards, after binding to an
excess of protein A-Sepharose beads (Zymed Laboratories Inc.) using a ratio of 0.5 ml of medium/20 µl of settled beads and elution twice by sample buffer.
To determine the proportion of the glypican-Fc fusion protein that was
synthesized in a glycanated form, 293 cells in a six-well plate were
transfected with the fusion protein construct, and after 24 h
cells were washed twice with a short term labeling medium
(methionine/cysteine-free Dulbecco's modified Eagle's medium supplemented with 1% ITS+) followed by incubation for
1 h with the same medium. The medium was subsequently changed for
4 h to labeling medium containing 125 µCi of
[35S]methionine/cysteine. The glypican-Fc fusion protein
was purified from the conditioned medium by adsorption to protein
A-Sepharose beads, and an aliquot of the beads was digested with
heparitin-sulfate lyase (EC 4.2.2.8, Seikagaku America, Rockville, MD)
at a concentration of 50 milliunits/ml in 0.1 M Tris
buffer, pH 7.2, for 3 h at 37 °C and compared with another
aliquot incubated with buffer alone. (Enzyme assays using bovine kidney
heparan sulfate as substrate and measurement of unsaturated
disaccharide products by their absorption at 232 nm demonstrated that
the heparitinase retains >85% of its initial activity after 6-h
incubation under these conditions.) Both aliquots were boiled in sample
buffer; proteins were separated by SDS-PAGE on a 5% gel; and after
soaking for 30 min in 200 ml of 1 M sodium salicylate, the
gel was dried and bands were quantitated by fluorography using the
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Pools of conditioned medium were later thawed and concentrated by
pressure ultrafiltration on an Amicon PM-30 membrane in a stirred cell.
After reclarification by centrifugation (30 min, 27,000 × g), the glypican-Fc fusion protein was bound to protein A-Sepharose beads with gentle mixing at 4 °C overnight, using a
ratio of 1 mg of fusion protein (in 2-5 ml of concentrated medium)/ml of settled beads. The beads were thoroughly washed with cold PBS followed by 50 mM Tris-buffered saline, pH 8.0, containing
1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. After
re-equilibration in 0.1 M phosphate buffer, pH 8.0, the
glypican-1 fusion protein was cross-linked to the protein A (17) at
room temperature using a 30-fold molar excess of dimethyl pimelimidate
(Pierce) added four times at 10-min intervals. The coupling reaction
was terminated by washing the beads with 1 M Tris-glycine
buffer, pH 7, followed by PBS, and the beads were stored in PBS
containing 0.02% sodium azide. Before each use of the affinity matrix,
any free fusion protein was removed by washing with 1 M
NaCl in 50 mM PBS followed by 0.1 M glycine,
and the beads were then again equilibrated in 50 mM
PBS.
Affinity Chromatography of Rat Brain Extracts--
In an initial
experiment to determine which subcellular fractions might be enriched
in glypican-1 ligands, brains of 30- to 130-day-old Sprague-Dawley rats
were homogenized using a Teflon-glass tissue grinder in 4 volumes of
Tris-HCl buffer, pH 8.25 at 4 °C, containing 0.15 M NaCl
and protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 50 mM EDTA, and 100 mM 6-aminohexanoic acid). The homogenate was centrifuged
for 10 min at 1,000 × g, and the supernatant (S1) was
saved. The pellet was resuspended in the original volume of buffer,
recentrifuged, and the pellet saved as P1. The S1 fraction was
centrifuged for 30 min at 45,000 × g, the pellet (P2)
saved, and the supernatant centrifuged for 1 h at 200,000 × g to yield a soluble fraction (S3) and pellet (P3). The P2
and P3 pellets were combined to give a mitochondrial/microsomal fraction (P2/3). The P1 and P2/3 pellets were resuspended in the original Tris-HCl buffer (2.75 ml/g original brain) containing 1%
CHAPS, 0.2 M NaCl, 1 mM EDTA, 10 µM pepstatin A, and 10 µM leupeptin and
extracted with stirring overnight at 4 °C. After centrifugation for
1 h at 200,000 × g, the supernatants were saved as fractions S4N (a crude "nuclear" extract) and a membrane extract (S5M), respectively.
Later studies aimed at the isolation and amino acid sequencing of
ligands used a simplified procedure. Brains of 30-40-day-old rats were
homogenized in 4 volumes of 25 mM PBS, pH 7.2, containing 5 mM EDTA, 100 µM phenylmethylsulfonyl
fluoride, 10 µM leupeptin, and 10 µM
pepstatin. The homogenate was centrifuged for 10 min at 1,000 × g and washed once. The washed P1 pellet was then extracted by stirring overnight at 4 °C in PBS with protease inhibitors as
described above but with the addition of 1% CHAPS, using 2.75 ml/g
brain, and the extract was centrifuged for 2 h at 200,000 × g. The resulting supernatant (designated S3N) was then used for affinity chromatography, after filtration through a pre-column of
Sepharose CL-4B beads to remove any remaining particulate material or
proteins that bind nonspecifically to the Sepharose.
Beads containing the glypican-Fc fusion protein were equilibrated in 50 mM PBS and mixed overnight with the brain extract at
4 °C using gentle agitation, in a ratio of 10 ml of brain extract (3.6 g of brain)/ml of beads. Unbound proteins were removed by centrifugation followed by four washes (3 bead volumes each) with PBS.
The beads were then transferred to a column, and bound proteins were
eluted with 1 M NaCl. (Initial trials demonstrated that no additional protein was released by subsequent elution with 50 mM diethylamine, pH 11.5, in 0.15 M NaCl or by
0.1 M glycine, pH 3.) Proteins eluted with 1 M
NaCl were concentrated by adsorption to StrataClean resin (Stratagene)
using 10 µl of beads/10 ml of eluate and released by boiling in
SDS-containing sample buffer before use for SDS-PAGE (18).
Peptide Sequencing by Mass Spectrometry--
Proteins in the 1 M NaCl eluate from the glypican-1 affinity column were
electrophoresed on several lanes of an 8% 1-mm minigel, silver-stained
using a protocol suitable for subsequent sequencing (19), and stored at
4 °C. A major band with an apparent molecular size of ~200 kDa was
excised from two to three lanes of the gel and digested in
situ with trypsin (19). The complete tryptic peptide mixture was
desalted and concentrated (20) on an Eppendorf Geloader pipette tip
(Brinkman) packed with Poros RII resin (PE Biosystems, Framingham, MA)
and eluted with 2 µl of 50% methanol in 5% formic acid into a
nano-electrospray sample needle. The unfractionated digest was analyzed
by nano-electrospray (21) on a QTOF mass spectrometer (Micromass,
Manchester, United Kingdom), and partial or complete sequences from 16 peptides were obtained by tandem mass spectrometry (22). Amino acid
sequences of nine or more residues from 10 different peptides were used
to search protein or EST data bases for matches.
cDNA Library Screening--
The peptide sequences obtained
by mass spectrometry were compared with the nucleotide sequences in the
nonredundant GenBankTM data base (EST Division), translated
in all six reading frames using the NCBI BLAST server
(http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast?Jform=1). Two
translated EST sequences matched perfectly to our peptides 3 and 8 and
were designated P3EST and P8EST, respectively. P3EST was amplified by
PCR from a rat brain stem/spinal cord cDNA library (Stratagene)
using primers designed from the EST sequence (sense, 5'-CCATCGATGCACCTCAGAGTTCTTCAGCTC-3'; antisense,
5'-GCTCTAGACAAGTTGTTCGAGTGGAGTCG-3') and subcloned in pBluescript.
P8EST could not be amplified from any of our three available rat brain
cDNA libraries but was amplified from a neonatal mouse brain
cDNA library (Stratagene) using primers designed from the EST
sequence (sense, 5'-CCATCGATTCCTTGACGTGGCATCCCTG-3'; antisense,
5'-GCTCTAGACTCTATCACAGCTGTCCCCG-3') and subcloned in pBluescript.
The P3EST sense and antisense primers were also used for PCR screening
of amplified pools from a 6-week rat brain and an adult rat brain
stem/spinal cord cDNA library (Stratagene). Plaques from
PCR-positive pools were screened using a 400-base pair
XbaI/ClaI restriction fragment of
pBluescript/P3EST that was labeled in the presence of
[
-32P]CTP (18 mCi/ml) using the Klenow fragment of DNA
polymerase I, after purification by agarose gel electrophoresis and
QIAEX extraction (Qiagen Inc., Chatsworth, CA).
Plaques were transferred to nitrocellulose filters, and DNA was
immobilized by baking in vacuo. Filters were hybridized
overnight at 42 °C and washed three times at room temperature with
2 × SSC, 0.1% SDS and twice at 65 °C with 0.2 × SSC,
0.1% SDS. Supernatants of positive plaques were first screened by PCR
using the antisense primer of P3EST in combination with T3 or T7 vector
primers, and clones showing the largest insert sizes were subjected to
a second screening. Individual plaques were lifted and the plasmids
isolated by in vivo excision.
Northern Analysis--
pBluescript/P3EST and pBluescript/P8EST
were linearized by ClaI digestion and transcribed into
digoxigenin-labeled antisense RNA with T3 RNA polymerase (Promega)
using the GENIUS 4 RNA labeling kit (Roche Molecular Biochemicals). The
resulting probes were used for hybridization with Northern blots and
for in situ hybridization histochemistry.
mRNA from rat tissues and rat C6 glioma cells was prepared using
the FastTrack mRNA isolation kit (Invitrogen Corp., San Diego, CA).
Hybridization, washing, and detection with alkaline
phosphataselabeled anti-digoxigenin antibodies were performed
as described previously (23).
In Situ Hybridization Histochemistry--
The
digoxigenin-labeled sense RNA probes were prepared as described above
except that plasmids were linearized by XbaI and transcribed
with T7 RNA polymerase. In situ hybridization histochemistry was performed as described by Engel et al. (24).
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RESULTS |
Affinity Chromatographic Isolation of Glypican-1
Ligands--
Initial experiments surveyed extracts prepared from
several subcellular fractions of brain (see "Experimental
Procedures") for the presence of glypican-1 ligands, which were
detected predominantly in the "crude nuclear fraction." This
fraction was therefore used for all subsequent studies. Specific
ligands were considered to be those proteins that bound only to the
glypican-Fc fusion protein affinity beads, but not to protein
A-Sepharose beads that did not contain the fusion protein. SDS-PAGE
analysis and silver staining revealed a major specific ligand with an
apparent molecular size of ~200 kDa (Fig.
1A), and a second band with a
slightly slower mobility could frequently also be resolved (data not
shown).

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Fig. 1.
Identification of specific glypican-1 ligands
by affinity chromatography. A, silver staining of 10%
SDS-PAGE gels showing two proteins, migrating at ~200 and ~22 kDa,
that were isolated from rat brain using a glypican-Fc fusion protein
affinity column, but do not appear in the 1 M NaCl eluate
when the same brain extract was applied to protein A-Sepharose.
B, digestion of the fusion protein affinity beads with
heparitinase (+) abolished binding of the 200-kDa protein, whereas
there was no effect of incubating the fusion protein beads under the
same conditions with buffer alone ( ).
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Methionine/cysteine labeling of the glypican-Fc fusion protein secreted
by transfected 293 cells followed by SDS-PAGE before and after
heparitinase treatment showed that approximately 40% of the fusion
protein is glycanated (data not shown). These results indicate that in
comparison with endogenous glypican-1 in rat brain and C6 glioma cells
(14), the addition of heparan sulfate chains in transfected 293 cells
lagged considerably behind the high level of expression of the
glypican-1 core protein. Interaction of the 200-kDa ligand with the
glypican-1 affinity matrix is at least partially mediated by the
heparan sulfate chains, insofar as binding is abolished after treatment
of the beads for 5 h with heparitinase (Fig. 1B).
However, the 200-kDa band was not detected in eluates of brain proteins
bound to heparin-agarose using identical conditions (data not shown).
Another specific ligand with an apparent molecular size of ~22 kDa
was also detected, and at least five major bands were seen in the
40-70 kDa range (Fig. 1A), but since these latter proteins bound equally well to protein A-Sepharose beads that did not contain the fusion protein they probably represent rat immunoglobulins or other
nonspecific ligands. No sequence could be obtained from the 22-kDa band
after transfer to a ProBlott membrane, indicating that it was
N-terminally blocked.
Peptide Sequences Deduced from Mass Spectrometric Data--
The
200-kDa band stained poorly with Coomassie Blue and sufficient amounts
could not be obtained for transfer to a membrane for N-terminal Edman
sequencing or for protease treatment and high performance liquid
chromatography fractionation of peptides. We therefore used
nano-electrospray collision-induced dissociation-tandem mass
spectrometry (nano-electrospray CID-MS/MS), a more sensitive peptide
sequencing technique that is capable of yielding useful sequence at the
femtomole level from single silver-stained polyacrylamide gel bands
(21). When applied to an unfractionated tryptic digest derived from
several lanes of the 200-kDa protein, MS/MS spectra for 17 peptides
were generated (Fig. 2A). From
these data, 16 complete or partial peptide sequences could be deduced
(Table I). The MS/MS spectrum of the
doubly charged [M + 2H]2+ peptide P8 ion is shown in Fig.
2B. The m/z 756.4 value for this ion
corresponds to a molecular weight of 1510.6 for the peptide. The
spectrum shows a nearly complete set of C-terminal sequence ions
(labeled yn) beginning with fragment
y1 that contains only Arg and ending with
fragment y10 that includes all of the residues
up to and including Tyr. Although this amount of sequence was
sufficient to identify the 200-kDa protein (see below), additional sequence information can be obtained by interrogating the mass difference between the highest observable yn ion and the molecular mass of the peptide. This difference corresponds to the
sum of the in-chain masses of any N-terminal amino acids not yet
accounted for by the MS sequencing (most often the first two residues).
In the example shown (Fig. 2B), the difference of 227 Da can
correspond to only three possible pairs of amino acids (Table I,
considering Leu and Ile as a single possibility, and assuming no
posttranslational modifications). In this case, the combination
N,X (X = L) matched the first two residues
for the hit from the data base (Fig. 3).
At low m/z, several N-terminal sequence ions
(labeled bn) are also evident, and the m/z value of the b2 ion
confirms the residual mass of the first two N-terminal residues
(b2 = 227 + H).

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Fig. 2.
Peptide sequencing of the 200-kDa SDS-PAGE
band by nano-electrospray mass spectrometry. A, ES-MS
spectrum of the unfractionated total tryptic digest after microcolumn
desalting. This spectrum is a mixture of predominantly single, doubly,
and triply charged peptide ions. Peptide ions marked with either or
* were sequenced by CID-MS/MS. B, CID-MS/MS spectrum of the
doubly charged ion at m/z 756.4 (*). The
10-residue partial sequence shown in the inset was
determined from the yn series labeled on the
spectrum (see text for details) and was used to search the dbEST
expressed sequence tag data base (see Ref. 25 for nomenclature).
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Table I
Peptide sequences obtained by nano-electrospray tandem mass
spectrometry
For P1-10, the number in parentheses at the beginning of the sequence
is the mass of the residual N-terminal amino acids (usually two
residues) that could not be individually identified. Combinations of
amino acids that will fit this mass are shown below the sequence,
separated by commas to indicate that their order is unknown. In the
sequences, X stands for either leucine or isoleucine, and
glutamine residues (Q) are distinguished from lysine (K) based on the
assumption that if this residue was lysine trypsin should have cut
there. For P11-16, the sequences were assigned by fitting the peptide
fragment ion data to the Slit protein sequences (Fig. 3). C* indicates
acrylamide-modified cysteine.
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Fig. 3.
Alignments of peptide sequences obtained from
the 200-kDa glypican-1 ligand with those of Slit proteins. The
numbering refers to amino acids in rat Slit-1/MEGF4. M8EST represents
the sequence that was amplified by PCR from a mouse brain cDNA
library using primers designed from the mouse EST sequence. Peptides
derived from the 200-kDa glypican-1 ligand are numbered as in Table I,
and their sequences are underlined (except for peptide 9).
Peptide 6 corresponds to the human keratin sequence (data not shown)
and was presumably a result of contamination, whereas peptide 9 does
not match perfectly with any reported protein and may represent either
a new member of the Slit family or reflect amino acid
polymorphism.
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Using 3-4 residues of sequence data together with mass information
(residual N- and C-terminal masses and peptide molecular weights), we
generated short MS-derived peptide sequence tags (26) for the 16 peptides. These were searched against the current nonredundant protein
data bases revealing that except for peptide 6, which is derived from
keratin, there was no significant identity with previously identified
proteins. Ten of the MS/MS spectra yielded sequences of nine or more
amino acids (two of the peptide sequences differed by only a single
amino acid), which we used to search EST data bases. This approach
found ESTs from mouse embryo and myotube that matched peptides P3 and
P8 (GenBankTM accession numbers AA396603 and AA645364, respectively).
Cloning of the 200-kDa Ligand (Rat Slit-2)--
Using the EST
sequences for primer design and rat and mouse brain cDNA as
templates, we amplified by PCR the corresponding rat and mouse
sequences (designated P3EST and P8EST, respectively) and used the P3EST
as a probe to screen a rat brain cDNA library. The longest clone
obtained from screening (designated 1131, GenBankTM
accession number AF141386) and the P8EST amplified from a mouse brain
cDNA library show 91-93% amino acid identity to human Slit-2
(27), indicating that our partial sequences represent rat and mouse
homologues of Slit-2 (Table II). Because
some of our peptide sequences match better with MEGF4/Slit-1 than with Slit-2 (Fig. 3), it would appear that the peptide sequences in the
200-kDa gel band are derived from more than one Slit protein and that
glypican-1 can bind to both Slit-1 and Slit-2 and possibly also to
other related proteins.
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Table II
Comparison of sequence similarities among members of the slit protein
family
Based on sequence comparisons, it is likely that both clone 1131 obtained by cDNA library screening using rat P3EST as a probe, and
the P8EST amplified by PCR from a mouse brain cDNA library,
represent the Slit-2 protein, whereas the two recently reported genes
designated MEGF4 and MEGF5 (Ref. 28) appear to be rat homologues of
Slit-1 and Slit-3, respectively. Numbers in parentheses indicate the
number of amino acids or nucleotides in the sequences that were used
for comparison. High degrees of identity are noted in bold, and the
prefixes h and r in the column headings refer to the human and rat Slit
genes. The GenBankTM accession numbers for the rat and human Slit/MEGF
proteins are: rat Slit-1, AB017170; rat Slit-2 (clone 1131), AF141386;
rat MEGF4, AB011530; rat MEGF5, AB011531; human Slit-1, AB017167; human
Slit-2, AB0177168; human Slit-3, AB017169.
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Rothberg et al. (16) compared the LRRs and the conserved N-
and C-terminal sequences surrounding the LRRs of the
Drosophila Slit protein with other proteins containing LRRs.
Similarity was found in several proteins involved in adhesive events
such as the oligodendrocyte-myelin glycoprotein and the Toll protein of Drosophila, as well as with other proteins involved in
extracelluar protein-protein interactions such as the platelet
glycoproteins IX, Ib
, and Ib
and small leucine-rich proteoglycans
including decorin, biglycan, and fibromodulin. These conserved
sequences flanking the LRRs can also be found in our partial sequence
of rat Slit-2 (Fig. 4).

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Fig. 4.
Deduced amino acid sequence of clone
1131. The putative signal peptide is shown in italics
and potential N-glycosylation sites by
arrowheads. The P3EST sequence is indicated in
bold, and the sequences of peptides 1 and 3 are
boxed. The underlined sequences represent the
LRRs, which match the first three of four LRRs of the Slit/MEGF family,
and the conserved N- and C-terminal sequences surrounding the LRRs are
indicated by open and filled bars,
respectively.
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Tissue Distribution and Cellular Sites of Synthesis of
Slit-2--
P3EST and P8EST were used as templates to transcribe
digoxigenin-labeled riboprobes for use in Northern analysis and
in situ hybridization histochemistry. Northern analysis
showed that both probes hybridized with 7.6- and 8.5-kb bands present
in rat brain and C6 glioma cell mRNA (Fig.
5), confirming that both peptides (and
ESTs) are derived from the same gene. Non-nervous tissues, including
skeletal muscle, showed no message with the exception of lung, possibly
due to the presence of bronchial smooth muscle.

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Fig. 5.
Expression of Slit-2 in rat tissues.
Northern blots of mRNA from 7-day and adult brain, C6 glioma cells,
and adult liver, heart, spleen, kidney, lung, and muscle were probed
with digoxigenin-labeled P3EST and P8EST RNA transcripts.
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In the hippocampus at 1 month postnatal, Slit-2 mRNA is present in
CA1 and CA3 pyramidal neurons and in granule cells of the dentate gyrus
(Fig. 6A), and consistent with
the previously reported expression of glypican-1 in cerebellum (9),
Slit-2 mRNA is seen in cerebellar granule cells (Fig.
6B). Although there is a weaker signal in white matter,
which is probably in oligodendrocytes, glial cells do not appear to be
a major source of Slit-2. In cerebrum at embryonic day 19 (E19), Slit-2
mRNA is seen primarily in the cortical plate and subplate (but not
in the intermediate cortical layer), as well as in the thalamic nuclei,
hippocampal formation, caudate putamen (Fig. 6C), and in the
subcommissural organ (data not shown). In spinal cord at E13 and E16,
Slit-2 mRNA is present predominantly in the ependymal and mantle
layers, the floor plate, and dorsal root ganglia (Fig.
7A), and in E16 brain Slit-2
mRNA is detected in the trigeminal ganglion and the ventricular
zone, including the entire ganglionic eminence (Fig. 7B).
Slit-2 expression can be also seen in the embryonic retina (Fig.
7C) and optic stalk (data not shown).

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Fig. 6.
Localization of Slit-2 mRNA in rat
postnatal hippocampus and cerebellum and embryonic cerebrum.
In situ hybridization histochemistry of P28 rat hippocampal
formation (A) shows mRNA in neurons of the dentate gyrus
(DG) and the CA1-CA3 regions. In P8 cerebellum
(B) the mRNA is primarily present in granule cells of
both the external (arrow) and internal
(arrowhead) granule cell layers. In E19 cerebrum
(C) the mRNA is seen primarily in the cortical plate
(CP) and subplate (arrows), as well as in the
ventricular zone (VZ), hippocampal formation (H)
and the caudate putamen (CA). Note that the intermediate
cortical layer (ICL) does not show Slit-2 message.
Bars, 300 µm.
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Fig. 7.
Cellular sites of synthesis of Slit-2 in the
embryonic rat nervous system. In E13 spinal cord (A)
Slit-2 mRNA is present predominantly in the ependymal layer
(EL) and mantle layer (ML) and in the dorsal root
ganglion (DRG). At E16 (B) Slit-2 mRNA is
detected in the ventricular zone of the brain (arrows),
including the entire ganglionic eminence (GE) and in the
trigeminal ganglion (TG). Slit-2 expression can also be seen
in the retina at E16 (C). Bars: A, 100 µm; B, 500 µm; C, 200 µm.
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DISCUSSION |
By affinity chromatography of rat brain extracts on a matrix
containing a glypican-Fc fusion protein, we identified a 200-kDa ligand
whose peptide sequences did not match any sequences then available in
the data bases. During the course of our cloning of this ligand, other
data became available indicating that the 200-kDa SDS-PAGE band
contained peptide sequences derived from at least two proteins (rat
Slit-1 and Slit-2) that are mammalian homologues of the
Drosophila Slit protein. This overlapping of protein bands
is not surprising insofar as all five of the human and rat Slit
proteins that have been cloned up to now have very similar amino acid
sequences and differ in size by only 11 or fewer (out of ~1,500)
amino acids.
The Drosophila Slit protein is expressed by midline glia and
is distributed along commissural axons. Reduction in slit expression results in a disruption of the developing midline cells and the commissural axon pathways. The presence of a putative signal peptide and the lack of a transmembrane domain together with other structural features indicate that the Slit proteins are secreted extracellular matrix proteins. Although Slit was not detected in a Tris-buffered saline extract of rat brain, like other extracellular matrix components it may be tightly bound to other cell surface or extracellular proteins
and require detergents or dissociative conditions for extraction.
The amino acid sequence of the Slit proteins can be divided into four
domains. These consist of four N-terminal LRRs, seven to nine epidermal
growth factor-like repeats (seven in Drosophila and nine in
vertebrates), a motif with a high degree of identity to agrin, laminin,
and perlecan (designated the ALPS domain, Ref. 29), and a C-terminal
cysteine-rich domain. LRRs are found in a number of intracellular and
extracellular proteins and contribute to protein-protein interactions
and cell adhesion (30, 31). The epidermal growth factor-like motif has
been identified as an extracellular binding domain involved in cell
adhesion and receptor-ligand interactions (32), the ALPS domain is
responsible for protein-protein interactions and self-aggregation of
agrin, laminin, and perlecan (for a review, see Ref. 29), and the
cysteine-rich domain is considered to be essential for dimerization of
proteins such as von Willebrand factor (33). It is likely that the Slit proteins function to link multiple ligands and thereby mediate cell
interactions. In view of the complex domain structure of the Slit
proteins and their heparitinase-sensitive interactions with glypican-1,
it will be important to identify which protein domain(s) may also be
involved in this binding.
During our MS-based sequencing experiments, we attempted CID-MS/MS on a
2,011-Da peptide from the 200-kDa protein tryptic digest. Although we
were unable to generate any sequence-specific fragment ions, the
peptide readily lost neutral fragments characteristic of tyrosine
phosphorylation (34). These preliminary data therefore suggest that
Slit proteins may be phosphorylated.
We demonstrated that two major rat Slit-2 mRNA species (8.6 and 7.5 kb) are expressed in adult brain and lung and in rat C6 glioma cells
and that the localization of this mRNA in the postnatal hippocampal
formation and developing cerebellum is similar to that of glypican-1.
In contrast to these results, Northern analysis of human Slit-2 showed
a single 8.5-kb mRNA expressed predominantly in adult spinal cord
but also in fetal lung and kidney, and a 9-kb rat MEGF5/Slit-3 message
was seen in brain, whereas a major 5.5-kb mRNA and a minor 9.5-kb
species of human Slit-3 were found in adult endocrine tissues but were
not detectable in brain (27). Although human Slit-1 has the same
expression pattern as that of rat Slit-1, there may be species
differences in the tissue distribution and alternative splicing of
Slit-2 and Slit-3 in humans as compared with rodents. It is also
noteworthy that whereas Drosophila Slit is expressed only by
glia, rat MEGF4/Slit-1 and Slit-2 are predominantly neuronal products
(Ref. 27 and Figs. 6 and 7).
In adult brain, rat MEGF4/Slit-1 is expressed in the hippocampus,
cerebral cortex, and olfactory bulb but not in the cerebellum (27),
where we detected Slit-2 mRNA. Because our various peptide sequences match both Slit-1 and Slit-2, it would appear that at least
two Slit proteins were isolated by our affinity chromatographic procedure and that glypican-1 functions in nervous tissue may be
mediated by its differential interactions with two or more members of
the Slit protein family. Recent genetic and biochemical studies of
Drosophila and mammals demonstrated that Slit proteins bind
Robo, a repulsive guidance receptor on growth cones (35-37). At least
one of the mammalian Slit proteins, Slit-2, is a repulsive molecule for
olfactory bulb axons (37), embryonic spinal motor axons (35), and
developing forebrain axons (38). Interestingly, a 140-kDa N-terminal
fragment of Slit-2 was purified from a tissue extract that stimulates
the elongation and branching of sensory axons and shown to be
responsible for these effects, whereas full-length Slit-2 does not have
this activity (39). Although it was also demonstrated that heparin can
release Slit-2 from the cell surface (35), we found that Slit proteins
could not be isolated from our brain extract when a heparin-agarose
matrix was substituted for the glypican-Fc fusion protein affinity
column. Because the 140-kDa N-terminal fragment of Slit-2 was not
detected in our affinity column eluate, it is possible that glypican-1
binds full-length Slit proteins via both its core protein and the
heparan sulfate chains and that this interaction regulates the
proteolysis and thereby the generation of biologically active fragments
of Slit proteins. The identification of interactions between glypican-1 and Slit proteins therefore not only provides the most direct evidence
yet available for an involvement of glypican-1 in nervous tissue
development, but also suggests a possible regulatory mechanism underlying the dual functionality of Slit proteins.