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J. Biol. Chem., Vol. 278, Issue 26, 24164-24173, June 27, 2003
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, Interacts with Neuronal Receptors and Promotes Neurite Outgrowth*
From the
Laboratoire de Neurobiologie du
Développement et de la Régénération, CNRS Centre
de Neurochimie, 67084 Strasbourg, France, ¶Max
Planck Institute for Medical Research, 69120 Heidelberg, Germany, and
||Department of Cell Morphology and Molecular
Neurobiology, Ruhr-University, 44780 Bochum, Germany
Received for publication, November 18, 2002 , and in revised form, March 27, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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provides a new component in cell-cell and cell-extracellular matrix signaling
events in which these proteins have been implicated. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although phosphacan occurs in the CNS as a large CSPG (>800 kDa)
(4); it is in fact a secreted
splice variant of an even larger transmembrane receptor protein tyrosine
phosphatase (RPTP), RPTP-
(6), also known as PTP-
(-zeta) (7). Hence phosphacan
corresponds to the entire extracellular part of the long RPTP- receptor
(the relative structures of the proteins are illustrated in
Fig. 1). These proteins are
characterized by a carbonic anhydrase-like (CA) domain at their extracellular
N terminus. A third splice variant, the short RPTP- receptor form,
contains the same intracellular phosphatase domains as the long receptor, but,
unlike phosphacan and the long RPTP-, it is distinguished by the absence
of an 850-amino acid highly glycosylated sequence, the IS region, which
possesses many of the predicted glycosaminoglycan (GAG) attachment sites.
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In the CNS, phosphacan and the RPTP- receptors have punctual
spatiotemporal expression patterns that suggest potential roles for these
proteins in various developmental processes, including cell migration
(8), differentiation
(9), synaptogenesis
(10), synaptic function
(11), and myelination
(12)
(13), and phosphacan is also
up-regulated upon wounding and regeneration in the CNS
(14–16).
In vitro functional studies have provided evidence for such roles in the development and maintenance of the CNS, and there have been a number of reports of the effects of phosphacan on process outgrowth from neuronal cultures. These have shown that the proteoglycan can either promote or inhibit neurite outgrowth dependent upon the neuronal type tested and the conditions under which it is presented. For example, we have shown previously that phosphacan has outgrowth-promoting properties on mesencephalic and hippocampal neurons (4), whereas it is inhibitory for laminin-promoted outgrowth from neonatal dorsal root ganglion explants (5).
Biochemical studies have demonstrated a number of potential binding partners for phosphacan, both in the ECM, such as the tenascins, TN-C (17, 18) and TN-R (19), and on the cell surface, such as the immunoglobulin cell adhesion molecules (IgCAMs), F3/contactin (20), and NrCAM (21). In addition, a number of growth factors can bind to phosphacan, including bFGF (22) and HB-GAM (19). Interaction sites on phosphacan have been characterized at three levels (13). First, there are the long negatively charged CS GAG polymer chains, which can bind, for example, to TAG-1/Axonin-1 (23), and the cytokines, amphoterin (19), midkine (24), and HB-GAM/pleiotrophin (19, 25). Second, on the core glycoprotein, there are other classes of glycosylation, represented by oligosaccharides carrying epitopes such as HNK-1 (sulfated glucuronic acid) and Lewis-X (sialyl-Lewis-X) (5), which have also been implicated in IgCAM interactions (17). Third, there is the primary protein sequence, which includes the CA, and a fibronectin type III-like (FNIII) domain, but the remaining 80% of the molecule bears no strong homology to other characterized protein data base structures.
Here we report the identification and characterization of a novel short
isoform of phosphacan. This new isoform corresponds to the first third of
phosphacan, and although it is glycosylated, it is not a proteoglycan. It is
not as readily extracted from brain tissue as phosphacan, perhaps because of
stronger interactions with the cell surface. A recombinant protein
corresponding to the new isoform sequence can, like phosphacan, also promote
neurite outgrowth from cortical neurons. Hence regulated expression of this
protein may introduce an extra level of complexity to the proposed functional
interactions of phosphacan and the RPTP- receptor forms during CNS
development and regeneration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polyclonal sera were all from rabbit. KAF13 was raised against the purified
DSD-1-PG (4) and has been used
to clone all forms of RPTP- from mouse brain expression libraries
(5), KAF14 was raised against
purified TN-C from post-natal mouse brains
(31), polyclonal
F3/contactin-1367 was raised against amino acids 37–50 (peptide
KGFG-PIFE-EQPINT) of F3/contactin coupled to keyhole limpet hemocyanin
(32), and anti-PSI was raised
against the purified recombinant GST-PSI protein described below using
standard immunization protocols
(4).
Antibody Screening of cDNA Expression Libraries—A mouse
brain cDNA expression library was screened using the polyclonal antibody,
KAF13, at 1 µg/ml as described previously
(5). The library used was
BALB/c neonatal whole brain oligo(dT) and random-primed Uni-ZAP XR l
(Stratagene). Screening 3 x 106 recombinant phages, yielded
four positive clones, corresponding to bases 1144–1509 and
1032–3591 of phosphacan/long RPTP- and 1186–7850 of short
RPTP-. The fourth cDNA clone contained the truncated ORF corresponding
to a shorter form of phosphacan. This begins at base 1276 and ends with a
termination codon at 1792. This is followed by a 3'-UTR of 1988 bases. A
Marathon cDNA amplification kit (Clontech, Heidelberg, Germany) was used to
generate P0 and P7 total mouse brain cDNA libraries from 1 µg of
poly(A)+ RNA. Using PCR amplification of this cDNA, bands from the
ORF to 3'-UTR were cloned and sequenced (primers were as follows: 1
(sense), 5'-ATG CGA ATC CTG CAG AGC TTC CTC; 1700 (sense), 5'-GCC
TCC TTA AAC AGT GGC T; 1891 (antisense), 5'-CTA TCC AGC TGA AGA GTC ATC
GGC; 2120 (antisense), 5'-ACT TAG AAC TGG TGC GGA C).
Northern Blot Analysis—Poly(A)+ RNA was prepared
from 1 g of mouse brain from different developmental stages using a Fast-Track
2.0 mRNA isolation kit (Invitrogen). It was separated on formaldehyde/1%
agarose gels and then blotted by capillary action onto Hybond nylon membrane
(Amersham Biosciences). Blots were pre-hybridized for 1 h and then hybridized
overnight at 50 °C for DNA probes and at 65 °C for riboprobes.
Prehybridization was in hybridization buffer without probe (7% SDS, 50%
deionized formamide, 5x SSC, 50 mM sodium phosphate, pH 7,
0.1% N-lauroyl sarcosine, 2% blocking reagent (Roche Applied
Science)). Washing for DNA probes was in three changes of wash buffer (40
mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA) at 68
°C for 40 min, and for riboprobes, at 65 °C, 2 x 5 min in
2x SSC (where 1x SSC = 0.15 M NaCl, 15 mM
sodium citrate, pH 7), and in 0.1x SSC, 3 x 15 min. Signals were
revealed with Hyperfilm (Amersham Biosciences) and amplifying screens at
–80 °C. The DNA probe for the 5'-ORF of all RPTP-
isoforms (1–1785; see Fig.
1) was labeled with [32P]dCTP by random priming.
Because of homology in the 3'-UTR with the complementary 28 S rRNA
sequence, it was not possible to employ a DNA probe without a strong
cross-reaction with rRNA, because sequences from the sense strand hybridize to
28 S rRNA. Hence, it was necessary to employ an antisense riboprobe that only
hybridized to the sense strand of the PSI transcript. This corresponded to
bases 2653–3784 of the 3'-UTR of the PSI transcript and was
synthesized with incorporation of radioactive 
-35S-UTP
(Amersham Biosciences) using T7 RNA polymerase (MBI Fermentas GmbH, St Leon
Rot, Germany) and the linearized pBluescript II plasmid (Stratagene)
containing the PSI cDNA. A riboprobe against -actin was used as an
indicator of relative RNA concentrations (bases 739–970).
Tissue Fractionation—P7 mouse brains were homogenized in
different buffers to test the efficiency of different extraction conditions.
Protease inhibitors were used throughout (1 mM phenylmethylsulfonyl
fluoride, 1 µM trypsin inhibitor, 0.1 µM
Aprotinin, 1 µM pepstatin, 5 µM leupeptin, 1
µM antipain, 1 mM benzamidine, 1 mM EDTA).
Initially, 20 brains were homogenized in PBS on ice with a mechanical dounce.
Nuclei and insoluble debris were removed by centrifugation at 600 x
g for 20 min. Half of this supernatant was detergent-solubilized by
addition of 1% Nonidet P-40 and mixing at 4 °C for 1 h. The remaining
600-g PBS supernatant was ultracentrifuged at 100,000 x g for 1
h at 4 °C to precipitate PBS-insoluble material including total cellular
membranes. This PBS-insoluble pellet was subsequently resuspended in 1%
Nonidet P-40/PBS. As an alternative to detergent-solubilization with Nonidet
P-40, 10 brains were homogenized in 60 mM
n-octyl--D-glucopyranoside, 50 mM Tris,
pH 8, 50 mM sodium acetate and then centrifuged at 100,000 x
g for 1 h at 4 °C. Finally, for urea extraction, 10 brains were
homogenized in 8 M urea, 10 mM sodium acetate, pH 6, and
then centrifuged at 100,000 x g for 1 h at 4 °C. Protein
determination used the DC assay kit (Bio-Rad).
Deglycosylation Studies—Protein fractions were incubated for
4 h at 37 °C in 40 mM Tris, pH 8, 40 mM sodium
acetate with 0.4 units/100 µg peptide N-glycosidase F (EC
3.5.1.52
[EC]
; Roche Diagnostics), 2 milliunits/100 µg keratanase
(endo--galactosidase, EC 3.2.1.103
[EC]
; Seikagaku, Kogyo, Tokyo), or 20
milliunits/100 µg chondroitin ABC lyase (EC 4.2.2.4
[EC]
; Roche Molecular
Biochemicals).
Partial Protein Purification and Protein Sequencing—50 P7
mouse brains were homogenized in 1% Nonidet P-40/PBS (10 mM
KH2PO4, 10 mM
Na2HPO4-2H2O, pH 7.4, 150 mM NaCl)
in the presence of protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1.5 µM antipain, 1 µM
ortho-phenanthroline, 1 µM pepstatin, 1
µM aprotinin, 1 µM leupeptin, 1 mM
benzamidine, 2 mM EDTA). The homogenate was centrifuged at 20,000
x g for 15 min at 4 °C and then ammonium sulfate was added
to the supernatant to a final concentration of 1.5 M (35%
saturation). Precipitated proteins were removed by centrifugation at 20,000
x g for 1 h at 4 °C followed by filtration through
0.45-µm filter units. This supernatant was loaded onto a methyl HIC
(hydrophobic interaction chromatography)
column (Bio-Rad) in a Biologic Duo-Flow gradient chromatography system
(Bio-Rad), equilibrated with 1.5 M AmSO4/PBS. The column
was washed until the baseline was attained and then eluted with a linear
gradient from 1.5 to 0 M AmSO4/PBS. The 90-kDa protein
eluted at around 1 M AmSO4. The eluate was diluted by
addition of 2x binding buffer (20 mM Tris, pH 7.4, 0.5
M NaCl, 1 mM MnCl2,1mM
CaCl2) and then loaded onto a concanavalin A-Sepharose affinity
chromatography column (Amersham Biosciences). The column was washed with 20
volumes of binding buffer before being eluted with elution buffer (0.5
M methyl--D-mannopyranoside, 20 mM
Tris, pH 7.4, 0.5 M NaCl). Sample buffer was added to this eluate,
which was then separated on 10% SDS-PAGE. The proteins were transferred by
electroblotting onto ProBlott PVDF membrane (Applied Biosystems). Bands were
revealed by Ponceau Red coloration, and the 90-kDa band was confirmed by
alignment with an adjacent strip, which was revealed using immunodetection
with pAbs KAF13 and anti-PSI. The N-terminal protein sequence of the 90-kDa
KAF13-positive band was determined by automatic Edman degradation on an
Applied Biosystems 473A microsequencer.
Developmental Expression Blot—Whole brains from mice at
different developmental stages were homogenized on ice in PBS with protease
inhibitors. The homogenate was centrifuged at 600 x g for 20
min at 4 °C and then the supernatant was further centrifuged at 100,000
x g for 1 h at 4 °C. The pellet was resuspended in PBS/20
mM n-octyl--D-glucopyranoside with
protease inhibitors.
Preparation of Recombinant PSI Protein—The recombinant PSI
protein was generated by expression in Escherichia coli of a plasmid
vector containing the PSI cDNA sequence fused in-frame to GST. The PSI cDNA
was amplified by PCR using primers 5'-ATG CGG ATC CTG CAG AGC TTC C
(which introduces a BamHI restriction site just after the initiation
codon by changing the "A" at the sixth base into a
"G") and 5'-CTA TCC AGC TGA AGA GTC ATC GGC (which includes
the termination code in the PSI transcript). The PCR cDNA product was purified
from an agarose gel and digested with BamHI before being ligated into
the plasmid vector, pGEX-5X-3 (Amersham Biosciences), which had been
pre-digested with the restriction endonucleases, BamHI and
SmaI. Competent E. coli cells (TOP10F'; Invitrogen)
were transformed, and clones were selected. The correct insertion of the PSI
cDNA sequence into the vector was verified by DNA sequencing of the entire
construct. For recombinant protein expression, the plasmid-bearing bacterial
clone was grown in LB broth supplemented with 50 µg/ml ampicillin at 37
°C to an A595 nm of 0.8, when protein expression was
induced by addition of isopropyl-1-thio--D-galactopyranoside
to a final concentration of 0.05 mM, and the culture was
transferred to 24 °C for a further 3 h. Bacteria were pelleted and then
lysed in PBS/1% Nonidet P-40 with 50 µg/ml lysozyme. The PSI-GST
recombinant protein was purified by affinity chromatography using a HiTrap
glutathione column (Amersham Biosciences). The GST control protein was
produced in the same way using bacteria transformed with the same plasmid
vector but without insert.
GST Pull-down—Purified GST and GST-PSI protein was incubated
with glutathione-agarose beads (2 µg/10 µl pre-swollen beads; Amersham
Biosciences) in PBS/1 mM dithiothreitol/0.5% Nonidet P-40 for 1 h
at room temperature with mixing and then washed three times with PBS. 20 µl
of beads were incubated with the 1 mg (2 µg/µl) P7 PBS-insoluble 100,000
x g brain fraction in PBS, 1 mM dithiothreitol, 0.5%
Nonidet P-40, 0.5% n-octyl--D-glucopyranoside, 1
mM EDTA, plus protease inhibitors at room temperature with mixing
for 1 h, and then placed on ice for 30 min before washing with 3 x 1 ml
of ice-cold PBS. The beads were finally boiled in SDS-PAGE sample buffer, and
the proteins were separated on 10% SDS-PAGE gels before transfer onto PVDF
membrane.
Immunoprecipitation—Antibodies (2 µg) were added to 2 mg
(2 µg/µl) of P7 mouse brain PBS-insoluble 100,000 x g
fraction solubilized in 50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 40 mM glucose, 1%
n-octyl--D-glucopyranoside, 0.3% polyoxyethylene
sorbitan monolaurate (Tween 20) plus protease inhibitors and incubated at 4
°C with mixing overnight. Subsequently, 50 µl of 50% pre-swollen bead
slurry was added for a further 1 h of mixing at 4 °C; protein A-Sepharose
(Sigma) was used to precipitate the rabbit polyclonal antisera, and anti-rat
Ig-Sepharose (Sigma) was employed for the rat antibodies. The beads were then
precipitated by centrifugation and washed by three rounds of resuspension and
precipitation in radioimmune precipitation assay buffer (Tris-buffered
saline/1% Nonidet P-40/0.1% SDS/0.5% deoxycholate) before being boiled in
SDS-PAGE sample buffer. Proteins were separated on 10% SDS-PAGE and
transferred to PVDF membrane before immunodetection. For
coimmunoprecipitation, the 100,000 x g PBS-insoluble mouse
brain fraction solubilized in 50 mM Tris-HCl, pH 7.4, 150
mM NaCl, 40 mM glucose, 1%
n-octyl--D-glucopyranoside, 0.1% polyoxyethylene
sorbitan monolaurate plus protease inhibitors was incubated with 2 µg of
mAb 324 (anti-L1) or mAb 4–121 (anti-F3/contactin) overnight with mixing
at 4 °C. A 50-µl 50% slurry of anti-mouse IgG-agarose (for
anti-F3/contactin; Sigma-Aldrich) or anti-rat IgG-agarose (for anti-L1;
Sigma-Aldrich) were then added and incubated with mixing for 2 h at 4 °C.
The beads were precipitated by centrifugation and then resuspended and
reprecipitated three times with 20 mM Tris, pH 7.4, before being
boiled with SDS sample buffer. Eluted proteins were separated on 10% SDS-PAGE
and electroblotted to PVDF membranes.
Phosphacan Purification—Phosphacan was purified from detergent-free physiological saline-buffered brain lysates from post-natal day 7 to 14 mice as described previously (4) using a combination of affinity chromatography with the mAb, 473HD, bound to Sepharose resin and anion-exchange chromatography (4). It was quantitated using the protein assay (Bio-Rad) and by the determination of uronic acid equivalents (33).
Cell Culture—Neuronal cell cultures were established from embryonic day 17 (E17) mouse brains. The cerebral hemispheres were dissected in PBS, and the meninges were removed. Dissociation was achieved by addition of 0.25% trypsin at 37 °C for 10 min, followed by passage through a sieve with 48-µm pores. The resulting cell suspension was plated on coverslips at a density of 7,000 cells/cm2 in minimal Eagle's medium (Invitrogen) supplemented with the N2 mixture, namely 5 µg/ml insulin, 20 nM progesterone, 100 µm putrescine, 30 nM selenite (34), 1 mM pyruvate, 0.1% (w/v) ovalbumin, and 100 µg/ml transferrin (Sigma-Aldrich). The cultures were kept in a humidified atmosphere with 5% CO2 at 37 °C.
Neurite Outgrowth Assays—E17 mouse cortical neurons were
plated on supports coated with different substrates; glass coverslips were
treated with 15 µg/ml poly-L-lysine in 0.1 M borate
buffer, pH 8.2, for 1 h at 37 °C. The coverslips were then washed with
water and dried. Purified phosphacan (50 µg/ml uronic acid equivalents) or
recombinant proteins (50 µg/ml) were coated for 4 h at 37 °C. After
coating, the coverslips were washed three times with PBS. After 24 h of
culture, neurons were fixed with 4% paraformaldehyde for 15 min. After
permeabilization with 0.1% Triton X-100 for 3 min and a blocking step with 3%
bovine serum albumin in PBS, cells were stained with a mAb directed against
the neuronal marker, 3-tubulin (mouse; Sigma; 2-h incubation in 3%
bovine serum albumin/PBS) and then revealed with a cy3-conjugated antibody
(goat anti-mouse-cy3; Jackson Immunoresearch Laboratories; 30-min incubation
in 3% bovine serum albumin/PBS). The coverslips were then mounted on slides in
Mowiol. Neurons were observed using a DMRB microscope (Leica), and pictures
were taken with an axiocam camera (Zeiss). A first parameter considered was
the fraction of process-bearing cells (with at least a process longer than one
neuronal cell body) from at least 100 neurons per experiment, chosen at
random. Subsequently the neurite lengths of the longest neurite on each
process-bearing neuron was measured, and morphometric analysis was performed
using the ImageTool software (University of Texas Health Science Center, San
Antonio, TX). Three independent experiments were performed, and the data were
analyzed with the Kyplot software (Kyence Inc.) using one-way analysis of
variance statistics ( = 0.05), followed by a Tukey test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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—The cDNA
corresponding to phosphacan was cloned previously
(5) from a mouse cDNA
expression library using KAF13, a pAb raised against the entire proteoglycan
purified from post-natal mouse brain. In addition to clones corresponding to
phosphacan and the long and short receptor forms of RPTP-, the antibody
also recognizes a clone expressing a novel truncated form of the extracellular
domain.
In this cDNA clone, the ORF is identical to the other three isoforms up to
base +1787 but then terminates at +1792 with a stop codon
(Fig. 2). It encodes a protein
of 597 residues. The ORF is followed by a 2-kb 3'-UTR that has homology
with rRNA sequences on the complementary strand and finishes with a putative
polyadenylation signal. This 3'-UTR is different from those for
phosphacan and for the two RPTP- receptor transcripts. The 5'-end
of the clone was confirmed by reverse transcriptase PCR analysis employing
primers from the 5'-end and from the 3'-UTR to generate the
complete ORF region.
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By comparison with the three known splice variants of RPTP-, the
truncated protein, which we designate PSI, represents 37% of phosphacan and
the extracellular part of the long RPTP- receptor form and 78% of the
extracellular portion of the short RPTP- receptor
(Fig. 1). The PSI protein
shares the same N-terminal region, with the signal peptide (first 23 residues)
followed by the CA and FNIII domains present in the other forms plus an
additional 188 residues of the S region. This latter region lies between the
FNIII domain and the splice site for the short receptor form, which occurs at
residue 762.
The expression of the PSI transcript was investigated by Northern blot
analysis of poly(A)+ RNA from E15 and P0 mouse brains, detected
with probes against the ORF and 3'-UTR regions
(Fig. 3). In addition to
previously characterized bands at around 6.5, 8, and 9.5 kb, corresponding,
respectively, to the short RPTP- receptor, phosphacan, and the long
RPTP- receptor transcript
(35), a band is detected with
the ORF probe at around 4 kb, which as predicted is also hybridized by the
probe against the 3'-UTR of the PSI transcript.
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In Vivo Expression of a Protein Corresponding to the Phosphacan Short
Isoform Transcript—Previously, we have worked with phosphacan, the
soluble secreted proteoglycan isoform of RPTP-. This protein can be
readily extracted from brain tissue using detergent-free physiological buffers
(4). It was originally
characterized by the presence of a chondroitin sulfate epitope, DSD-1, which
is recognized by the mAb, 473HD (hence the original name, DSD-1-PG). As is
shown in Fig. 4a),
most of the 473HD-bearing proteoglycan is present in the PBS-soluble 100,000
x g supernatant (molecular mass > 500 kDa), although there
is also material in the PBS-insoluble 100,000 x g pellet
fraction, which contains all of the cell membranes. This protein band
corresponds to the transmembrane, long RPTP- receptor form, which
contains the whole of the phosphacan sequence in its extracellular half and is
also a CSPG. Analysis of these brain extracts with an antibody against TN-C
(Fig. 4c), another
major component of the neural ECM, reveals a similar profile, the majority of
the secreted glycoprotein being extracted with PBS.
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KAF13, the anti-phosphacan pAb employed for the expression cloning of the
PSI cDNA, was used to analyze Western blots of these brain extracts for the
corresponding protein (Fig.
4b). Phosphacan and the long RPTP- are visualized
as large proteoglycan species (>500 kDa) in the PBS-soluble and insoluble
fractions, respectively. Protein species around 250 and 200 kDa are present in
all fractions, although these are considerably larger than the size of the
predicted PSI protein (67 kDa), and because they are present in the
PBS-soluble fraction, they are unlikely to correspond to the short RPTP-
transmembrane receptor form. Instead it has been suggested that they represent
less glycosylated forms of the phosphacan core protein
(36). Based on its amino acid
sequence, the PSI protein should be secreted into the extracellular
environment, but there was no evidence of a smaller protein species <180
kDa recognized by pAb KAF13 in this fraction. However, a smaller
KAF13-positive protein band around 90 kDa was detected in brain tissue
extracted with detergent and urea-containing buffers
(Fig. 4b, open
arrow). This 90-kDa protein could be extracted with the detergents,
Nonidet P-40 or octylglucoside, but was absent from the PBS-soluble fraction,
being precipitated by centrifugation at 100,000 x g. It was
also possible to extract this 90-kDa protein with urea, suggesting that it was
associated with the PBS-insoluble fraction, which includes the cell membranes,
through non-lipid molecular interactions. Sequence analysis using several
computer logarithms (e.g. Prosite
(37) and
Glycosylphosphatidylinositol Modification Site Prediction
(38)) did not indicate the
presence of any known lipid binding domains in the PSI protein. In addition,
F3/contactin, a receptor protein that is associated with the cell membrane
through lipid interactions (glycosylphosphatidylinositol anchor) was also
found in the detergent extracts and the PBS-insoluble 100,000 x
g fraction, but, unlike the 90-kDa protein, it was not found in the
urea extract (Fig.
4d).
The 90-kDa PSI Protein Is Glycosylated, but It Is Not a
Proteoglycan—To further test whether the 90-kDa protein corresponds
to the PSI cDNA, the presence of glycosylation was investigated using
glycosidases (Fig. 5). To
determine the presence of GAG chains, protein fractions were treated with GAG
lyases. Digestion of the PBS-insoluble 100,000 x g fraction
with chondroitinase ABC (ChABC) removes the CS GAGs from the CSPGs. The
"core protein" of the long RPTP- receptor form, recognized
by pAb KAF13 (indicated by a bar in
Fig. 5), now migrates as a
smaller, although still heavily glycosylated, species around 300–400
kDa. The mAb, 3F8, recognizes an epitope on phosphacan and the long
RPTP- receptor (but not on a keratin sulfate variant of phosphacan,
called phosphacan-KS (6,
9,
30) or on the short
RPTP- receptor (39)) and
detects both the undigested long RPTP- receptor at the top of the
separating gel and the core protein of long RPTP- following digestion of
the CS GAGs with ChABC (presumably the 3F8 epitope is to some extent masked by
the CS GAGs, because the signal is much stronger for the core protein), but it
does not detect any other protein in this fraction. Hence, the PSI protein
does not seem to bear the 3F8 epitope. Keratanase digestion (to remove keratin
sulfate GAG chains) does not appear to result in any significant
deglycosylation of the long RPTP- receptor. Meanwhile the 90-kDa protein
(arrow) detected with KAF13 shows no shift on either ChABC or
keratanase treatment, and hence, as predicted for PSI, it does not appear to
be a proteoglycan. However, the presence of other carbohydrate modifications
on PSI is probable, because the N-terminal region of phosphacan is
glycosylated (17), and there
is a difference between the migration of the 90-kDa protein on SDS-PAGE and
the calculated size from the primary PSI sequence (around 67 kDa). To test for
the presence of N-linked carbohydrates, the membrane fraction was
treated with peptide N-glycosidase F. As expected, this results in a
significant reduction in the size of the 90-kDa protein detected by KAF13 by
around 15–20 kDa, indicating that the 90-kDa band is indeed a
glycoprotein (Fig. 5). This
indicates that without sugars, the 90-kDa protein migrates at around 70 kDa,
which would be the expected size of the PSI protein sequence. A weaker band
around 190 kDa is also shifted down by N-glycosidase F treatment and
may correspond to the short RPTP- receptor form, which is not a
proteoglycan and does not carry the 3F8 epitope but does contain the entire
PSI sequence in its extracellular part.
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To further confirm the identity of the 90-kDa protein, a new polyclonal antisera was raised against a bacterially expressed recombinant PSI protein. This anti-PSI sera also clearly recognized the 90-kDa protein detected by the anti-phosphacan sera, KAF13 (Fig. 6a). In addition, it appears that the HNK-1 carbohydrate epitope is also present on the 90-kDa protein. This sugar has been found on a range of extracellular proteins in the CNS, including CAMs, TN-C and TN-R, and we have previously shown it to be present on phosphacan (5). As further confirmation of the recognition of the 90-kDa HNK-1 positive protein by the anti-phosphacan and anti-PSI sera, immunoprecipitation experiments were performed. These showed that both the anti-phosphacan and anti-PSI sera recognized the 90-kDa protein precipitated from brain extracts by the anti-HNK-1 mAb and that the 90-kDa band precipitated by both anti-PSI and anti-phosphacan carried the HNK-1 epitope (Fig. 6b). Hence, the 90-kDa band precipitated by HNK-1 is recognized by both anti-phosphacan and anti-PSI, whereas HNK-1 recognizes the 90-kDa protein precipitated by both of these polyclonal sera.
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Purification and Protein Analysis Confirms the Identity of the 90-kDa
Band as Phosphacan Short Isoform—To confirm the identity of the
90-kDa protein band, it was further enriched from brain detergent extracts,
and protein sequence was obtained. Given the presence of the other splice
variants of phosphacan/RPTP-, which share common epitopes with PSI, it
was necessary to follow the purification steps by Western blot analysis of
fractions using both pAb KAF13 and anti-PSI. Following a series of pilot
assays, a relative enrichment of the 90-kDa protein was obtained using a
combination of hydrophobic interaction and concanavalin A lectin-affinity
chromatography. The protein was finally separated on SDS-PAGE and transferred
to sequencing grade PVDF membrane. Based on alignment with the KAF13-positive
band, the 90-kDa protein corresponding to the candidate isoform was excised
and subject to Edman degradation, which yielded an N-terminal sequence,
XXRQQRKLVEEI. This corresponds to the predicted start of all of the
RPTP- splice variants following cleavage of the 23-amino acid signal
peptide (6). Thus, in
conclusion, we have found a protein that has the biochemical properties
predicted for the protein product of the PSI transcript.
Developmental Expression of the Phosphacan Short Isoform
Protein—A developmental profile of PSI protein expression in mouse
brain indicates that it is already present by E16 and that, like phosphacan
(5,
40), its expression levels
rise steadily to plateau in the first and second weeks post-natal, before
decreasing a little in the adult (Fig.
7). This peak of expression correlates with the phase of
myelination in the developing CNS, a process in which phosphacan/RPTP-
has been implicated (12,
41,
42). There are several other
bands seen on the blot at higher molecular masses (around 180 kDa and >400
kDa). These could correspond to the receptor forms of RPTP- that may
also show developmental regulation, both with respect to their relative levels
of expression and also in terms of the degree of glycosylation present on the
core proteins (5,
40).
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Binding Partners for Phosphacan Short Isoform—The
association of the secreted PSI protein with the PBS-insoluble 100,000 x
g brain fraction suggests that it is complexed with material in this
fraction. The 100,000 x g fraction contains all of the cellular
membranes, and hence it is possible that PSI is retained in this fraction
because of interactions with membrane-bound receptor proteins. A number of
neuronal receptor proteins have already been identified for phosphacan,
including the IgCAMs, NCAM, NrCAM, L1/NgCAM,
(19,
21) F3/contactin
(20), and TAG-1/Axonin-1
(23). In addition, studies
with recombinant protein constructs corresponding to different parts of the
phosphacan protein sequence have shown that the CA domain can bind to
F3/contactin (20) and can also
be immunoprecipitated as a complex with a third transmembrane protein, Caspr
(42), whereas the S region up
to residue 630 binds to NCAM, NrCAM, and L1/Ng-CAM
(21). Because the PSI protein
sequence contains the entire CA and FNIII domains and 188 of the 223 amino
acids (84%) of the S region used in the mapping studies, a similar interaction
profile might be expected. Immunoprecipitation studies of brain membrane
extracts suggest that the PSI protein can indeed be co-precipitated with L1
and F3/contactin (Fig.
8a). Further evidence for such interactions came from
binding experiments with a recombinant PSI protein. The PSI protein was
expressed as a GST recombinant construct in E. coli and attached to
glutathione-agarose beads prior to incubation with mouse brain extracts. After
washing the beads, the proteins that had been "pulled down" were
assessed on Western blots. These showed that, compared with the GST control
protein, there was an interaction of the GST-PSI protein with both
F3/contactin and L1 (Fig.
8b). Hence, these results confirm the previous reports of
interactions between the N-terminal domains of phosphacan/RPTP- and the
IgCAMs, F3/contactin (20) and
L1 (21), and support the
possibility that, in addition to phosphacan and the extracellular domains of
the receptor forms of RPTP-, the PSI protein might also interact in
vivo with such cell membrane receptor molecules. In addition, these
results indicate that the interactions between the PSI protein and these cell
adhesion molecules can occur in the absence of glycosylation.
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PSI Recombinant Protein and Phosphacan Promote Neurite Outgrowth from Cortical Neurons—One of the most studied functional effects of phosphacan has been its influence upon process outgrowth from neurons, a process that it may influence as an ECM component in differentiating cortical layers and along axon tracts, as well as in CNS lesions. It has been shown previously (43) that phosphacan can promote outgrowth from cortical neurons and that the N-terminal domains of phosphacan can also promote outgrowth, some of these effects being mediated via the IgCAMs, F3/contactin and NrCAM (20, 21). Based upon these studies, it seems likely that PSI can also affect neurite outgrowth. To investigate this, the effects of a recombinant PSI protein on outgrowth from embryonic (E17) mouse cortical neurons were compared with those obtained with the purified phosphacan proteoglycan.
The PSI protein was expressed as a GST recombinant construct and purified using glutathione affinity chromatography. As can be seen in Table I and Fig. 9, there was a significant promotion of neurite outgrowth by the PSI-GST protein relative to both the poly-L-lysine control and the GST protein alone, although the promotion on the phosphacan substrate was more robust. It is possible that the native phosphacan can exert stronger effects than the PSI construct because of the presence of additional promontory signals that may occur either in the much larger phosphacan protein sequence or in the extensive additional glycosylation, including the GAG chains, present on the proteoglycan. Nevertheless, it appears that the PSI protein sequence is capable of promoting neurite outgrowth from cortical neurons and that this effect can be obtained without eukaryotic post-translational modifications.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
The novel PSI transcript was cloned from a brain cDNA expression library using
anti-phosphacan sera, and its expression in vivo was confirmed by
Northern blot hybridization. Using the same antisera, we have searched brain
extracts for the protein corresponding to the translation product of the PSI
transcript and have identified a 90-kDa glycoprotein that fulfils the expected
biochemical criteria. The deglycosylated protein has the predicted size and
N-terminal peptide sequence. It is also recognized by antisera raised against
the recombinant PSI protein and bears the HNK-1 sugar epitope, which is also
found on phosphacan. Hence, it appears that the 90-kDa protein does indeed
correspond to the product of the PSI transcript.
However, ECM proteins can be subject to proteolysis, and an alternative
explanation for the 90-kDa protein we have characterized might be that it is a
proteolytic breakdown product of phosphacan/RPTP-. There are several
reasons, besides the existence of the PSI transcript, why this is unlikely to
be the case. For a start, an analysis of the phosphacan protein sequence for
potential cleavage sites by known proteases (PeptideCutter program
(44)) indicates that it could
be digested into relatively small peptides, especially in the N-terminal
region. In addition, the only reported in vivo demonstration of
proteolytic cleavage of phosphacan is for the tissue plasminogen activator and
plasmin (45), where it was
shown that I125-radiolabelled phosphacan could be digested by pure
plasmin in vitro, but no digestion product was observed in the range
of 40 to 150 kDa. Moreover, a clear demonstration of in vivo
proteolysis of phosphacan compared with transgenic mice null for either tissue
plasminogen activator or plasminogen was only observed in the hippocampus
following sclerotic changes induced by kainite injections
(45). Hence, there does not
seem to be a constitutive large scale cleavage of phosphacan under
non-pathological conditions, unlike the situation that is readily observed for
another prominent CNS-specific CSPG, neurocan. Neurocan has been shown to be
subject to a specific processing generating large N- and C-terminal fragments
(46). However, this processing
is developmentally regulated and can be readily observed both in vivo
and in vitro, even occurring in astrocytic cell cultures that have
been extensively supplemented with protease inhibitors
(47).
In addition, although it has been suggested previously (48) that a 180-kDa protein species that reacts with anti-phosphacan sera may be a proteolytic product, this was because it was not recognized by the mAb 3F8. We observe similar bands migrating around 200–250 kDa (Fig. 4b), and another explanation might be that the 180-kDa band corresponds to a form of mostly unglycosylated phosphacan (36) in which the 3F8 epitope is either hidden by a small oligosaccharide, or, indeed, it has even been suggested that 3F8 may itself be a sugar epitope (40). In addition, this 180-kDa band was found in cell-conditioned media (48), unlike the 90-kDa band that we have shown to be in the PBS-insoluble fraction. Hence, on balance, it seems unlikely that the 90-kDa protein that we have characterized results from a proteolytic cleavage event.
Molecular Interactions of PSI and Phosphacan—The
identification of a novel isoform of phosphacan/RPTP- provides a new
component in the cell-cell and cell-ECM signaling events in which these
proteins have been implicated. Compared with ECM structures in other tissues,
the ECM of the CNS appears to have a relatively "loose"
supramolecular organization. As such, many ECM components can be readily
extracted from the CNS using physiological buffers, whereas the use of
chaotropic reagents such as urea and guanidium hydrochloride is often
necessary to dissociate ECM components from structures in other tissues
(1). Phosphacan is a large CSPG
that can be quantitatively recovered from brain tissue using detergent-free
physiological buffers (4). By
contrast, the smaller PSI protein remains associated with the PBS-insoluble
material, which includes the total cell membrane fraction. The PSI protein can
be solubilized from this fraction using either detergent or urea, suggesting
that, although it is strongly associated with this fraction, it is not
directly associated with lipids. In addition, there is no evidence of any
lipid binding domains in the PSI sequence. Consequently, it appears likely
that the PSI protein is bound with PBS-insoluble proteins in this fraction.
Based on previous reports of binding partners for phosphacan, several
membrane-bound members of the IgCAM superfamily might account for such
interactions. Thus, the transmembrane IgCAMs, L1/NgCAM, NrCAM, and NCAM-180,
and the glycosylphosphatidylinositol-anchored IgCAMs, F3/contactin and
TAG-1/Axonin, have all been shown previously to bind to phosphacan
(20,
21,
23,
49). The PSI protein comprises
the CA and FNIII domains and half of the S region, and as such it could also
be involved in the interactions that have already been shown for these parts
of phosphacan. Hence, it has been shown that F3/contactin binds to the CA
domain (20), whereas NCAM,
L1/NgCAM, and NrCAM have all been shown to interact with the S region
(21). Indeed of the neuronal
IgCAMs found to interact with phosphacan, only TAG-1/Axonin-1, which binds to
phosphacan via the CS GAG chains
(23), interacts principally
with parts of the molecule that are absent from the PSI protein.
Here we demonstrate that both the native and recombinant PSI protein can interact with L1/NgCAM and F3/contactin, and thus it seems likely that both the PSI protein and phosphacan could interact with the same receptor proteins at sites that are common to both isoforms. Therefore, it is possible that where the PSI protein and phosphacan are coexpressed they may compete for common receptors.
The retention of PSI in the PBS-insoluble fraction of post-natal brain under conditions in which phosphacan is readily extracted suggests that in fact the PSI protein may be more tightly bound to such membrane receptors than phosphacan. This weaker association of phosphacan with the membrane fraction could also be because of additional binding interactions with PBS-soluble factors via other sites present on the phosphacan molecule. Hence binding interactions have been described between the CS GAG chains on phosphacan and growth factors such as HB-GAM/pleiotrophin (19, 25), midkine (24), and amphoterin (19), and perhaps such molecular binding interactions could modify binding interactions at other sites on the proteoglycan. In addition, the high negative charge and steric bulk of the CS GAG chains may in themselves represent factors that lower the relative affinity of phosphacan for the membrane compared with the PSI protein.
Phosphacan Short Isoform and Neurite Outgrowth Promotion—Previously we have shown that the phosphacan proteoglycan can have opposing effects upon neurite outgrowth dependent upon the neuronal lineage, which may be mediated either via the CS GAG chains (4) or the core glycoprotein (5). Similarly, promotion was found from E16 cortical neurons on a phosphacan substrate that was neutral in the same study for outgrowth from E16 thalamic neurons (43), whereas phosphacan was inhibitory for outgrowth from E17 cerebellar neurons (25) and P6-P8 retinal ganglion cells (50).
In this study, we have found that a recombinant PSI protein can promote neurite outgrowth from E17 cortical neurons. Previous studies have implicated both the GAG chains and the glycosylated core protein (following either enzymatic digestion or as a result of eukaryotic expression of recombinant constructs), but evidence is presented here that, even in the absence of any glycosylation or other eukaryotic post-translational modification, there are interaction sites on the PSI protein that can promote neurite outgrowth.
Studies with recombinant Fc fusion proteins containing the different domains of phosphacan have demonstrated previously (20) that the CA domain can support neuronal adhesion and neurite outgrowth by binding to F3/contactin on the surface of neurons (20), and NrCAM has been shown to promote outgrowth via interactions with the S region. Both of these effects could be blocked by specific antibodies directed against F3/contactin and NrCAM (20, 21). Our co-immunoprecipitation and GST pull-down studies indicate that both the native and recombinant PSI protein can, as predicted, interact with L1/NgCAM and F3/contactin, and hence it is possible that the outgrowth promotion observed is because of binding interactions between neural IgCAM receptors and the PSI protein. Phosphacan is subject to extensive glycosylation, with up to 16 predicted N-glycosylation and 41 potential O-glycosylation sites. By comparison, PSI, which lacks the large 850-amino acid IS region and the C-terminal half of the S region, contains only eight of the predicted N-glycosylation and three of the potential O-glycosylation sites, and it is not substituted with GAG chains. Consequently, the range of possible interactions of the PSI protein is reduced compared with phosphacan, and this might explain the weaker outgrowth promotion observed in our study. Nevertheless, it seems that even without carbohydrate modifications, the PSI protein sequence can still promote process outgrowth from cortical neurons.
Phosphacan Short Isoform and Phosphacan as Regulators of the Tyrosine
Phosphatase Activity of the RPTP- Receptor Forms—It
has been suggested that phosphacan/RPTP- isoforms are involved in a
complex in vivo neuron-glia cross-talk via interactions with other
receptor molecules and ECM proteins
(13). The expression of the
PSI protein could also intervene in such processes by competing selectively
for ligands. A similar relationship has already been proposed for interactions
between the secreted phosphacan proteoglycan and the transmembrane receptor
forms of RPTP-. Hence extracellular sites common to the different
isoforms could compete for similar extracellular ligands and thereby modulate
the nature of the cytoplasmic tyrosine phosphatase signal from the RPTP-
receptor forms (51).
Alternatively, they could interact in either cis or trans
with other cell surface receptor molecules, affecting the respective signaling
responses of these molecules
(21). Thus, our identification
of a novel truncated isoform of phosphacan representing 78% of the shorter
extracellular region of the short RPTP- receptor protein could be a
complementary element to the competitive ligand model that has already been
proposed for the larger extracellular domains of phosphacan and the long
RPTP- receptor. In addition, although both phosphacan and the long
RPTP- receptor are modified by the addition of CS GAG chains, the PSI
protein and the short RPTP- receptor are not proteoglycans. Hence, the
expression of PSI suggests the possibility that both of the RPTP-
receptor forms have their own respective complementary secreted forms and that
these could serve as competitive regulators of their ligand interactions and
consequently of their intracellular enzymatic activity.
A possible functional interaction between the different isoforms of
phosphacan/RPTP- might occur during myelination, because it has been
shown that the CA domain, common to all four isoforms, can interact with
F3/contactin in a neuronal signaling complex that has been localized in
vivo at paranodal junctions between axons and paranodal loops of
myelinating glia (42). It has
also been shown recently (52)
that the recombinant CA domain can block the localization of this complex to
the paranodes, suggesting that the complex may be targeted via ECM
interactions with phosphacan/RPTP- expressed by myelinating glia.
Additionally a significant role for phosphacan/RPTP- in myelination has
been suggested by studies on mice deficient for the RPTP- gene that
indicate that, although there is no gross abnormality in the overall brain
architecture, there is a fragility in the myelin sheaths of CNS neurons
(12), and there is impaired
recovery from demyelinating lesions in these mice
(41).
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FOOTNOTES |
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* This work was supported in part by the CNRS, the German Research Council
(DFG, Fa 159/11-1,2,3), the International Spinal Research Trust, and the
Association pour la Recherche contre le Cancer. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Awarded a Poste rouge from the CNRS during part of this work. To whom
correspondence should be addressed. Tel.: 33-3-88-45-66-53; Fax:
33-3-88-41-17-80; E-mail:
garwood{at}neurochem.u-strasbg.fr.
1 The abbreviations used are: ECM, extracellular matrix; CNS, central nervous
system; CSPG, chondroitin sulfate proteoglycans; RPTP, receptor protein
tyrosine phosphatase; CA, carbonic anhydrase-like; GAG, glycosaminoglycan; TN,
tenascin; CAM, cell adhesion molecule; FNIII, fibronectin type III-like; mAb,
monoclonal antibody; pAb, polyclonal antibody; GST, glutathione
S-transferase; PSI, phosphacan short isoform; ORF, open reading
frame; UTR, untranslated region; P, post-natal day; PBS, phosphate-buffered
saline; PVDF, polyvinylidene difluoride; E, embryonic day; ChABC,
chondroitinase ABC; CS, chondroitin sulfate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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