From the Rheinische
Friedrich-Wilhelms-Universität Bonn, Institut für
Physiologische Chemie, Nussallee 11, D-53115 Bonn,
§ Universitätsklinikum,
Heinrich-Heine-Universität Düsseldorf, Neurologische
Klinik, Moorenstrasse 5, D-40225 Düsseldorf,
Anatomisches Institut, Nussallee 10, D-53115 Bonn, and
** Biologisch-Medizinisches Forschungszentrum,
Heinrich-Heine-Universität Düsseldorf,
Universitätsstrasse 1, D-40225 Düsseldorf, Germany
Received for publication, October 4, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Chondroitin sulfate proteoglycans are
structurally and functionally important components of the extracellular
matrix of the central nervous system. Their expression in the
developing mammalian brain is precisely regulated, and cell culture
experiments implicate these proteoglycans in the control of cell
adhesion, neuron migration, neurite formation, neuronal polarization,
and neuron survival. Here, we report that a monoclonal antibody against
chondroitin sulfate-binding proteins from neonatal rat brain recognizes
collapsin response mediator protein-4 (CRMP-4), which belongs to a
family of proteins involved in collapsin/semaphorin 3A signaling.
Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate
affinity columns, and CRMP-specific antisera co-precipitated
chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were
found to be localized immuno-histochemically in overlapping
distributions in the marginal zone and the subplate of the cerebral
cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is
strongly expressed in the upper cortical plate of neonatal rat where
cell death is abundant. Therefore, naturally occurring cell death is a
plausible mechanism that targets CRMPs to the extracellular matrix at
certain stages of development. In summary, our data indicate that
CRMPs, in addition to their role as cytosolic signal transduction
molecules, may subserve as yet unknown functions in the developing
brain as ligands of the extracellular matrix.
The extracellular matrix
(ECM)1 of the developing
brain has a unique composition from lecticans, tenascins, laminins,
and hyaluronic acid (1-5). Lecticans are proteoglycans that carry
chondroitin sulfate (CS) side chains on core proteins encompassing a
N-terminal hyaluronan binding domain and a C-terminal lectin domain.
Four different members of the lectican protein family are known
(neurocan, aggrecan, versican, and brevican) (1). Their
multidomain composition enables lecticans to interact with multiple
cell surface molecules and diffusible ligands (see below) (6,
7). Chondroitin sulfates are glycosaminoglycans composed of repeated
glucuronic acid-[ The functions of chondroitin sulfate proteoglycans (CS-PGs) in neural
tissue can be categorized into effects on cell adhesion, cell
migration, neurite formation, neuron polarization, synaptic modulation/plasticity, and neuron survival (for reviews, see Ref. 6 and
references therein and Ref. 11). These effects often critically depend
on glycosaminoglycans or may even be attributed solely to
glycosaminoglycan chains. Many of the listed functions, however, have
been discovered using in vitro experiments that were
designed in a way that the proteoglycan or glycosaminoglycan component
became limiting in the assays. In vivo, on the other hand,
there is marked structural redundancy of ECM components (1, 3) if one
considers for example the existence of four different lecticans (see
above). Thus, phenotypes of knockout animals lacking a single
ECM protein are often rather mild (for example in knockouts for
neurocan (12) and tenascin-C (13, 14)). Inactivation of enzymes
involved in CS biosynthesis, on the other hand, could lead to more
severe phenotypes since CS is a component of multiple ECM proteins. In
the chondroitin-6-sulfate transferase knockout mouse, however, no major
CNS pathology was found (15). C-6-S may be replaced by C-4-S, and
expression data suggest that C-4-S is probably more important in the
developing brain. Nevertheless, the importance of CS in vivo
is underscored impressively by the observation that after local
treatment with chondroitinase ABC, which degrades C-4-S, C-6-S, and
dermatan sulfate, regeneration of functional neurites in the adult
spinal cord is enabled (16). Thus, CS-PGs are considered to contribute to the inhibition of regenerative responses in the adult mammalian nervous system.
The current insight into the mechanisms of how CS acts on neurons is
still rudimentary. Binding partners of CS-PGs at the plasma membrane
include sulfatide and several (eventually
glycosylphosphatidylinositol-anchored) cell adhesion molecules
of the Ig family such as N-CAM, L1, TAG-1, and F3/contactin (for a
review, see Ref. 6). The signaling events exerted after binding of
CS-PGs to these molecules are unknown. On the other hand, CS-PGs bind a
variety of soluble ligands including growth factors like bovine
fibroblast growth factor and oligomeric glycoproteins of the ECM-like
tenascins (6). Interestingly, CS is a critical component of a molecular
scaffold to which diffusible molecules are bound that convey inhibitory or promoting actions, e.g. on the adhesion of thalamic
neurons and the formation of neurites (17). The identity of these
diffusible molecules, however, is as yet unknown.
To elucidate the molecular basis of the neurotrophic actions of
chondroitin sulfate, we previously fractionated protein extracts from
neonatal rat brain on a chondroitin sulfate affinity column and used
the eluted binding proteins to generate monoclonal antibodies (18). One
of these antibodies, termed mAb-9, recognizes a 65-kDa protein with
laminar expression in the neocortex, which parallels the expression of
CS. The protein is present in both the fraction of soluble proteins and
in the particulate fraction of neonatal rat brain. The aim of the
present study was to identify this chondroitin sulfate-binding protein
and to characterize its interaction with glycosaminoglycans. We show
that mAb-9 recognizes a soluble protein that is present in the cytosol,
termed collapsin response mediator protein-4 (CRMP-4). This protein and
its relatives interact with chondroitin sulfate, and they are released
from the cytosol of neurons to the extracellular space most probably
after cell death. This may explain why CRMP-4 was found to be
co-localized with chondroitin sulfate in the developing neocortex of
rat brain in regions where naturally occurring cell death is prevalent.
Unless otherwise stated, chemicals were from Serva (Heidelberg,
Germany), Sigma, Roche Molecular Biochemicals, or Merck. ZERO Blunt vector for PCR cloning was from Invitrogen, and pQE-30 vector for
bacterial expression of histidine-tagged proteins was from Qiagen
(Hilden, Germany).
SDS-PAGE and Western Blots--
SDS-PAGE was performed on
5-15% gradient slab gels (Bio-Rad Protean II) or 10% mini-gels
(Bio-Rad MiniProtean) (19). Gels were stained by silver (20), Coomassie
Blue, or zinc/imidazole (21). For Western blotting, proteins were
transferred to nitrocellulose in a semi-dry blotting apparatus
(Bio-Rad) according to Kyhse-Andersen (22). After blocking with 3%
nonfat dry milk powder, 1% bovine serum albumin in Tris-buffered
saline containing 0.05% Tween 20 (TBST), the first antibody incubation
was performed for 1 h at room temperature in TBST. Bound
antibodies were visualized after binding of peroxidase-labeled
secondary antibodies with enhanced chemiluminescence (ECL kit, Amersham Biosciences).
Protein Preparation--
Whole brains from neonatal Wistar rats
were shock-frozen in liquid nitrogen and homogenized in a Dounce
homogenizer in 10 mM HEPES, pH 7.4, 2 mM
MgCl2 (HEPES buffer) containing 2 mM Pefabloc, 1 mM leupeptin, and 1 mM pepstatin. The
homogenate was centrifuged at 10,000 × g for 15 min,
and the supernatant was subsequently centrifuged at 100,000 × g for 1 h to obtain soluble protein. Forty milligrams
of this protein material were loaded on a 1-ml Mono Q fast protein
liquid chromatography column (Amersham Biosciences), washed with 10 column volumes of loading buffer (HEPES buffer), and eluted stepwise
with 5 column volumes of HEPES buffer containing 100, 300, 500, and
2000 mM NaCl. One-milliliter fractions were collected and
analyzed by SDS-PAGE, silver staining and Western blot with mAb-9.
Western positive fractions were concentrated (Centricon10, Millipore)
and loaded on a 25-ml Superose-12 column (Amersham Biosciences). Again,
1-ml fractions were collected and analyzed by Western blotting. For
mass spectrometry, proteins were prepared according to the method of
Gevaert et al. (23). Briefly, the positive fractions were
concentrated by ultrafiltration and separated by SDS-PAGE. After
staining with zinc/imidazole the protein bands corresponding to the
Western signal of the monoclonal antibody were excised. After
destaining in 5% citric acid for 15 min and another 3 times for 15 min
in deionized water and incubation in SDS-PAGE sample buffer containing
0.1% SDS, 10% glycerol, 50 mM dithiothreitol, 12 mM Tris/HCl, pH 6.8, and 0.1% bromphenol blue for 1 h, the gel was cut into small pieces (5 × 5 mm) and loaded on top
of a concentration gel (5% acrylamide, 0.26% bisacrylamide, 125 mM Tris/HCl, pH 6.8, and 0.1% SDS) inside of a Pasteur
pipette (length, 145 mm). The pipette was transferred to an
isoelectro-focusing unit (Bio-Rad), and the gel pieces were carefully
overlayered with running buffer (50 mM Tris, 190 mM glycine, 0.1% SDS). Electrophoresis at 250 V was
continued until the bromphenol blue approached the lower edge of the
pipette. The gel was removed from the Pasteur pipette and stained with
Coomassie Blue, and the sharp blue protein band in the lower part of
the pipette was excised, destained, and stored at In-gel Digestion for Nanospray-ESI-MS--
The destained gel
piece was washed twice in digestion buffer (10 mM
NH4HCO3) for 15 min and twice for 15 min in
digestion buffer/acetonitrile 1:1 (v/v). The gel piece was re-swollen
with 2 µl of protease solution (trypsin at 0.05 µg/µl in
digestion buffer), and after 20 min another 10 µl of digestion buffer
was added. After digestion overnight at 37 °C the supernatant was collected and dried down to about 0.5 µl. For nanospray ESI-MS 2 µl
of 70% formic acid were added, and this solution was used in 0.5-µl aliquots.
ESI-MS--
ESI-MS was done as described (24) using a TSQ 7000 Triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA)
equipped with the standard ESI source or an in-house constructed
nanospray source. The ESI-voltage was between 0.6 and 1.1 kV for
nanospray. Mass spectra were acquired with a scan speed of 1000 Da/s.
For ESI-MS/MS analysis, argon at a pressure of 3 millitorr was used as
collision gas. Data acquisition and evaluation were done on a DEC work
station using the ICIS software, version 8.2.1. Peptide mass
calculation was done with the BIOWORKS software, version 8.2.1.
Peptide Mass Fingerprint Data Base Search--
Peptide masses
obtained from in-gel digestion were used for searching the
SwissProt.r34 data base with MS-FIT
(falcon.ludwig.ucl.ac.uk/msfit.htm). The standard parameters were:
species Rattus norvegicus, molecular mass 40-100 kDa,
tryptic digest with a maximum of 1 missed cleavage site. Peptide masses
were assumed to be monoisotopic, and cysteine was assumed to be not
modified. The allowed mass error was set at 0.1%.
PCR Amplification of CRMP-cDNAs--
First-strand cDNA
was amplified after reverse transcription of 3 µg of a mixture of
E17/P4 rat brain RNA using Superscript reverse transcriptase
(Invitrogen) according to the manufacturer's instructions using an
oligo-dT primer. After digestion with RNase H one-tenth of the reaction
was used as template for PCR amplification by Pfu polymerase
(Stratagene, La Jolla, CA) using 94 °C for 90 s, 55 °C for
45 s, 72 °C for 2 min for 30 cycles. The following primer sequences were used: CRMP-1(5'), 5'-ATGTCTCATCAGGGGAAGAAGAG-3', CRMP-1(3'), 5'-TATCTGGCGCATCTGAGGTCAACC-3', CRMP-2(5'),
5'-ATGTCTTATCAGGGGAAGAAAAAT-3', CRMP-2(3'),
5'-GCAGGCCTAGGAGCTTTAGCCCAG-3'), CRMP-3(5'),
5'-ATGTCCTTCCAAGGCAAGAAGAGCATTCCCCGGATA-3', CRMP-3(3'),
5'-TGCCAGACCCCAAGTCTAAGAAAG-3', CRMP-4(5'), 5'-CAGAATCGCCACCATGTC-3', CRMP-4(3'), 5'-GAGGGCTTAACTCAGGGA-3'. The PCR products were
cloned into the ZERO Blunt vector and sequenced using an ABI Prism 310 Genetic Analyzer.
Overexpression of CRMPs--
Bacterial overexpression in
Escherichia coli was performed using the Qiaexpress kit
(Qiagen) according to the instructions of the manufacturer. The coding
regions of the four CRMP-cDNAs were re-amplified using primers
containing restriction sites for SalI and HindIII
and the pCR Blunt vectors containing the CRMP inserts as templates. The
following primer sequences were used: CRMP-1(5')Sal,
5'-ACCTAGCGTCGACACATAGAAGGTAGAATGTCTCATCAGGGG-3'; CRMP-1(3')Hind,
5'-CGCCGCAAGCTTTATCTGGCGCATCTGAGG-3'; CRMP-2(5')Sal, 5'-ACCTAGCGTCGACACATAGAAGGTAGAATGTCTTATCAGGGG-3'; CRMP-2(3')Hind, 5'-CGCCGCAAGCTTGCAGGCCTAGGAGCTTTA-3'; CRMP-3(5')Sal,
5'-ACCTAGCGTCGACACATAGAAGGTAGAATGTCCTTCCAAGGC-3'; CRMP-3(3')Hind,
5'-CGCCGCAAGCTTTGCCAGACCCCAAGTCTA-3'; CRMP-4(5')Sal, 5'-ACCTAGCGTCGACACATGTCCTACCAGGGCAAGAAG-3'; CRMP-4(3')Hind,
5'-CGCCGCAAGCTTACTCAGGGATGTGATGTTAGA-3'. The PCR products were
gel-purified, digested with SalI and HindIII, and
ligated into the pQE-30 expression vector, thereby incorporating a 5'
extension of the cDNA coding for a His6 tag.
M15[pRep] bacteria were transformed, and ampicillin/kanamycin
resistant strains were analyzed for CRMP cDNA inserts and sequenced
to verify ligation sites and the PCR products. Positive strains were
grown in Luria Bertani medium containing kanamycin and ampicillin, and
expression was induced by 2 mM
isopropyl-1-thio- Antiserum Production, Affinity Purification of Antibodies, and
Biotinylation of IgG--
Antibodies raised in rabbit against the
synthetic peptides CRMP-4-pep (FDLTTTPKGGTPAGSTRGSPTRPN, rCRMP-4
residues 504-527) and CRMP-Fam-pep (SFYADIYMEDGLIKQIGDN, rCRMP-4
residues 30-48) were obtained from Pineda Antiköper
Service, Berlin, Germany. CRMP-1-pep
(YEVPATPKHAAPAPSAKSSPSKHQ, rCRMP-1 residues 504-527), CRMP-2-pep-a
(CEVSVTPKTVTPASSAKTSPAKQQ, rCRMP-2 residues 504-527), CRMP-2-pep-b
(GIQEEMEALVKDHGV, rCRMP-2 residues 147-161), CRMP-3-pep (HEVMLPAKPGSGTQARASCSGKIS, rCRMP-3 residues 496-519) were synthesized as described (25) and coupled to keyhole limpet hemocyanin as described
(26) via additional N-terminal cysteine residues. The
peptide-keyhole limpet hemocyanin conjugates were used to immunize New Zealand White rabbits (Lammers, Euskirchen, Germany). For
the first immunization 200 µg of peptide dissolved in Freund's complete adjuvant (Sigma) was injected subcutaneously. Each animal was
boosted twice at intervals of 4 weeks with the same amount of antigen
in incomplete Freund's adjuvant (Sigma). For the immunization protocol, special permission according to Section 8 of the German Law
on the Protection of Animals had been obtained from the
Bezirksregierung Köln. All rabbit antisera were used at a
dilution of 1:10,000 for Western blotting. Monospecific IgG was
purified by immunoaffinity chromatography with the peptides immobilized
on thiol-Sepharose (Amersham Biosciences) as described (26). Purified
IgG was ultra-filtered into 50 mM NaHCO3, pH
8.5. After the addition of 10 µl of 150 mM NHS-Biotin
(Pierce) the antibody solution was incubated at 4 °C overnight. IgG
was then separated from the unreacted free biotin by size exclusion
chromatography on NAP10 columns (Amersham Biosciences).
Glycosaminoglycan Affinity Chromatography--
Chondroitin
sulfate was coupled to EAH-Sepharose (Amersham Biosciences) as
described (18). As control columns, heparin Hitrap, SP Hitrap,
and CM Hitrap 1-ml columns (Amersham Biosciences) were used.
Approximately 10 mg of soluble neonatal rat brain proteins obtained
after ultracentrifugation of postnuclear supernatants at 100,000 × g for 1 h were filtered (0.45 µm) and
chromatographed on 1-ml analytical columns using an Äkta Explorer
equipment (Amersham Biosciences). Runs on CS columns and control
columns were carried out in parallel, taking advantage of the
column-scouting routine of the Unicorn 3.1 software using sequential
step elution with PBS containing 300 mM NaCl, 750 mM NaCl, 2 M NaCl, and 4 M
guanidinium hydrochloride (GuaHCl) as described (18). To remove the
salt from the eluent fractions and to concentrate the proteins, they were precipitated with acetone ( Immunoprecipitation--
1-ml aliquots of soluble neonatal rat
brain proteins prepared as described above were incubated with 1 µl
of the different peptide-specific CRMP antisera for 1 h at 4 °C
and with 10 µl of protein A-agarose (Calbiochem) for an additional
hour at 4 °C with continuous rocking. The precipitates were
collected by centrifugation, washed 3 times in phosphate-buffered
saline, dissolved in 100 µl of Laemmli buffer, and analyzed by
Western blotting with the biotinylated CRMP-Fam antibody or CS56
(Sigma) against CS.
Immunohistochemistry--
Before brain dissection, animals were
perfused with standard mammalian Ringer's solution, pH 7.4, followed
by 3.7% formaldehyde. Brains were post-fixed for 16 h and washed
extensively in tap water. After dehydration in a series of increasing
ethanol concentrations, brain tissue was embedded in paraffin, and
10-µm sections were cut on a microtome (Leica HM 355 S). Sections
were mounted on Histobond slides, dried for 2 days at 37 °C, and
used for immunohistochemistry. First, sections were deparaffinized and
hydrated by decreasing concentrations of ethanol in H2O.
Afterward, sections were incubated in boiling 2× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) for 20 min. After equilibrating in PBS, sections were treated with 1%
H2O2 in PBS for 10 min to remove endogenous
peroxidase activity. After permeabilization with 0.5% Triton X-100 in
PBS for 10 min, sections were washed with PBS and incubated in a 4% bovine serum albumin, PBS solution for at least 30 min. First, antibodies to CRMP-4 were used in a dilution of 1:1000, and for CS56
(Sigma), in a dilution of 1:200 (all in 4% bovine serum albumin, PBS).
Sections were incubated at 4 °C overnight. After washing, sections
were treated with the secondary biotinylated antibody diluted 1:200 in
4% bovine serum albumin, PBS, washed again, and incubated in a
streptavidin-peroxidase complex (ABC-kit, Vector). After a 1-h
incubation, sections were washed intensively and stained with
diaminobenzidine (0.05% in Tris-buffered saline). Counterstaining was
done with hemalum (Mayers hemalum, 1:6 dilution in
H2O, Merck) for 5 min followed by several washes in
H2O and a final wash in tap water. Pictures were taken with
a digital camera (Polaroid DMC Ie, Cambridge, UK) connected to a Zeiss
Axioskop 2 (Jena).
Cell Culture--
NTera-2 precursor cells (Stratagene) were
grown in Dulbecco's modified Eagle's medium/F-12 supplemented with
10% fetal calf serum (FCS), L-glutamine, and
penicillin/streptomycin. Cell death was induced by feeding the cells
with medium without FCS after repeated washings with serum-free medium.
Conditioned medium was harvested after 3 days and centrifuged at
1000 × g for 15 min to remove floating cells and
debris. For Western blot analysis, conditioned medium was subjected to
Q-Sepharose (Hitrap 1-ml column, Amersham Biosciences) chromatography
to capture CRMPs using elution with a linear gradient from 150 mM to 1 M NaCl in phosphate buffer.
Primary neocortical neurons were prepared as described (27) and plated
onto poly-D-lysine-coated 10-cm cell culture Petri dishes
(Falcon) or 6-well cell culture plates (Sarstedt). Cultures were
incubated at 37 °C in humidified 10% CO2, 90% air for
16-24 h and analyzed by phase contrast microscopy. To visualize the morphology of nuclei, cultures were fixed for 10 min in 4%
paraformaldehyde, washed with PBS, and stained briefly with
4,6-diamidine-2-phenylindole (100 µg/ml in PBS). Pictures were taken
with a digital camera (Axiovision, Zeiss) connected to a Zeiss Axiovert
100M, and images were processed with the Axiovision software (Zeiss,
Göttingen, Germany).
Analytical Procedures--
Protein determination was performed
with the Bradford assay or the detergent-compatible protein assay (both
purchased from Bio-Rad) using bovine serum albumin as the standard.
Lactate dehydrogenase activity was determined as described (28).
Purification of CRMP-4 from Neonatal Rat Brain--
The monoclonal
antibody mAb-9 used in this study recognizes a 65-kDa protein that is
abundant in the soluble fraction of neonatal rat brain (18). From this
material the 65-kDa protein was captured on a Mono Q anion exchange
column (Fig. 1). When steps of increasing ionic strength were applied to the column, about 30% of the
cross-reacting protein was eluted with 200 mM sodium
chloride, and the remaining 70% was released at 500 mM
sodium chloride according to Western blot analysis (Fig.
1C). Interestingly, the electrophoretic mobility of the
cross-reacting protein bands from the eluate fractions was slower in
comparison to the starting material (Fig. 1C). Because the
200 mM NaCl eluate contained a lower amount of
contaminating proteins than the 500 mM eluate, according to
silver-stained SDS-gels (Fig. 1B), fraction 7 from the Mono
Q column was subsequently fractionated using size exclusion
chromatography on a Superose 12 column (Fig.
2). From this column the cross-reacting
protein eluted after 11-12 ml, corresponding to a molecular mass of
~200 kDa (Fig. 2, A and C). Finally,
purification to homogeneity was achieved by preparative SDS-PAGE (data
not shown). In-gel digestion with trypsin yielded 22 peptides, which
were analyzed by ESI-MS. In the SwissProt.r34 rat sequence data base
(Table I) 10/22 peptide masses fitted to
a rat sequence homologous to "Turned on after division" protein of
64 kDa (TOAD-64 (29), also called collapsin response mediator protein-2
(CRMP-2 (30)). Extension of the search to the entire mammalian data
base (nrdb, EMBL Heidelberg), however, showed a more close match of
15/22 peptides to mouse CRMP-4 (mUlip, Unc33-like phosphoprotein (31)).
Furthermore, MS/MS analyses of three different peptides from the
spectrum confirmed that these were derived from the rat homologue of
CRMP-4. Because only a truncated cDNA sequence for rat CRMP-4 was
available in the data base, a chimeric sequence was assembled from the
available truncated rat CRMP-4 and mouse CRMP-4/mUlip. To this chimeric sequence 19 of the 22 obtained masses could be matched exactly (data
not shown). Thus, the mass spectrometric analyses indicate that mAb-9
recognizes collapsin response mediator protein-4 from rat brain.
Western Blot Analysis of Recombinant CRMPs with
mAb-9--
To confirm that mAb-9 recognizes CRMP-4 and to determine
the specificity of the antibody, the coding regions of rat CRMP-1-4 sequences were amplified by PCR, sequenced, and expressed as
N-terminally His-tagged fusion proteins in E. coli. All four
CRMPs were found in inclusion bodies. When equal amounts of the four
recombinant proteins were analyzed by Western blotting, all recombinant
proteins were detected with an anti-polyhistidine antibody (Fig.
3B). Recombinant CRMP-4
clearly reacted with mAb-9, whereas CRMP-2 was not detected even after
prolonged exposure of radiography films (Fig 3A). Moreover, CRMP-1 and -3 cross-reacted weakly with mAb-9 (Fig. 3A). In
summary, mass spectrometric experiments and the immunological analysis of recombinant CRMPs consistently demonstrated that mAb-9 recognizes an
epitope that is present on CRMP-4.
Characterization of Peptide-specific Antibodies against
CRMPs--
To distinguish different members of the CRMP family, we
produced peptide-specific polyclonal antisera. Peptides were designed from regions of the CRMP amino acid sequence that displayed marked divergence between the CRMPs. Furthermore, a sequence was selected that
is conserved among the CRMP family members to generate a pan-specific
antibody. After immunization of rabbits, five different antibodies were
obtained termed anti-CRMP-1 to -4 and anti-CRMP-Fam. Western blots of
the bacterially expressed recombinant CRMPs demonstrated binding of
anti-CRMP-Fam to all four recombinant CRMPs (Fig. 3C) and
mono-specific binding of the antibodies anti-CRMP-1, -2, and -4 to the
corresponding recombinant proteins (Fig.
4, A, B, and D). Anti-CRMP-3 strongly bound to recombinant CRMP-3 and
showed weak cross-reactions with CRMP-1 and -2 (Fig.
4C).
Interaction of CRMPs with Glycosaminoglycans--
The mAb-9 was
raised against chondroitin sulfate-binding proteins that were
solubilized with CHAPS from the particulate fraction of neonatal rat
brain. This raises the possibility that CRMPs could be cytosolic
associates of membrane-associated protein complexes that contain
receptors for ECM proteoglycans. In this case, soluble CRMPs might not
necessarily bind to chondroitin sulfate. Thus, we examined if CRMPs
from the soluble fraction of neonatal rat brain bind to chondroitin
sulfate-Sepharose columns. First, eluates from a
chondroitin-6-sulfate-Sepharose column were screened for the presence
of CRMPs using the peptide-specific antibodies. All four CRMPs bound to
the column (Fig. 5). Although substantial amounts of the bound CRMPs were released on washing of the column at
moderate stringency (Fig 5, lane 1 of each panel),
limited quantities of each CRMP remained on the column even after harsh washing with 2 M NaCl and were only eluted by the
chaotropic salt GuaHCl (Fig. 5, lane 4 of each panel).
Almost identical elution profiles of CRMPs were observed with a
chondroitin4-sulfate column (data not shown). As the control we
performed in parallel chromatographies on a heparin-Sepharose column
under exactly the same experimental conditions. Although all four CRMPs
bound quantitatively to the heparin column, they were eluted completely
by washing the column with 300 mM NaCl (Fig.
6, lane 1 of each panel), none
of the CRMPs was eluted with GuaHCl, as seen for both chondroitin
sulfate columns (Fig. 6, lane 4 of each panel). Taken
together, these chromatography profiles indicate a weak charge-mediated
interaction of CRMPs with heparin but tight binding of subclasses of
each CRMP to chondroitin sulfates.
To rule out the possibility that soluble CRMPs were retained on
chondroitin sulfate columns because they interact with putative contaminants of these glycosaminoglycans from their biological sources
(i.e. shark cartilage for C-6-S and bovine trachea for C-4-S), we carried out immuno-co-precipitation experiments as an
independent approach. Anti-CRMP4 and anti-CRMP-Fam precipitated detectable amounts of CRMPs (Fig.
7A). Western blot analysis
with the monoclonal antibody CS56 against chondroitin sulfate
demonstrated co-precipitation of characteristic smeary proteoglycan
bands after precipitation of soluble brain-derived proteins with
anti-CRMP4 and the anti-CRMP-Fam but not after incubation with a
preimmune serum (Fig. 7B). Thus, immuno-coprecipitation data
confirmed that soluble CRMPs bind to chondroitin sulfate.
Localization of CRMP-4 and Chondroitin Sulfate in Overlapping
Regions of the Cerebral Cortex--
To determine sites in brain tissue
where an interaction between CRMPs and chondroitin sulfate may take
place, we examined the immunohistochemical distribution of CRMP-4 and
chondroitin sulfate in the cerebral cortex of neonatal rat brain.
CRMP-4-positive cells were present in the upper part of the cortical
plate. Interestingly, within these cells nuclei were strongly stained
(Fig. 8, A and C).
Moreover, there was a fine reticular CRMP-4 staining without obvious
relationship to cellular structures in the marginal zone and a diffuse
labeling of the subplate and prospective white matter (Fig. 8,
A and C). Chondroitin sulfate, on the other hand,
was expressed as a reticular meshwork in the marginal zone (Fig. 8, B and D). Furthermore, the subplate was diffusely
stained. In summary, CRMP-4 and chondroitin sulfate were partly
expressed in the same regions of the neocortex. In agreement with
published data on the naturally occurring cell death in the developing
rodent brain (32-34), we found numerous pyknotic nuclei in the upper
cortical plate, immediately beneath the marginal zone corresponding to layer II of the mature neocortex (Fig. 8D,
arrowheads).
Release of CRMPs to the Extracellular Space--
The biochemical
interaction of CRMPs with chondroitin sulfate in vitro
obviously raises the question of under what conditions this segregation
might break down and in what compartment the interaction could become
relevant in vivo. The almost congruent reticular staining
patterns of CRMP-4 and CS in the marginal zone of the cerebral cortex
suggested that CRMPs may be released to the extracellular space,
e.g. after the programmed death of neurons. To substantiate
that CRMPs are released to the extracellular space, we screened cell
culture supernatants of neural cells for the presence of CRMPs. First,
in serum-free conditioned media of NTera-2 precursor cell cultures we
detected CRMP immunoreactivity after capture on a Q-Sepharose column
(Fig. 9A). The fact that no
CRMP-like molecules were detected in fetal calf serum (Fig.
9B) rules out the possibility that residual traces of FCS
are the source of CRMP-like immunoreactivity in these supernatants.
Because serum-starved cultures contained many dead cells on microscopic
examination and considerable activity of the cytosolic marker enzyme
lactate dehydrogenase (LDH) (Fig. 9C), we assume
that CRMP-like molecules possibly were released from the cytosol of
NTera-2 precursor cells that underwent cell death. Our data do not
exclude the possibility, however, that active mechanisms of release may
exist. Second, primary cultures of neocortical neurons were studied for
the release of CRMPs. These cultures had been characterized previously
and contained >95% microtubule-associated protein 2-positive neurons (11, 18, 27). When these neuron cultures were grown in the presence of
HEK293 cell-conditioned medium to provide trophic support, no lactate
dehydrogenase activity was detected in the supernatants of these
cultures. However, we observed that ~50% of cells underwent cell
death with compacted rounded cell morphology (Fig.
10, A and B) and
pyknosis of the nuclei (Fig. 10, C, D, and E) independent on the initial plating density. Supernatants
of these cultures contained CRMPs and their amount paralleled the extent of cell death (Fig. 10F). Almost all of the released
CRMP was CRMP-4 (Fig. 10G) since we were not able to detect
significant amounts of the other CRMPs in the supernatant even after
prolonged film exposure (data not shown). Taken together, these
experiments indicated that NTera-2 precursor cells and neocortical
neurons release CRMPs to the extracellular compartment and that
naturally occurring cell death may be a possible release mechanism.
Previously, we generated the monoclonal antibody mAb-9 against
chondroitin sulfate-binding proteins from neonatal rat brain. This
antibody recognizes a 65-kDa protein with laminar expression in the
cerebral cortex (18). In the present study, we identified this protein
as collapsin response mediator protein-4 (CRMP-4/Ulip (31, 35)) based
on mass spectrometric analysis of the purified protein and Western blot
analysis of recombinant CRMP-4. Furthermore, we obtained evidence that
CRMP1, -2, -3, and -4 interact with chondroitin sulfate proteoglycans.
The collapsin response mediator proteins form a family of five
homologues, the first of which (formerly called CRMP-62, now termed
CRMP-2) was identified by expression cloning as a signal transduction
molecule, mediating the growth cone collapse activity of semaphorin
3A/collapsin on peripheral sensory neurons (30). The rat orthologue
(called TOAD-64) was identified as a marker of differentiating
post-mitotic neurons re-expressed after nerve lesions in the adult
animal (29). CRMP-4 (mUlip) (31) was cloned in mouse as a
phosphoprotein cross-reacting with an anti-stathmin antibody and was
later identified in rat and human by homology screening (35, 36).
Recently, CRAM/CRMP-5 was cloned as a protein that interacts with
CRMP-3 (37), with a glycine transporter (38), and that cross-reacts with an anti-ZAP-70 antibody (39). CRMPs share sequence similarities with dihydropyrimidinase and with the gene product of the unc-33 gene
of Caenorhabditis elegans, which is involved in
axonal pathfinding (30).
Different phosphorylation states of CRMPs exist (31, 40) and may
account for slightly different electrophoretic mobilities of the
protein bands recognized by mAb-9 (Fig. 1C), since in the absence of phosphatase inhibitors like orthovanadate, the protein may
undergo dephosphorylation, which may markedly influence the apparent
molecular weight as determined by SDS-PAGE (41). Moreover, differences
in phosphorylation may explain why CRMP immunoreactivity eluted from
the Mono Q column in two distinct peaks (Fig. 1C). On the
other hand, taking into account that mAb-9 weakly cross-reacts with
CRMP-1 and CRMP-3 (Fig. 3A), the second peak could also
represent a different CRMP. Interestingly, on size exclusion
chromatography CRMP-4 migrated with a velocity corresponding to a
molecular size of ~200 kDa (Fig. 2, A and C).
This obvious discrepancy to the apparent Mr of
65 kDa, as determined by SDS-PAGE (Figs. 1C and 2C), could reflect the tendency of CRMPs to form
heterotetramers under native conditions (42).
In addition to their heterotetramerization, CRMPs interact with several
other proteins. In sensory neurons, CRMP-2 is phosphorylated on Thr-555
by Rho kinase upon stimulation of growth cone collapse by
lysophosphatidic acid (43). Although this lysophosphatidic acid-dependent signaling pathway does not depend on
semaphorin 3A, activity of phospholipase D2 that is inhibited by CRMP-2
was found to be regulated by semaphorin 3A in PC12 cells (44).
Furthermore, semaphorin 3A enhances tyrosine phosphorylation of CRMP-2
and CRMP-5 via Fes/Fps tyrosine kinase (45). Recently, it was shown that CRMP-2 binds to tubulin heterodimers and promotes microtubule assembly (46). Other interactions of CRMPs probably exist since CRMP-2
copurifies with dichlorophenol-indophenol oxidoreductase, aldolase C,
and glyceraldehyde-3-phosphate dehydrogenase from adult bovine brain,
suggesting complex formation of these proteins (47). All interactions
of CRMPs mentioned so far involve proteins that are exposed to the
cytosol. In this study, however, we show that CRMPs interact with
chondroitin sulfates. Chondroitin sulfates represent an entirely novel
category of interaction partners for CRMPs, since they are
carbohydrates, and they are assumed to reside mainly in the
extracellular space. Similar to heparin, chondroitin sulfates are
polyanionic glycosaminoglycans, but they carry a lower density of
negative charges.
Interestingly, the elution patterns of CRMPs from heparin and
chondroitin sulfate columns differed markedly. Although soluble CRMPs
bound completely to a heparin column (Fig. 6) and were eluted quantitatively at moderate ionic strength (300 mM NaCl),
indicating a charge-mediated interaction of low affinity, only about
50% of each CRMP bound to chondroitin sulfate columns (Fig. 5).
This incomplete binding to CS columns could reflect competition of the
abundant endogenous brain-derived CS proteoglycans with the immobilized
glycosaminoglycan, as suggested by our immuno-coprecipitation experiments (Fig. 7B). Importantly, the CRMPs were not
completely recovered from the CS columns even after stringent washing
with buffer containing 2 M NaCl, as evidenced by the
presence of CRMPs in eluates obtained with buffer containing the
chaotropic salt guanidinium hydrochloride (Fig. 5, lane 4 in
each panel). Thus, certain CRMP forms obviously engage in high affinity
interactions with chondroitin sulfates that were not observed on
heparin and which cannot be attributed solely to ion exchange effects.
Chondroitin sulfates are abundant in the extracellular matrix of the
developing brain (1), whereas CRMPs as proteins without secretory
leader peptides are assumed to exist well separated in the cytosol
(30). The immunohistochemical fine reticular or diffuse staining
patterns of CS in the marginal zone and subplate of the cerebral cortex
(Fig. 8, B and D) are in agreement with published
data (9, 48, 11) and presumably represent extracellular localizations
of the glycosaminoglycan. No cytoplasmic CS was detected in the present
study, and in the literature, evidence for cytoplasmic proteoglycans in
the CNS is limited to the adult stage (49). On the other hand, CRMP-4
was found (i) in cells of the cortical plate mainly in nuclei, (ii) in
the marginal zone with a reticular staining pattern very similar to the
CS-staining, and (iii) in the subplate and prospective white matter
(Fig. 8, A and C). The nuclear localization of
CRMP-4 is consistent with reports on the targeting of a GFP-CRMP-1
fusion protein to nuclei in lung cancer cells (50) and of
CRMP-2-positive nuclear inclusions after overexpression in Neuro2A
cells (51). The similar reticular staining pattern of CS (Fig. 8,
B and D) and CRMP-4 (Fig. 8, A and
C) in the marginal zone, however, suggest that CRMP-4 may be
present in extracellular compartment of the cerebral cortex. Taking
into account that some proteins lacking a secretory leader peptide are
targeted extracellularly (e.g. bovine fibroblast growth factor in MG-63 cells (52)), release of CRMPs to the extracellular space may occur under particular circumstances. Obviously, it is
difficult to prove rigorously that a protein is located in the
extracellular space based on immunohistochemical analysis of brain
tissue. An important argument for the existence of extracellular CRMPs
is the presence of these proteins in cell culture supernatants of
NTera-2 precursor cells (Fig. 9A) and of neocortical neurons (Fig. 10, F and G). CRMPs were detected in the
extracellular compartment, concomitant with cell death, as judged from
the release of the cytosolic marker lactate dehydrogenase in the
NTera-2 precursor cultures (Fig. 9C) and the presence of
condensed, pyknotic chromatin (Fig. 10, C, D, and
E) in ~50% of the primary neocortical neurons, indicating
naturally occurring (programmed) cell death. Naturally occurring cell
death is a widely distributed phenomenon during the development of the
central nervous system (33) and is found in the perinatal rat neocortex
mainly in the future layers II/III (32, 34). In these layers we found
particularly strong cellular CRMP-4 staining (Fig. 8, A and
C) and numerous pyknotic nuclei (Fig. 8D,
arrowheads). Moreover, the non-cellular reticular CRMP-4 immunoreactivity in the marginal zone toward the pial surface and in
the cortical plate is consistent with the formation of diffusion
gradients of released CRMP-4 away from the zone of neuron death.
Binding to CS proteoglycans may help to shape and stabilize gradients
of diffusible CRMPs (53). On the other hand, CS could participate in
the control of CRMP release from dying neurons since it inhibits the
death of neocortical neurons in vitro (11). Thus, CS could
be an important regulator of the release and distribution of
extracellular CRMPs in the cerebral cortex. On the other hand, collapsin/semaphorin 3A could regulate the release of CRMPs from dying
cells since it has been shown to promote the apoptosis of certain
neuron classes (54).
The function of extracellular CRMPs could relate to the establishment
of contacts with afferents, since the spatiotemporal pattern of
naturally occurring cell death in the neocortex correlates with the
arrival and settlement of cortical afferents at the different cortical
levels (32). Interestingly, Emerling and Lander (17) obtained evidence
that CS-bound soluble cues dramatically influence the growth of
thalamic neurites within the cerebral cortex. Thus, it is tempting to
speculate that CRMPs may belong to these CS-bound cues. However,
screening for functional effects of purified CRMP applied in cell
culture paradigms and careful analysis of CRMP transgenes will help to
clarify the as yet unknown physiological roles of extracellular CRMPs
in the future.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1,3]-N-acetylgalactosamine disaccharide units that are linked by [
1,4]-glycosidic bonds into
long unbranched chains with molecular masses of up to 50 kDa and more (8). With respect to the position of sulfate esters at the
galactosamine, CSA (C-4-S, containing C-4-sulfate) and CSC (C-6-S,
containing C-6-sulfate) are distinguished. The expression of these
proteoglycan core proteins is precisely tuned during brain development
(9). Moreover, the disaccharide composition of CS is regulated (9,
10).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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20 °C until
further analysis. Aliquots of this material were analyzed by Western
blotting to confirm hat the desired protein had been excised.
-D-galactopyranoside. Cells were lysed
in Tris/phosphate buffer containing 8 M urea, and
expression of recombinant His-tagged CRMP proteins was tested by
Western blot with an anti-RGS-His antibody (Qiagen).
20 °C) and washed twice with 80%
ethanol (4 °C) before Western blot analysis with the
peptide-specific antibodies against CRMPs.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (56K):
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Fig. 1.
Anion exchange chromatography of soluble
brain proteins. Forty mg of soluble protein extract from neonatal
rat brains was chromatographed on a Mono Q column. A,
chromatogram of proteins eluted with a step gradient as described under
"Material and Methods." Silver staining (B) and Western
blot (C) with the mAb-9 of Mono Q fractions. The fraction
marked with an asterisk was used for gel filtration
chromatography. Bars indicate the position of apparent
molecular weight marker bands (in kDa). L, load;
F, flow-through
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[in a new window]
Fig. 2.
Gel filtration chromatography. Fraction
7 from the Mono Q chromatography was subjected to gel filtration
chromatography on a Superose 12 column. A, chromatogram.
Antigen containing fractions are marked with a bar, and the
positions of the molecular weight standards (in kDa) is indicated by
arrows. Silver staining of SDS-PAGE (B) and
Western blot (C) with the mAb-9. Bars indicate
the position of apparent molecular weight marker bands (in kDa).
MS-Fit search results of the peptide masses obtained after digestion of
the unknown protein, recognized by mAb-9 with trypsin
, relative mass differences between observed and
predicted peptides; "Start" and "End" refer to the position of
the peptides in the TOAD-64 sequence; "Peptide sequence," the amino
acid sequences are given in single-letter code (residues in parentheses
refer to the amino acids immediately preceding or following the
peptides, respectively); "Modifications," oxidation of methionine
was the only allowed protein modification (IMet-ox). The 12 unmatched
masses are 732.8, 803.8, 923.0, 946.0, 1032.0, 1066.2, 1258.2, 1447.4, 1711.9, 2032.0, 2043.1, and 2929.0. The matched peptides cover 17%
(98/572 amino acids) of the protein.
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Fig. 3.
Western blot analysis of bacterially
expressed CRMPs with mAb-9 and a pan-specific antibody.
Recombinant CRMPs were produced in E. coli and separated by
SDS-PAGE. After transfer to nitrocellulose Western blot analysis with
mAb-9 (A), an anti-His-tag antibody (B), and
anti-CRMP-Fam (C) were carried out. The monoclonal antibody
recognizes recombinant CRMP-4 (A, lane 4) and
slightly cross-reacts with CRMP-1 and -3 but not CRMP-2.
View larger version (35K):
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Fig. 4.
Western blot analysis of bacterially
expressed CRMPs with peptide-specific antibodies. Western blots of
bacterially expressed CRMPs were probed with rabbit antisera raised
against peptides corresponding to amino acid sequences specific for
CRMP1-4 (A-D). Specificity of the antiserum is given on
the right. Bars on the left indicate
the position of apparent molecular weight marker bands (in kDa).
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Fig. 5.
Chondroitin sulfate affinity chromatography
of soluble CRMPs from rat brain. Soluble proteins (10 mg) from
neonatal rat brain were loaded on a chondroitin-6-sulfate-Sepharose
column, washed with 300 mM NaCl (1) and eluted
with 750 mM NaCl (2), 2 M NaCl
(3), or 4 M GuaHCl (4). All fractions
were tested in Western blot with the different mono-specific CRMP
antisera as indicated on the right. L, load; F,
flow-through. Bars on the left indicate the
position of apparent molecular weight (MW) marker bands (in
kDa).
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[in a new window]
Fig. 6.
Heparin affinity chromatography of soluble
CRMPs from rat brain. Soluble proteins (10 mg) from neonatal rat
brain were loaded on a heparin-Sepharose column, washed with 300 mM NaCl (1), and eluted with 750 mM
NaCl (2), 2 M NaCl (3), or 4 M GuaHCl (4). All fractions were tested in
Western blot with the different mono-specific CRMP antisera as
indicated on the right. L, load; F, flow-through.
Bars on the left indicate the position of
apparent molecular weight (MW) marker bands (in kDa).
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Fig. 7.
Immuno-co-precipitation of CRMPs and
chondroitin sulfate. Soluble proteins from neonatal rat brain were
precipitated with the peptide-specific anti-CRMP antisera as indicated
above the lanes or preimmune serum (P) and analyzed by
Western blotting with the biotinylated pan-specific anti-CRMP-Fam
antibody (A) or the chondroitin sulfate-specific CS56
antibody (B). Positions of apparent molecular weight marker
bands are indicated on the left. (B, control lane
with soluble brain proteins).
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Fig. 8.
Immunohistochemistry of chondroitin sulfate
and CRMP-4 in the neocortex of newborn rat. A, low
power micrograph of the cerebral cortex after staining with anti-CRMP-4
(brown). Nuclei were counterstained with hemalum
(blue). Bar, 50 µm. B, staining with
CS56. C, high power micrograph of the pia mater, marginal
zone, and upper cortical plate (later layers II/III) stained with anti
CRMP-4. Bar, 20 µm. D, section corresponding to
C stained with CS56. Arrowheads, pyknotic nuclei.
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Fig. 9.
Release of CRMP-like molecules from NTera-2
precursor cells. A, Q-Sepharose chromatography of
serum-free NTera-2 precursor cell-conditioned medium eluted with a
linear NaCl gradient (150 mM to 1 M NaCl).
Top, chromatogram. Bottom, Western blot analysis
with the pan-specific anti-CRMP-Fam antibody of chromatography
fractions (L, load; F1 and F2,
flow-through fractions; A2 to B6, eluate
fractions). mAU, milliabsorbance units. Bars
indicate the position of chromatography peaks. B, Western
blot analysis of Dulbecco's modified Eagle's medium containing 10%
fetal calf serum (FCS) and soluble brain proteins from
neonatal rat (P1 brain) using anti CRMP-Fam. C,
lactate dehydrogenase (LDH) activity in serum-free
conditioned media of NTera-2 precursor cells and 10% FCS as indicated
below (mean of triplicate determinations, error bar:
S.D.).
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Fig. 10.
Release of CRMP-4 from primary neocortical
neurons. A, phase contrast image of neocortical
neurons on days in vitro 1 grown in low density
(plating density 25,000/cm2). Bar, 100 µm.
B, phase contrast image of a high density culture (plating
density 75,000/cm2). Bar, 100 µm.
C, 4,6-diamidine-2-phenylindole staining of low density
culture. Bar, 20 µm. D,
4,6-diamidine-2-phenylindole staining of high density culture.
Bar, 20 µm. E, comparison of the frequencies of
pyknotic nuclei per visual field (0.14 mm2) in
4,6-diamidine-2-phenylindole-stained low and high density cultures
(counts were done in triplicate per condition. Error bars
are S.D.). F, Western blot (WB) analysis with
anti CRMP-Fam of cell culture supernatants from low density culture
(low), high density culture (high), and HEK-293
cells (HEK) as indicated above the
lanes. The positions of apparent molecular weight standards
are shown on the right. G, Western blot analysis
with anti-CRMP-4 of high density culture supernatant (high)
and soluble proteins from neonatal rat brain (brain) as
indicated above the lanes. The positions of
apparent molecular weight standards are shown on the right
(in kDa).
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Norbert Rösel and Werner Tomberg for expert technical assistance, Mario Sitzia for help with the handling of animals, Prof. H. E. Meyer and Dr. D. Immler (Bochum) for MALDI-TOF and ESI-MS analyses, and Dr. Uwe Rauch (Lund) for valuable comments on the manuscript. We thank Prof. Hans Werner Müller for support in the beginning of the project.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Ka 762/2-1 (to J. K. and U. J.), BONFOR Grant O161.0008 (to J. K.), and BONFOR Grant O167.0004 (to S. L. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally.
§§ Present address: Institut für Anatomie und Zellbiologie, Martin-Luther-Universität Halle-Wittenberg, Grosse Steinstrasse 52, 06097 Halle, Germany.
¶¶ To whom correspondence should be addressed: Institut für Physiologische Chemie, Nussallee 11, D-53115 Bonn, Germany. Tel.: 49-228-734744; Fax: 49-228-732416; E-mail: kappler@institut.physiochem.uni-bonn.de.
Present address: Fakultät für Chemie und
Mineralogie, Biotechnologisch-Biomedizinisches Zentrum, Johannisallee
29, D-04109 Leipzig, Germany.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M210181200
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; CS, chondroitin sulfate; CRMP, collapsin response mediator protein; CS-PG, CS proteoglycan; FCS, fetal calf serum; PBS, phosphate-buffered saline; mAb, monoclonal antibody; ESI-MS, electrospray ionization-mass spectrometry; GuaHCl, guanidinium hydrochloride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight spectroscopy.
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