From the Max-Delbrück-Centrum für
Molekulare Medizin, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany, ¶ Fachbereich Biologie,
Universität Konstanz, D-78457 Konstanz, Germany, and
Friedrich Miescher Institut, P. O. Box 2543, CH-4002 Basel, Switzerland
Received for publication, August 9, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Recently, we described a novel chick neural
transmembrane glycoprotein, which interacts with the extracellular
matrix proteins tenascin-C and tenascin-R. This protein, termed CALEB,
contains an epidermal growth factor-like domain and appears to be a
novel member of the epidermal growth factor family of growth and
differentiation factors. Here we analyze the interaction between CALEB
and tenascin-C as well as tenascin-R in more detail, and we demonstrate
that the central acidic peptide segment of CALEB is necessary to
mediate this binding. The fibrinogen-like globe within tenascin-C or -R enables both proteins to bind to CALEB. We show that two isoforms of
CALEB in chick and rodents exist that differed in their cytoplasmic segments. To begin to understand the in vivo function of
CALEB and since in vitro antibody perturbation experiments
indicated that CALEB might be important for neurite formation, we
analyzed the expression pattern of the rat homolog of CALEB during
development of retinal ganglion cells, after optic nerve lesion and
during graft-assisted retinal ganglion cell axon regeneration by
in situ hybridization. These investigations demonstrate
that CALEB mRNA is dynamically regulated after optic nerve lesion
and that this mRNA is expressed in most developing and in one-third
of the few regenerating (GAP-43 expressing) retinal ganglion cells.
A complex network of molecular interactions regulates the
differentiation of the nervous system. These interactions occur between
neural cells as well as between neural cells and their extracellular
surroundings, the extracellular matrix
(ECM)1 which is composed of a
complex agglomerate of glycoproteins and proteoglycans with diverse
functions (1). For example, some of these glycoproteins can support or
inhibit axonal growth (2, 3), and others may modulate the function of
proteins and peptides that are synthesized and released by neurons and
glial cells (4). One such example of the latter is the binding of
acetylcholine receptor-inducing activity (ARIA, a specific isoform of
neuregulin-1) to the glycosaminoglycan portion of proteoglycans due to
charged interactions with its amino-terminal portion (5). The EGF-like domain of ARIA that mediates its biological activity can be released by
proteolysis from the transmembrane precursor and might become tethered
to the ECM of developing synapses (6). Due to regulated proteolysis,
the active EGF-like domain of ARIA might become accessible to the ErbB
receptor tyrosine kinases and induce the synthesis of acetylcholine
receptor subunits at the neuromuscular junction (7-10).
In a screen to isolate novel molecules presumed to be involved in
nervous system differentiation, we identified the transmembrane protein
CALEB (chicken acidic leucine-rich
EGF-like domain containing brain protein) (11).
We described three different forms of CALEB of molecular masses of 200, 140, and 80 kDa, all of which contain an EGF-like domain, which is most
similar to the EGF-like domains of the members of the EGF family of
transmembrane growth and differentiation factors. In addition to the
EGF-like domain, a very acidic peptide segment is present in the
extracellular part of all the three CALEB components. The 140- and
200-kDa components of CALEB additionally comprise an amino acid
sequence enriched in leucines and prolines and potential attachment
sites for chondroitin sulfate chains. CALEB is expressed in synapse and
axon-rich areas in the developing nervous system, and in
vitro antibody perturbation experiments revealed a participation
of CALEB in neurite formation in a permissive growth environment. CALEB
is able to interact both with tenascin-C (TN-C) and tenascin-R (TN-R),
members of the tenascin family of ECM proteins. These are large
glycoproteins composed of a cysteine-rich region at the amino terminus,
multiple EGF-like repeats of the tenascin subtype, several fibronectin
type III (FNIII)-like repeats, and a single carboxyl-terminal globe
similar to the globular parts of the Our previous investigations demonstrated that CALEB can interact with
TN-C and TN-R, but it has not been defined which regions of CALEB or
the tenascins are required (11). Here we report that the acidic peptide
segment of CALEB is necessary for binding to TN-C and TN-R.
Furthermore, we show that the fibrinogen-like module of TN-C and TN-R
is responsible for the interaction with CALEB. We have investigated
whether other isoforms of CALEB are generated in the chick, and we
compared these sequences with mammalian homologs. In all species
examined, two isoforms of CALEB exist, which are generated by
alternative pre-mRNA splicing. These splicing events lead to two
different cytoplasmic tails of CALEB. Since our previous in
vitro investigations suggested a participation of CALEB in neurite
formation, we examined the expression pattern of the rat homolog of
CALEB during development, after optic nerve lesion, and during
graft-assisted axon regeneration in the retina of adult rats.
Constructs and cDNA--
CALEB constructs C1, C2, and C3
were generated using PCR and the CALEB cDNA (11). For construct C1,
C2, and C3, the upstream primers
5'-cacactagtcgagatcatcgacgttgactactac-3',
5'-cacactagtggccgatttctaccccaccacctcc-3', and
5'-cacactagtgccggagaacagcagtgagtgccg-3' were used, respectively. The
downstream primer in all cases was
5'-cacctcgaggtcggtgacgatggcctcgcagcg-3'. PCR products were introduced
into vector pDELF-1 (33) using SpeI and XhoI
sites. mCALEB/NGC constructs mC2 and mC3 were generated using
mCALEB/NGC cDNA and PCR. For construct mC2 and mC3 the upstream primers were 5'-cacactagtgtctgatttctaccccaccacatcc-3'and
5'-cacactagttctcctgccctctcaaaagcctgg-3', respectively. The downstream
primer was 5'-cacctcgaggtccgtgatgatggactcacagcg-3' in both cases. PCR
products were subcloned into vector pDELF-1 using SpeI- and
XhoI-cloning sites.
The putative mouse homolog of CALEB was obtained by screening a
cDNA library of P20 mouse brain (Stratagene, Heidelberg, Germany) using a probe composed of the sequence that encodes the acidic peptide
segment, the EGF-like domain, and the transmembrane region of CALEB.
Different independent cDNA clones were obtained, which cover most
of the coding sequence but lacked the 5'-end. 5'-RACE technology using
the "5'-RACE System for Rapid Amplification of cDNA Ends, Version
2.0" (Life Technologies, Inc.) with the following modification was
employed to establish the 5'-end. 2 µg of poly(A)+ RNA
for cDNA synthesis was used instead of total RNA, and the "gene-specific primer" was 5'-cacactagtcactgcagccgatgcctctag-3'. PCR products of four independent reactions were cloned and sequenced. The presumed rat homolog of CALEB was amplified by RT-PCR from poly(A)+ RNA prepared from rat brain using the upstream
primers 5'-cacgaattctgggcagcttcccggggtcaccag-3' and
5'-cacgaattcaagccgcgcgcaccggcaacagc-3' and the downstream primer
5'-cacgaattctgcccgagaggagacaacagtccttc-3', all of which were derived
from the corresponding mouse sequence. Several independent amplification products were cloned and sequenced. A cDNA clone encoding CALEBb, the isoform not described so far, was obtained by
screening an E16 chick brain library (34) using a probe, which is
composed of the sequence encoding the EGF-like domain, the
transmembrane region, and the cytoplasmic domain of CALEB.
Proteins and Antibodies--
TN-C and TN-R were isolated from
urea extracts of adult chicken brains by immunoaffinity chromatography
as described using mAb M1 and mAb 23-13, respectively (35, 36).
SDS-PAGE was performed with 7% acrylamide under reducing conditions
followed by Coomassie Blue staining (37). Western blots of CALEB
components were analyzed using mAb 4/1 to CALEB. Recombinant chick TN-C
variants (TN-C/190, FF
Purified TN-C and TN-R as well as the different recombinant proteins
were coupled to red fluorescent microspheres of 0.5 µm diameter
according to the manufacturer's protocol (Bioclean: Duke Scientific
Corp., Palo Alto, CA). Proteins to be coupled were used in a
concentration range of 20-50 µg/ml. The coupling reactions were
performed in phosphate-buffered saline for 2 h at 24 °C or overnight at 4 °C. Residual binding sites were blocked with bovine serum albumin.
For affinity chromatography using recombinant proteins
TNC/EFb Transfection of COS7 Cells and Microsphere Binding
Assay--
COS7 cells were transiently transfected using the
DEAE-dextran method as described previously (16, 38). At day 1 after transfection, the cells were trypsinized, washed, and replated on
collagen-coated 8-well chamber slides (Nalge Nunc International Corp.,
Naperville, IL). At day 2 after transfection, the supernatant was
removed and replaced by 200 µl of Dulbecco's modified Eagle's medium with 10% fetal calf serum. 1 µl of fluorescent microspheres was added per well. After a 2-h incubation at 37 °C, cells were washed, fixed, and stained for CALEB construct expression by indirect immunofluorescence using rabbit anti-human IgG, Fc Surgery and in Situ Hybridizations--
Surgery and preparation
of retinal explants were performed exactly as described (39). To
generate a cRNA probe, a 200-base pair fragment of mCALEB was amplified
by PCR using the mCALEB cDNA with the primers
5'-cacgaattcttggcaaagaagacagtgagcatg-3' and
5'-cacggatccccagctactgccacaacggc-3'. The generation of cRNA and the
procedure of the in situ hybridization were carried out as
detailed (39). For double in situ hybridizations, the
hybridization solution contained both a DIG- and a fluorescein-labeled
riboprobe (150 ng/ml each). The DIG-labeled probe is detected first
using anti-DIG-alkaline phosphatase reaction developed with BM purple. After the washing steps, the alkaline phosphatase is inactivated by
heat (80 °C for 20 min). The fluorescein-labeled riboprobe was then
detected by incubation with an alkaline phosphatase-conjugated antibody
to fluorescein followed by a color reaction with Fast Red (Roche
Molecular Biochemicals).
The Acidic Peptide Segment of CALEB Is Necessary to Mediate the
Binding to TN-C or TN-R--
In a screening procedure applied to
characterize novel binding proteins of known axon-associated
glycoproteins, we identified CALEB due to its interaction with the ECM
glycoproteins TN-C and TN-R (11). Further independent assays confirmed
the binding between CALEB and TN-C or TN-R. To map regions of CALEB
that might be responsible for these interactions, we used different
deletion constructs of the extracellular portion of CALEB cloned into
the pDELF-1 vector (Fig. 1). This vector
is a derivative of plasmid pSG5, which contains the SV40 early
promoter/origin of replication and carries the signal peptide sequence
of the neural cell adhesion molecule F11, a multiple cloning site, and
the CH2 and CH3 domains of human IgG1 followed by the
glycosylphosphatidylinositol anchor attachment signal of F11 (33). The
signal peptide of F11 results in a strong cell surface expression of
CALEB domains, and the constant domains of IgG1 allowed us to compare
the expression efficiency of the different CALEB constructs in COS7
cells by indirect immunofluorescence. Construct C1 contains amino acid residues 286 (EIIDV) to 476 (AIVTD) of CALEB (11) and therefore composes the acidic peptide segment and the EGF-like domain. In addition, it contains at its amino terminus a potential tyrosine sulfation motif, which might be important for binding TN-C or TN-R.
Construct C2 lacks this potential tyrosine sulfation motif, starts with
amino acid 338 of CALEB (ADFYP), and ends at position 476 as construct
C1. Construct C3 is composed of the amino acid sequence starting from
residue 411 (PENSS) and ending with residue 476 of CALEB. This
construct encodes the EGF-like domain but not the acidic peptide
segment of CALEB. These vectors were transfected into COS7 cells which,
after 2 days of cultivation, were incubated with red fluorescent
microspheres coated with purified TN-C or TN-R, respectively.
Transfected COS7 cells were identified using an antibody to the Fc The Fibrinogen-like Module of TN-C and TN-R Mediates the
Interaction with CALEB--
TN-C and TN-R are large ECM glycoproteins
composed of a cysteine-rich region at the amino terminus followed by
multiple EGF-like and FNIII-like domains. A fibrinogen-like module is
located at the carboxyl terminus (12-15). To identify domains of TN-C
and TN-R responsible for binding to CALEB, we expressed CALEB construct C1 (Fig. 1E), which does bind purified TN-C and TN-R, on
COS7 cells. We then coupled proteins derived from different TN-C
constructs (Fig. 2E) onto red fluorescent microspheres, and
we incubated the transfected COS7 cells with these microspheres.
Microspheres coated with recombinant TN-C/190, the smallest occurring
splice variant of TN-C in chick lacking the FNIII-like domains A-D,
are able to bind to COS7 cells that had been transfected with CALEB construct C1 (Fig. 2A). Microspheres coated with protein
FF CALEB Components Can Be Purified from Detergent Extract by Affinity
Chromatography Using EFn CALEB Is Generated in Two Isoforms and Is Related to Neuroglycan C
in Mouse and Rat--
Several members of the EGF family of
differentiation factors such as the neuregulins are generated as
multiple isoforms. We have therefore extensively screened for
alternative forms of CALEB in cDNA libraries of embryonic chicken
brain. By using a probe that encodes the EGF-like domain, the
transmembrane region, and a part of the cytoplasmic domain of CALEB, we
detected two isoforms of CALEB, designated as a and b forms, which
differed in their cytoplasmic tails. The a form is identical to our
original published sequence of CALEB (11), and the b form lacks the
eight carboxyl-terminal residues (REAQHRAL) and instead contains an
additional sequence of 50 amino acid residues (Fig.
4). As previously discussed by us the
EGF-like domain, the transmembrane segment, and most of the cytoplasmic
stretch of the a form of CALEB are related to neuroglycan C (NGC), a
chondroitin sulfate containing proteoglycan originally described with a
cluster of basic amino acid residues in the rat (40), whereas the
amino-terminal half and the carboxyl-terminal portion of rat NGC
appeared completely unrelated to CALEB (11). This comparison suggested
to us that a family composed of at least two members, CALEB and NGC,
might exist in the developing nervous system. However, extensive
screening in chick cDNA libraries as well as PCR studies and the
identification of the b form which extends the relationship of CALEB to
NGC did not result in further support of this interpretation. It
remained conceivable, however, that the two species chick and rat
diverged with respect to the number of CALEB-related genes or with
respect to the amino- and carboxyl-terminal segments of CALEB.
To distinguish between these possibilities and to study the function of
CALEB-related proteins in the mouse and rat, we screened a cDNA
library of P20 mouse brain with a probe that encompasses the acidic
peptide segment, the EGF-like domain, and the transmembrane region of
CALEB. In combination with the 5'-RACE technique we identified only two
open reading frames of cDNA sequences, which encode proteins highly
related to CALEB in the chick and identical to NGC in mouse ((41),
GenBankTM accession number AF133700). The putative
rat homolog of CALEB cloned by RT-PCR contains an acidic peptide
segment and is identical to the corrected sequence of rat NGC ((42),
GenBankTM accession number U33553). The CALEB
isoforms are aligned with the different isoforms of the putative mouse
and rat homologs of CALEB, which have been identified in Fig. 4.
In all three species two isoforms of CALEB exist, which differ with
respect to their cytoplasmic domains. When comparing these sequences it
is obvious that a large part of CALEB has been highly conserved during
evolution. In particular the acidic peptide segment, the EGF-like
domain, the transmembrane region, and most of the cytoplasmic domain
are highly related with regard to their amino acid sequences (Fig. 4).
The mammalian proteins, however, contain an insert of 27 amino acid
residues in their cytoplasmic tail that has not been detected in the
chick. Curiously, the amino-terminal peptide segment is different
between CALEB and its proposed species homologs in mouse and rat. The
reason for this is not clear, but we have no indication that this
difference could be due to an alternative pre-mRNA splicing event
nor that two different but similar genes might be involved.
The Fibrinogen-like Module of TN-R, Which Mediates the Interaction
to CALEB, Also Binds to an mCALEB/NGC Fusion Protein Containing the
Acidic Peptide Segment--
Our studies indicate that the putative
species homologs of CALEB in mouse (mCALEB/NGC) and rat (rCALEB/NGC)
contain an acidic peptide segment close to the EGF-like domain as was
established for CALEB. To analyze whether the acidic segment of
mCALEB/NGC is also important in binding to TN-R, we transfected COS7
cells with mCALEB/NGC constructs that encode the EGF-like domain either joined to (Fig. 5C,
construct mC2) or lacking (Fig. 5C,
construct mC3) the acidic peptide stretch. As detailed above
for CALEB, COS7 cells were then tested for their ability to bind
microspheres coated with EFn rCALEB/NGC mRNA Is Present in Retinal Ganglion Cells (RGCs) of
Embryonic, Postnatal, and Adult Rats--
Our previous published
in vitro studies indicated that CALEB might be important for
neurite formation in a permissive growth environment. To extend these
studies we made use of an in vivo model system that analyzes
the importance of proteins for RGC axon growth and regeneration in rats
(43, 44, 52). This includes analysis of mRNA expression during RGC
development, after optic nerve lesion (ONL), and during RGC axon
regeneration following sciatic nerve transplantation (45). Earlier
results have shown that only some of the mRNAs and proteins
involved in axon growth in the embryo are (re-)expressed during
regeneration (i.e. L1, F11/F3, Gap-43), whereas other
mRNAs and proteins are down-regulated directly after lesion
(i.e. TAG-1; the netrin receptors DCC, UNC5H1, and UNC5H2
(39, 46-48)) and are not re-expressed in axon-regenerating RGCs.
Against this background, we first determined the presence of rCALEB/NGC
mRNA in embryonic, postnatal, and adult rats using in
situ hybridization. Changes following axotomy or axotomy followed by a sciatic nerve graft (grafted rats) were evaluated subsequently.
In situ hybridization with an antisense cRNA probe of
rCALEB/NGC resulted in staining of the RGC layer in E17 rat embryos, and of RGCs at P0 and P15 (Fig. 6,
A-C) indicating that RGCs produce rCALEB/NGC mRNA
during the time of RGC axon growth and target contact formation. Adult
rats continued to express rCALEB/NGC mRNAs (Fig. 6D).
The density of cells carrying the in situ hybridization signal was markedly reduced in the adult compared with retinae at
P15.
RGCs Dynamically Regulate the Expression of rCALEB/NGC mRNA
after Optic Nerve Lesion--
To analyze whether rat RGCs regulate the
synthesis of rCALEB/NGC mRNA in response to ONL, in situ
hybridization experiments were performed using retinae between 2 and 28 days after crush. Labeled RGCs were counted in 10 1-mm2
quadrants of 8 pie-shaped segments per retina (at least 4 retinae per
mRNA) and averaged (Fig.
7A). In the unlesioned adult
rat retina ~800 RGCs per mm2, which is roughly 30-50%
of all RGCs present (39, 43, 49-51), synthesize rCALEB/NGC mRNA. 5 days after crush (dac), when the number of RGCs begins to decline (to
~75% of those present normally) due to lesion-induced cell death,
the number of rCALEB/NGC synthesizing RGCs decreases to ~120 cells
per mm2 which amounts to roughly 10% of cells present. In
other words, RGCs down-regulate rCALEB/NGC mRNA. By 14-28 dac only
~10-15% of the RGCs survive (39, 43, 49-51), and counts of cells
synthesizing rCALEB/NGC mRNA indicate that the mRNA is
synthesized by the majority of the surviving cells (Fig.
7A).
To determine whether axon-regenerating RGCs express rCALEB/NGC
mRNA, the retinae of grafted rats (28 days after surgery) were subjected to the in situ hybridization procedure. Counts of
rCALEB/NGC mRNA producing RGCs in grafted rats were performed as
before (in 10 1-mm2 quadrants of 8 pie-shaped segments per
retina in 3 retinae per mRNA; Fig. 7B). RGCs, able to
regenerate an axon into the sciatic nerve graft, express GAP-43
mRNA (39). rCALEB/NGC mRNA synthesizing RGCs represent a
fraction (approximately one-third) of RGCs, which produce GAP-43
mRNA (Fig. 7B). This is confirmed by double in situ hybridization with cRNA probes of rCALEB/NGC and GAP-43
(three retinae) showing the existence of RGCs that produce both
rCALEB/NGC mRNA and GAP-43 mRNA (data not shown). This
indicates that at 28 days after grafting, a subpopulation of RGCs with
axons in the graft produce rCALEB/NGC mRNA thus implying either
that axon regeneration occurs in the presence and absence of rCALEB/NGC or that rCALEB/NGC mRNA is transiently expressed by all
axon-regenerating RGCs at earlier time points but is more rapidly
down-regulated than GAP-43 mRNA.
To further our understanding of the molecular functions of CALEB,
in this report we have investigated the interaction between CALEB and
TN-C or -R, have characterized isoforms of CALEB in chick and rodents,
and have studied its in vivo expression in an axonal
regeneration model system. For binding analysis of CALEB, we have used
deletion mutant polypeptides expressed on the surface of cells (CALEB)
or generated by a eukaryotic expression system (TN-C or TN-R). As
discussed elsewhere these methods have been proven to be reliable for
analysis of the interactions of several multidomain transmembrane and
extracellular matrix proteins (16, 18, 30-32) and thus appropriate for
use in beginning to dissect the molecular function of CALEB. In the
design of the deletion constructs, we were guided by the following
characteristics of the extracellular portion of CALEB. 1) Our
previously published binding studies were performed with material from
embryonic chicken retina in which the 80-kDa polypeptide dominates. 2)
The amino-terminal segment, which is highly enriched in the regularly
spaced amino acids leucine and proline (LP motif), appears to be less
conserved, and in the chick is only found in the 140- and 200-kDa
components but not in the 80-kDa components. 3) The acidic segment and
the EGF-like domain are highly conserved between chick, rodents, and humans and are expressed in the three CALEB forms (80, 140, and 200 kDa) identified to date. The amino acid sequence of the EGF-like module
that is closely located to the transmembrane segment is similar to the
corresponding sequences of members of the EGF family of transmembrane
growth and differentiation factors (9) that are known to bind to ErbB
receptor tyrosine kinases (10). Although it is currently not known
whether the EGF-like domain of CALEB might interact with the ErbB
proteins, our studies demonstrate that the EGF-like domain is not
sufficient to bind to TN-C or TN-R. In contrast the acidic peptide
segment located amino-terminally to the EGF-like domain is important
for TN-C or TN-R binding in chick and mouse. Similar acidic peptide
segments have been described only in a few other transmembrane
proteins; two of these, the We demonstrated here that the fibrinogen-like module at the carboxyl
terminus of TN-C or TN-R, which is the most highly conserved domain
within all tenascin family members, is important for binding to CALEB.
Whether or not the cysteine-rich segments of TN-C/R are necessary for
binding, in addition to the fibrinogen-like domain, cannot be deduced
from these experiments. However, CALEB was eluted by EDTA from an
affinity column containing immobilized EFn At present the function of the interaction between CALEB and TN-C or
TN-R has not been defined. By analogy to the EGF-like domain of ARIA, a
member of the EGF family of growth and differentiation factors, which
might become tethered to the ECM via its Ig-like domain after
proteolytic liberation from its transmembrane precursor (6), it is
conceivable that TN-C or TN-R also immobilize a putative soluble
fragment of CALEB containing the EGF-like domain and the acidic peptide
segment in the ECM. However, so far such a proteolysis product of CALEB
has not been detected. Alternatively, CALEB might be a cellular
receptor for TN-C and TN-R.
We have shown previously that in an in vitro test system Fab
fragments of antibodies to CALEB inhibit neurite formation (11). To
begin to understand whether CALEB might be important for axonal growth
in vivo, we analyzed the expression of the putative rat homolog of CALEB, rCALEB/NGC, in the rat retina which serves as a model
system to study axon regeneration in the mammalian central nervous
system (43, 45, 49). This system has been frequently used to follow the
temporal expression pattern of a number of different mRNAs encoding
cell surface proteins implicated in axon growth after ONL (39, 46). In
rats receiving a sciatic nerve transplant a fraction of RGCs (not more
than 5%) are capable of regenerating an axon into the graft. In the
developing rat retina, rCALEB/NGC mRNA is generated by most RGCs
which is in agreement with previous observations that CALEB is present
in the embryonic chick optic fiber layer (11). In the adult rat,
however, only ~30-50% of the RGCs produce rCALEB/NGC mRNA. It
is currently not known whether these cells represent a subpopulation of
RGCs or whether this expression pattern is a reflection of a dynamic
and transient synthesis of rCALEB/NGC mRNA. After ONL, the number of RGCs synthesizing rCALEB/NGC mRNA declines until 5 dac. During this period, most of the RGCs survive indicating that after loss of
target innervation rCALEB/NGC synthesizing RGCs down-regulate their
rCALEB/NGC-encoding mRNA. However, 5 days after ONL, when the
quantity of RGCs begin to decline due to cell death, the number of RGCs
producing rCALEB/NGC mRNA did not decline further suggesting that
at 14-28 dac most of the surviving RGCs continue to synthesize rCALEB/NGC mRNA. One possible interpretation for this temporal pattern of rCALEB/NGC mRNA production by RGCs might be that those RGCs, which express rCALEB/NGC encoding mRNA during the period after ONL, are somehow more resistant to cell death. This question awaits further investigations. The described time course for rCALEB/NGC mRNA synthesis after ONL is different from the temporal expression pattern described for other mRNAs examined so far. For example, TAG-1, an axon-associated cell adhesion molecule, as well as the netrin
receptors are expressed by adult rat RGCs, but their mRNA is
down-regulated and lost after ONL (39, 46). Almost all RGCs synthesize
L1 mRNA before ONL, and the number of RGCs producing this type of
mRNA decreased in parallel with the loss of RGCs due to cell death
(39). GAP-43 mRNA is not generated by adult RGCs, but the
expression of its mRNA is up-regulated in the first 5 days after
ONL and then subsequently declines (47, 48). GAP-43 is thought to be
important for axon extension and sprouting (63), and in grafted rats
most RGCs, retrogradely labeled with horseradish peroxidase used to
identify RGCs that regenerate an axon, synthesize GAP-43 mRNA. In
contrast, only one-third of those RGCs in grafted rats, which generate
GAP-43 mRNA, also synthesize rCALEB/NGC mRNA. Expression of
rCALEB/NGC mRNA in developing and in a fraction of
axon-regenerating RGCs in adults suggests a role for CALEB in the
differentiation process of RGCs including axon formation. Additional
indications on the in vivo function of CALEB might result
from the analysis of mCALEB/NGC-deficient mice generated by homologous recombination.
In summary, the data presented in this report increase our
understanding of the molecular function of CALEB. We established that
the acidic peptide segment of CALEB is necessary for binding TN-C and
TN-R and that the fibrinogen-like module of TN-C and TN-R is
responsible for mediating the interaction with CALEB. Furthermore,
CALEB-like proteins that are binding partners for TN-C and TN-R are
present in mouse and rat, and we have shown that the same segment in
mCALEB/NGC and CALEB is involved in binding the fibrinogen-like domain
of TN-R. We analyzed the expression of rCALEB/NGC following optic nerve
lesion and during graft-assisted axon regeneration, and we found that
RGCs dynamically regulate the synthesis of rCALEB/NGC mRNA in
response to lesion and that a subfraction of RGCs express CALEB
mRNA when regenerating an axon.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
chains of fibrinogen
(12-15). TN-C and TN-R associate to hexamers and trimers,
respectively, mediated by heptad repeats within the amino-terminal
cysteine-rich segment. For TN-C, several variants have been reported
that are generated by alternative splicing of the FNIII-like repeats
whereas TN-R contains two sites of alternative splicing. Multiple
ligands have been described for TN-C and TN-R including the cell
surface proteins F11, axonin-1, neurofascin, RPTP
/
, the
2 subunit of voltage-gated sodium channels, and
different types of integrins (16-25). Furthermore, both TN-C and TN-R
can bind to other ECM glycoproteins and proteoglycans such as neurocan,
phosphacan (a specific isoform of RPTP
/
), versican, brevican,
fibronectin, as well as to heparin (26-28). In addition, the
alternative spliced segment TnfnA-D is able to interact with annexin
II (29). Many functional features have been ascribed to TN-C and TN-R
in the nervous system. For example, TN-C and TN-R contain both adhesive
and counteradhesive sites for cell attachment. TN-C is able to
stimulate neurite outgrowth (30), and TN-R has been shown to enhance
neurite growth mediated by other substrates and to modulate the
cellular receptor usage that is responsible for mediating the outgrowth
effect (31, 32).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, EFb
, and
EFn
) were cloned as detailed elsewhere and expressed in
stably transfected HT 1080 cells (American Type Culture Collection,
Manassas, VA) (28, 30). The encoded recombinant proteins were purified
from the conditioned medium of the HT 1080 cells. The chick TN-R
construct EFn
(TN-R) corresponding to EFn
(TN-C) was cloned into the vector pFLAG-CMV-1 (Sigma) and expressed in
COS7 cells (American Type Culture Collection). For this, the cysteine-rich region of TN-R together with a His tag was amplified using PCR with the primers
5'-gcgaattcacatcaccatcaccatcacgaggggggcctggccaactgc-3' and
5'-gggagatctacggcacgactccaggct-3' and cloned into pFLAG-CMV-1 using
EcoRI and BglII. In the next step the
fibrinogen-like domain of TN-R was amplified by PCR using the primers
5'-gggagatctggtggccgtgtcttt-3' and 5'-cgcggatcctcagagctgtagggaccg-3'
and introduced into this modified vector using BglII and
BamHI. Recombinant protein was purified from conditioned
medium using nickel-nitrilotriacetic acid-agarose (Qiagen, Hilden,
Germany). The purity of proteins was analyzed by SDS-PAGE followed by
silver staining.
and TNC/EFn
immobilized to
CNBr-activated Sepharose, embryonic day 20 (E20) chick brains were
homogenized in homogenization buffer (250 mM sucrose in 20 mM Hepes, pH 7.2, with protease inhibitor mixture). The
10,000 rpm pellet was solubilized in homogenization buffer containing
2% Chaps. After centrifugation (100,000 × g), half the volume of the supernatant was passed over the TNC/EFn
and the TNC/EFb
columns, respectively. Both columns were
washed with homogenization buffer containing 2 mM
CaCl2, 2 mM MgCl2, and 0.5% Chaps.
Columns were eluted with the same buffer without cations but with 10 mM EDTA.
fragment
(Dianova, Hamburg, Germany). Binding reactions were analyzed with a
confocal microscope (MRC1000, Bio-Rad or Zeiss LSM, Oberkochen, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
portion of human IgG1, followed by a secondary antibody, which contains
a green fluorochrome. Red/yellow fluorescence indicates binding of the
microspheres to the transfected cells. As shown in Fig. 1, microspheres
coated with TN-C bind to COS7 cells transfected with CALEB construct C1
(Fig. 1A), or with construct C2 (Fig. 1C), but do
not bind to COS7 cells transfected with construct C3 (Fig.
1D). No binding was observed to untransfected COS7 cells or
to COS7 cells transfected with the pDELF plasmid on which the IgG1
domains without CALEB sequences are expressed (Fig. 1B). These observations further demonstrate the specificity of the assay.
The same results were obtained when using microspheres coated with TN-R
instead of TN-C (Fig. 2F and
data not shown). These results demonstrate that the acidic peptide
segment of CALEB is required for binding between CALEB and the
tenascins, whereas the EGF-like domain of CALEB alone or the potential
tyrosine sulfation motif is not important for this interaction.
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Fig. 1.
Binding of TN-C to COS7 cells expressing
different CALEB constructs. The CALEB mutant polypeptides shown in
E were generated using the pDELF-1 vector that drives cell
surface expression of these proteins in COS7 cells by the SV40 promoter
and the signal peptide of the neural cell recognition molecule F11. The
CALEB constructs were fused to the CH2 and CH3 domains of human IgG1
followed by the glycosylphosphatidylinositol (GPI) anchor
attachment signal of F11. Red fluorescent microspheres loaded with TN-C
were tested for binding to CALEB fusion proteins expressed on COS7
cells. Transfected cells were detected by their green fluorescence
after labeling with an antibody that recognizes the human IgG-Fc
portion of the fusion proteins followed by a fluorescein
isothiocyanate-coupled secondary antibody. Red fluorescent microspheres
loaded with TN-C bind to COS7 cells that express CALEB construct C1
(A) or C2 (C), both of which contain the acidic
peptide segment as well as the EGF-like domain of CALEB. These
microspheres do not bind to cells transfected with the control plasmid
lacking any CALEB encoding sequences (B). D shows
that TN-C-loaded microspheres are not able to interact with COS7 cells
expressing CALEB construct C3, which contains the EGF-like domain of
CALEB but lacks the acidic peptide segment. E summarizes the
binding results and shows the different constructs in a schematic
manner. TN-R reveals the same binding pattern as TN-C (Fig.
2F and data not shown). Bar, 50 µm.
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Fig. 2.
Binding of recombinant TN-C
(A-E) or TN-R (F-H) mutant
polypeptides to CALEB fusion protein expressed on COS7 cells. In
these experiments COS7 cells were transfected with CALEB construct C1
(see Fig. 1). The transfected cells were incubated with red fluorescent
microspheres loaded with different recombinant proteins encoded by TN-C
or TN-R constructs (see E and H). A
reveals that recombinant TN-C (TN-C/190) binds to CALEB-transfected
COS7 cells. Neither protein derived from construct FF of
TN-C (B), which lacks the FNIII-like repeats as well as the
fibrinogen-like module (Fg), nor from the construct
EFb
of TN-C (C), which does not contain both
the EGF-like segment and the fibrinogen-like module, bind to
CALEB-transfected cells. However, recombinant protein encoded by
construct EFn
of TN-C, which contains the cysteine-rich
domain (Cys-rich) as well as the fibrinogen-like module, but not the
EGF-like repeats or FNIII-like repeats, does bind to COS7 cells that
were transfected with CALEB construct C1 (D). F
and G show the binding of purified TN-R and the polypeptide
derived from construct EFn
of TN-R, respectively. Both
interact with COS7 cells transfected with CALEB construct C1. The
results are summarized in E and H. Bars, 50 µm.
(TN-C), which lacks both the FNIII-like domains as
well as the fibrinogen-like module of TN-C, do not bind to COS7 cells
transfected with CALEB construct C1 (Fig. 2B). The same
result was observed for microspheres coated with EFb
(TN-C), which composes the cysteine-rich region and the FNIII-like domains 1-5 and 6-8 of TN-C but lacks the fibrinogen-like module (Fig. 2C). In contrast, recombinant protein derived from a
construct that encodes only the cysteine-rich region and the
fibrinogen-like globe of TN-C (EFn
(TN-C); Fig.
2E) is able to mediate the binding to transfected COS7
cells, when coupled to red fluorescent microspheres (Fig. 2D). On the basis of these findings using TN-C mutant
polypeptides, we generated a similar deletion mutant of TN-R containing
the cysteine-rich segment and the fibrinogen-like module, and we coated microspheres with the recombinant protein (EFn
(TN-R);
Fig. 2H). These beads were found to bind to COS7 cells that
were transfected with CALEB construct C1 (Fig. 2G). In
summary, our investigations indicate that the fibrinogen-like module of either TN-C or TN-R mediates the interaction with the acidic peptide segment of CALEB.
(TN-C)--
To confirm the
importance of the fibrinogen-like globe in binding of TN-R or TN-C to
CALEB, we prepared affinity columns with the recombinant polypeptides
EFn
or EFb
of TN-C mentioned above. Equal
amounts of a detergent extract from E20 chick brain as indicated by
Coomassie Blue-stained gels in Fig.
3A were passed over both
columns (load). After washing, the columns were eluted with 10 mM EDTA, and the resulting fractions (load (L),
flow-through (F), wash (W), and eluate
(E)) were separated on SDS-PAGE, blotted to nitrocellulose,
and probed with mAb 4/1 to CALEB (Fig. 3B). Two
immunopositive bands (arrows) characteristic for CALEB from
embryonic brain could be detected in the load. The flow-through of
column EFn
(TN-C) contained a small amount of
immunopositive CALEB bands, whereas most could be detected in the
eluate. In contrast no CALEB components were observed in the eluate of
the column EFb
(TN-C) but were present in the
flow-through. The EDTA elution step followed by 1 M NaCl
did not result in elution of any additional CALEB components (data not
shown). Taken together, these results demonstrate that the
fibrinogen-like domain of TN-C or TN-R mediates the interaction to
CALEB and support the binding studies using COS7 cells.
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Fig. 3.
CALEB from brain detergent extract binds to
immobilized recombinant protein encoded by construct EFn
of TN-C. A detergent extract of chicken brain (embryonic day 20)
was passed over columns containing immobilized protein derived from
either construct EFb
of TN-C (TNC/EFb
) or
EFn
of TN-C (TNC/EFn
). After washing with
homogenization buffer (see "Materials and Methods") containing 2 mM CaCl2, 2 mM MgCl2,
and 0.5% Chaps, the columns were eluted with 10 mM EDTA in
homogenization buffer. L indicates the detergent extract
that was applied to the column (load); F indicates the
flow-through; W indicates the wash; and E
indicates the eluate. Samples were resolved by 7% SDS-PAGE and stained
with Coomassie Blue (A) or probed on Western blot with the
mAb 4/1 specific for CALEB (B). Molecular mass markers
(M) are indicated to the left. The two bands,
which were recognized by the mAb 4/1, are marked by
arrows.
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Fig. 4.
Alignment of CALEB isoforms and its putative
species homologs in mouse (mCALEB/NGC) and rat (rCALEB/NGC). A
novel isoform of CALEB was identified in the developing chick nervous
system that contains an additional segment of 50 amino acid residues at
the carboxyl terminus (form b). The two forms of CALEB were aligned
with the two forms identified in rodents. The putative mouse homolog of
CALEB was cloned by screening a mouse cDNA library and by the RACE
technique, whereas the putative rat homolog of CALEB was obtained by
RT-PCR. The rodent forms were found to be identical to NGC. The b-form
in mouse and rat contains an additional sequence of 27 amino acid
residues (additional peptide) in the cytoplasmic domain. The
splice site occurs at the same position in all three species. Identical
amino acid residues are boxed. Sequences of the acidic
peptide segment, the EGF-like domain, the transmembrane region
(TM), and the cytoplasmic domain are
underlined.
(TN-R) (see Fig.
2H). Similar to the results obtained with CALEB constructs
C2 and C3 (see Fig. 1), only COS7 cells that were transfected with
construct mC2 bound microspheres (Fig. 5A). In contrast, microspheres coated with EFn
(TN-R) do not bind to COS7
cells transfected with construct mC3 (Fig. 5B) thus
indicating a similar mechanism of binding between TN-R and CALEB or its
putative species homolog mCALEB/NGC. This result extends their
structural similarity to a functional relationship.
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Fig. 5.
Binding of the fibrinogen-like module of TN-R
to different mCALEB/NGC constructs. COS7 cells were transfected
with two different constructs (mC2 and mC3), derived from the cDNA
sequence encoding the putative mouse homolog of CALEB. The construct
mC2 resembles CALEB construct C2 (Fig. 1), whereas construct mC3 is
similar to CALEB construct C3 (Fig. 1). The transfected COS7 cells were
incubated with red fluorescent beads loaded with purified protein
derived from construct EFn (TN-R) (Fig. 2). This
polypeptide, which contains the cysteine-rich part and the
fibrinogen-like domain of TN-R, binds to COS7 cells transfected with
construct mC2 (A) but not with mC3 (B).
C summarizes the binding results. Bar, 50 µm.
GPI, glycosylphosphatidylinositol.
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Fig. 6.
Detection of rCALEB/NGC mRNA in the
developing and adult retina by in situ
hybridization. A, cross-section through the eye
of E17 rat embryos, with the lens at the top and the optic nerve
showing down. The in situ hybridization signals (see
arrowhead) in the RGC layer indicate the presence of
rCALEB/NGC mRNA. In situ hybridization signals were
detected in RGCs in segments of retina whole mounts from P0 rats
(B), P15 rats (C), and adult rats (D)
with antisense cRNA probes of CALEB. RGCs containing rCALEB/NGC
mRNA appear with dark somata and bright nuclei (see
arrows in D). The intensity of the in
situ hybridization signal varies over the RGC population.
Bar in A, 120 µm; bar in
D, 30 µm.
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Fig. 7.
Quantification of RGCs displaying in
situ hybridization signals with antisense cRNA probe of
rCALEB/NGC in the normal adult retina, in retinae after ONL, and in
grafted retinae. The height of the bars indicates the
average number of rCALEB/NGC cRNA-stained RGCs and the S.D.
A, the number of RGCs containing rCALEB/NGC mRNA changes
after optic nerve crush. Approximately 800 RGCs/mm2 in the
adult retina (adult) are positive for rCALEB/NGC mRNA.
That is 30-50% of all RGCs present (compared with data from Refs. 39,
43, and 49-51). This number decreases 2 and 5 days after optic nerve
crush (dac) to values below 10% when compared with the
total number of surviving RGCs at these time points. On day 14 and 28 after crush ~200 RGCs/mm2 are positive for rCALEB/NGC
mRNA. Compared with the total number of RGCs present at these time
points (data from Refs. 39, 43, and 49-51), more than 60% of the
surviving cells express mCALEB/NGC mRNA. B, very few
RGCs in grafted rats contain rCALEB/NGC mRNA. In these rats, a
small percentage of RGCs is able to regenerate an axon. Most of them
express GAP-43. Only one-third of the number of RGCs, which are
positive for GAP-43 mRNA (regenerating axons), also express
rCALEB/NGC mRNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amyloid precursor protein and the
fibroblast growth factor receptor 1 (53, 54), are expressed in the
nervous system. Our finding that the EGF-like domain of CALEB, which
shows similar sequence characteristics to those described for the
EGF-like domains of members of the EGF family of growth and
differentiation factors, does not bind to TN-C or TN-R implies that it
may interact with another currently unknown protein distinct from these
ECM glycoproteins. In this context it is of interest that the EGF-like
domain of CALEB and of its putative species homolog mCALEB/NGC is
encoded by two exons.2 The
first encodes the part of the EGF-like domain containing the first four
cysteine residues and the second encodes the remaining part of this
domain. This genomic structure points to an evolutionary relationship
between CALEB and several other members of the EGF family of growth and
differentiation factors (55-58).
suggesting
that the interaction between CALEB and TN-C/R is divalent ion-dependent or that the structural integrity of either
CALEB or EFn
is dependent on divalent ions. In this
context it should be noted that the fibrinogen-like globes of TN-C and
TN-R contain a segment that is related to EF-hand calcium-binding sites
identified in the
chain of fibrinogen, in thrombospondin, and in
calmodulin (59-61) and therefore might require divalent ions to fold
appropriately for binding. CALEB is not the only protein known to bind
to the fibrinogen-like domain of TN-C. For example, this domain also mediates the interaction with the ECM proteins neurocan and
phosphacan/RPTP-
/
which is enhanced by the presence of calcium
ions (19). Furthermore, this domain allowed lymphocyte rolling on TN-C
substrates (62), an effect mediated by a yet unknown receptor. In
addition, several integrins have been shown to interact with the
fibrinogen-like domain of TN-C including
2
1 on endothelial and
V
3 on Chinese hamster ovary cells (24,
25).
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ACKNOWLEDGEMENTS |
---|
We thank Marianne Brown-Lüdi, Frank-Peter Kirsch, Mechthild Henning, and Eva-Maria Stübe for technical help; Dr. Thomas Brümmendorf for providing the pDelf-1 vector and the chick E16 cDNA library; Drs. Philip Beesley (University of London) and Margret Moré for discussions and for critical reading of the manuscript; and Birgit Cloos for secretarial assistance.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Ra 424/3-1 (to F. G. R.) and by a grant from the Bundesministerium für Bildung und Forschung (to C. A. O. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF292101 and AF292102.
§ Present address and to whom correspondence may be addressed: Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-4558; Fax: 49-40-42803-4541; E-mail: sschumac@uke.uni-hamburg.de.
** To whom correspondence may be addressed: Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany. Tel.: 49-30-9406-3709; Fax: 49-30-9406-3730; E-mail: rathjen@mdc-berlin.de.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M007234200
2 S. Schumacher, M. Moré, and F. G. Rathjen, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: ECM, extracellular matrix; ARIA, acetylcholine receptor-inducing activity; CALEB, chicken acidic leucine-rich EGF-like domain containing brain protein; dac, days after crush; E, embryonic day; EGF, epidermal growth factor; FN, fibronectin; NGC, neuroglycan C; ONL, optic nerve lesion; P, postnatal day; RGC, retinal ganglion cell; RPTP, receptor protein-tyrosine phosphatase; TN-C, tenascin-C, TN-R, tenascin-R; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; RACE, rapid amplification of cDNA ends; PAGE, polyacrylamide gel electrophoresis; DIG, digoxigenin; mAb, monoclonal antibody; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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