From Molekulare Neurogenetik, Abteilung Neurochemie, Max-Planck-Institut für Hirnforschung, Deutschordenstrasse 46, D-60528 Frankfurt/Main, Germany
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ABSTRACT |
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The axonal guidance signal semaphorin D is a member of a large family of proteins characterized by the presence of a highly conserved semaphorin domain of about 500 amino acids. The vertebrate semaphorins can be divided into four different classes that contain both secreted and membrane-bound proteins. Here we show that class III (SemD) and class IV semaphorins (SemB) form homodimers linked by intermolecular disulfide bridges. In addition to the 95-kDa form of SemD (SemD(95k)), proteolytic processing of SemD creates a 65-kDa isoform (SemD(65k)) that lacks the 33-kDa carboxyl-terminal domain. Although SemD(95k) formed dimers, the removal of the carboxyl-terminal domain resulted in the dissociation of SemD homodimers to monomeric SemD(65k). Mutation of cysteine 723, one of four conserved cysteine residues in the 33-kDa fragment, revealed its requirement both for the dimerization of SemD and its chemorepulsive activity. We suggest that dimerization is a general feature of sema- phorins which depends on class-specific sequences and is important for their function.
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INTRODUCTION |
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The semaphorins are a large family of secreted and membrane-bound proteins that are involved in axonal navigation (1). To date, sequences of 15 vertebrate semaphorins have been published, and these can be divided into four classes (2-11) based on the similarity of their semaphorin domains and the presence of distinct sequence motifs in their COOH-terminal domains such as Ig homologies (classes III and IV), thrombospondin repeats (class V), and transmembrane segments (classes IV, V, and VI). The best studied vertebrate semaphorins are the murine SemD1 and its chick ortholog collapsin-1 (3, 6, 12-15). When added to cultures of dorsal root ganglia, both induce a rapid and reversible collapse of sensory growth cones (3, 12, 15). Gradients of SemD originating from aggregates of cells transfected with an expression vector repel sensory and sympathetic axons in collagen gel co-cultures, demonstrating that semaphorins have the ability to exclude axons from regions expressing these proteins (6, 13, 15).
The secreted class III semaphorins are synthesized as proproteins that are processed proteolytically to 95- or 65-kDa isoforms (designated 95k and 65k, respectively) at several conserved dibasic cleavage sites (15). Semaphorins SemA, SemD, and SemE act as repellents for specific populations of axons, and the potency of this repulsion is regulated by proteolysis (15). Cleavage of pro-SemD at a COOH-terminal processing site generates the 95k isoform (SemD95k)) and is required to activate its repulsive activity. Further cleavage of SemA, SemD, or SemE to a 65k form reduces their repulsive activities by at least 1 order of magnitude (15).
Semaphorins display specific and highly dynamic expression patterns in the developing nervous system as well as in non-neural tissues (2-6, 8, 9, 13, 14, 16-18). In vitro, specific subsets of spinal sensory afferents display a differential responsiveness to SemD which is regulated developmentally (5, 13, 14). It therefore has been proposed that SemD patterns spinal sensory innervation by dividing the spinal cord into dorso-ventrally organized subregions, one of which is accessible only to the prospective propioceptive fibers and excludes thermo- and nociceptive axons. The phenotype of mice homozygous for an inactivated semD gene supports this hypothesis (19). In addition, it reveals functions of semD in the differentiation of other tissues such as heart and skeleton.
Although the biological effects of semaphorins have been studied in some detail, the structural requirements for their function have not been analyzed to a similar extent. Here we show that the class III and IV semaphorins form homodimers linked by intermolecular disulfide bridges. Proteolytic cleavage of dimeric SemD(95k) results in its dissociation to monomeric SemD(65k). Mutation of a single cysteine residue in SemD both prevents dimerization and abolishes its repulsive activity. We propose that dimerization is an essential step in the maturation of the chemorepulsive guidance signal SemD and may have a similar functional importance for other classes of semaphorins.
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EXPERIMENTAL PROCEDURES |
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Expression Vectors and Transfection--
To express
recombinant semaphorins, cDNAs were cloned into the pBK-CMV
expression vector (Stratagene). An epitope-tag (Flag: DYKDDDDK) was
introduced between the signal peptide and semaphorin domain of SemD or
fused to the carboxyl terminus of SemB by polymerase chain
reaction as described previously (15). Cysteine to alanine mutations
(FlagSemDP1bC1: C567A and FlagSemDP1bC1: C723A) were introduced into
the FlagSemDP1b sequence using oligonucleotides including the intended
mutation (15) and verified by DNA sequencing. Amino acids
Ser580-Val772 were deleted to generate
FlagSemDCTD. To replace the COOH-terminal domain of SemD by the
hinge-CH2-CH3 (Fc) region from the human G1 immunoglobulin, FlagSemDFc
and FlagSemDP1bFc were constructed by replacing the sequences
corresponding to amino acids Ser580-Val772 by
oligonucleotides (5'-GGAACAGGTAAGTGGATCC-3') containing a splice donor.
The resulting semD sequence was cloned into pBK-Fc (generously provided by S. Heller), which contains a genomic
BamHI-NotI fragment encoding a splice acceptor
and the Fc region (20). In SemB
TCFlag, amino acids
His682-Ala760 were replaced by DYKDDDDKRS
using a polymerase chain reaction-based strategy.
Western Blot Analysis-- Human embryonic kidney 293 (HEK 293) cells (ATCC CRL 1573) were transfected by calcium phosphate co-precipitation (21) and the transfected cells grown in serum-free medium. Conditioned medium from transfected cells was collected 3 days after transfection and concentrated using Centriplus-30 concentrators (Amicon). Transfected cells were lysed in 400 µl of 2% (w/v) cholate (Sigma) in phosphate-buffered saline. Samples of recombinant proteins were separated by SDS-PAGE, transferred to nitrocellulose, and Western blots probed with the monoclonal anti-Flag antibody M2 (Kodak) or a polyclonal anti-COOH-terminal domain antiserum (anti-CTD) (15) using horseradish peroxidase-coupled anti-mouse or anti-rabbit IgG (Dianova) as secondary antibody in combination with the ECL detection system (Amersham Pharmacia Biotech) (15).
Cross-linking with Sulfhydryl-oxidizing Agents-- For cross-linking studies, recombinant proteins were expressed in HEK 293 cells, and conditioned media were concentrated using Centriplus-10 concentrators (Amicon). Concentrated media were supplemented with leupeptin (Sigma) to a final concentration of 10 µg/ml. Free thiols were acetylated by incubation of samples with iodoacetamide (Sigma) at a final concentration of 100 mM for 15 min at room temperature. Proteins were concentrated by methanol/chloroform precipitation (22), and the protein pellet was dissolved in 10 mM Tris/HCl, pH 7.5, supplemented with leupeptin. Dithiothreitol was added to a final concentration of 100 mM, and samples were incubated for 15 min at room temperature. The protein solution was precipitated as described above and the pellet dissolved in 10 mM Tris/HCl, pH 7.5. Aliquots of this solution were incubated with increasing concentrations of freshly dissolved (o-phenanthroline)2-Cu2+ for 30 min at room temperature. Cross-linking was terminated by the addition of 1 mM EDTA. Samples were taken after ultrafiltration, acetylation, and reduction with dithiothreitol and cross-linking, then they were dissolved in non-reducing sample buffer and analyzed by SDS-PAGE and Western blotting as described (15).
Gel Filtration Chromatography-- Gel filtration chromatography was performed on an Amersham Pharmacia Biotech XK 16/70 column with a Sephadex G-200 gel matrix (Sigma). Size standards (MW-GF-100, Sigma) were reconstituted in 50 mM Tris/HCl, pH 7.5, containing 50 mM NaCl as specified by the manufacturer. Recombinant proteins were expressed in HEK 293 cells, and concentrated conditioned media were loaded onto the column. Size standards and recombinant proteins were eluted at a constant flow rate of 0.2 ml/min, and 2.5-ml fractions were collected. Fractions were precipitated with trichloroacetic acid and analyzed by SDS-PAGE and Western blotting.
Co-culture Assay-- HEK 293 cells were transfected by calcium phosphate co-precipitation (21). Cell aggregates were formed essentially as described previously (6). Briefly, cells were treated with trypsin 5 h after transfection, washed with Eagle's minimum essential medium and 10% fetal calf serum (Life Technologies, Inc.), and resuspended in 0.4 ml of medium. Aggregates of HEK 293 cells were formed overnight in a hanging drop culture by placing drops of the cell suspension (20 µl) onto the lids of 35-mm dishes. Clusters of cells were harvested into medium and trimmed with tungsten needles for use in explant culture. Sympathetic ganglia from 9-day chick embryos were dissected into primary culture medium (23) and co-cultured with aggregates in a 10:1 mixture of collagen (Boehringer Mannheim) and Matrigel (Collaborative Research) in the presence of 50 ng/ml murine nerve growth factor (generously provided by H. Rohrer). After polymerization of the matrix for 60 min at 37 °C, primary culture medium was added, and the culture was incubated at 37 °C. Relative repulsive activities were determined as described previously (15).
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RESULTS |
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SemB and SemD Form Homodimers--
Semaphorins are characterized
by the presence of several conserved cysteine residues in both the
semaphorin domain and the COOH-terminal domain which may form intra- or
intermolecular disulfide bridges. To analyze this possibility we
investigated the biochemical properties of two semaphorins. Recombinant
SemB and SemD (Fig. 1) were expressed in
HEK 293 cells, and cell lysates or concentrated conditioned media were
analyzed by SDS-PAGE under reducing and non-reducing conditions
followed by Western blotting. We have shown previously that SemD is
processed proteolytically at several dibasic sequences (15). FlagSemD
is secreted as a 65-kDa protein from transfected HEK 293 cells, whereas
FlagSemDP1b contains a mutation in one of the cleavage sites (PCS1) and
consequently displayed a molecular mass of 95 kDa (Fig.
2, A and B). Under reducing conditions SemBFlag, FlagSemD, and FlagSemDP1b were detected at the expected molecular masses of 94, 65, and 95 kDa, respectively (Fig. 2A, lanes 2 and 3; Fig.
2B, lanes 1 and 3). The apparent molecular mass of FlagSemD changed only slightly under non-reducing conditions (Fig. 2B, lane 2), but SemBFlag and
FlagSemDP1b migrated at a molecular mass of about 190 kDa (Fig.
2A, lanes 5 and 6). This result
indicates that both SemB and SemD form homodimers that are linked by
one or several disulfide bonds. Because the 65-kDa isoform of SemD
behaved as a monomer under non-reducing conditions, it appeared likely
that the cysteine(s) responsible for dimerization of SemD reside within
the COOH-terminal domain. This is supported by the observation that the
COOH-terminal domain remained dimerized after proteolytic processing
(Fig. 2C). Efficient dimerization of SemB depended on the
presence of the transmembrane segment and/or the cytoplasmic domain, as
the majority of SemBTCFlag migrated as a monomer of 94 kDa after
deletion of its carboxyl-terminal end (amino acids
His682-Ala760; Fig. 2, lanes 1 and
4).
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Dimerization of SemD Requires Cysteine 723-- The different behavior of FlagSemD and FlagSemDP1b upon non-reducing SDS-PAGE indicates that the COOH-terminal domain of SemD is important for dimerization. To identify the residues that form intermolecular disulfide bonds we substituted four cysteine residues in the 33-kDa SemD COOH-terminal domain fragment which are conserved among all class III semaphorins by alanine. Three of these residues lie in the COOH-terminal domain and one at the carboxyl terminus of the semaphorin domain. Mutation of cysteines 598 and 650 had no effect on dimerization (data not shown), and their location in the Ig homology suggests that they might form an intramolecular disulfide bond similar to other peptide sequences of this type (24). Also, mutation of Cys567 did not change the molecular mass of SemD determined by SDS-PAGE under non-reducing conditions (Fig. 3, compare lanes 3 and 6). In contrast, mutation of Cys723 prevented the formation of dimers completely (Fig. 3, lanes 4 and 7).
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The 65- and 95-kDa Forms of SemD Differ in Their Ability to
Dimerize--
The size of the different recombinant proteins was
determined by gel filtration chromatography, to verify the molecular
mass observed by SDS-PAGE under non-reducing conditions (Fig.
5). The fractions were analyzed by
SDS-PAGE and Western blotting using an anti-Flag antibody, and the
protein concentration of the peak fractions was measured (Fig.
5A and data not shown). FlagSemD was processed to the 65-kDa
form and migrated with an apparent molecular mass of 70 kDa, consistent
with the expected size for an SemD monomer. The same fractions were
analyzed for the presence of the COOH-terminal domain by using the
anti-CTD antiserum raised against a fragment of the SemD COOH-terminal
domain. In Western blots this antibody revealed a protein that
migrated at a molecular mass of approximately 50 kDa, which probably
corresponds to a dimer of the 33-kDa COOH-terminal domain (Fig.
5B). SDS-PAGE under non-reducing conditions confirmed that
the COOH-terminal domain existed as a disulfide bond-linked dimer (data
not shown). When the semaphorin domain was expressed without the
COOH-terminal domain (FlagSemDCTD) it migrated at a size
similar to that of FlagSemD.
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Cys723 Is Essential for the Repulsive Activity of SemD-- To analyze the functional consequences of SemD dimerization, the repulsive activity of SemDP1bC1 (C567A) and SemDP1bC2 (C723A) was determined in a co-culture assay. Mutation of Cys560 resulted in a 7-fold reduction of repulsion compared with FlagSemD (Fig. 6A). In contrast, mutation of Cys723 almost completely abolished the repulsive activity of SemD; FlagSemDP1bC2 was almost 100-fold less active than FlagSemD (Fig. 6A). Similar results were obtained with constructs that did not contain a mutation in PCS1 (data not shown). Thus, Cys723 appears to be essential for the repulsive activity of SemD.
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DISCUSSION |
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The function of semaphorins as chemorepulsive axonal guidance signals has been analyzed in some detail (1, 3, 5-7, 12-15, 19, 25). However, the structural requirements for the activities of these proteins are still poorly understood. Here, we present evidence that two distinct semaphorins, the membrane protein SemB and the secreted SemD, form homodimers linked by intermolecular disulfide bonds when expressed in HEK 293 cells. Upon SDS-PAGE under non-reducing conditions, SemB and the 95-kDa form of SemD migrate with a molecular mass consistent with that of a dimer. A similar observation has been made for CD100, another class IV semaphorin (11, 26). Gel filtration chromatography of SemD(95k) also indicates a size of 190 kDa. Although semaphorins SemD and SemB both form dimers, the specific residues involved appear to be located at different positions. Dimerization of SemD depends on its COOH-terminal domain and is abolished by mutagenesis of Cys723. Cysteine residues are found at a similar position in other class III semaphorins, and it is likely that these will also form dimers. In contrast, SemB does not contain a cysteine residue at a corresponding position. Deletion of the putative membrane-spanning segment and cytoplasmic domain reduced the amount of SemB dimerization dramatically. Therefore, intermolecular interactions dependent on these sequences might precede the formation of disulfide bonds. Thus, although dimerization may be characteristic for semaphorins, the sequences responsible for it could be class-specific.
Mutational analysis of SemD revealed an important role of Cys723 not only in dimerization but also in its chemorepulsive activity. When analyzed in a co-culture assay, mutation C723A (FlagSemDP1bC2) almost completely abolished the repulsion of sympathetic axons. The inactivity of this mutant therefore is likely caused by its inability to form dimers as loss of both activities coincides. Thus, dimerization appears to be a prerequisite for SemD to display its repulsive activity. The function of Cys567 is less clear. Its mutation also resulted in a reduction in repulsive activity, and this residue may therefore be important for proper folding of SemD.
Previously, we have reported that proteolytic processing of SemD at
PCS1 to the 65-kDa form reduces its repulsive activity (15). Here we
show that cleavage not only removes the COOH-terminal domain but also
results in dissociation of SemD dimers. Determination of the molecular
mass of SemD by gel filtration chromatography showed that SemD(65k)
behaves as a monomer and does not remain associated with the dimerized
COOH-terminal domain. Thus, dissociation of SemD homodimers might
explain the reduced activity of SemD(65k) compared with SemD(95k).
However, monomeric SemD(65k) is still significantly more active than
FlagSemDCTD which is equivalent in sequence to the 65-kDa fragment
of FlagSemD. The COOH-terminal domain may be required as a co-factor in
addition to the semaphorin domain to activate putative SemD receptors
or, alternatively, to promote the adoption of an active conformation.
Artificial dimerization of the semaphorin domain by replacing its
COOH-terminal domain with the constant part of human IgG1 did not
result in active SemD and thus cannot substitute for the effects
mediated by the COOH-terminal domain. In this respect semaphorins
display a behavior different from that of another group of axonal
guidance molecules, the ephrins, which are activated by fusion to an Fc fragment (27, 28). In contrast, replacement of the SemD COOH-terminal domain by that of other class III semaphorins allows the synthesis of
at least partially active
SemD.2 Our results show that
dimerization mediated by the COOH-terminal domain is necessary but not
sufficient for the formation of active SemD (15). In addition,
processing of SemD at the carboxyl-terminal PCS3 and PCS4 is essential
for producing active SemD (15). Because FlagSemDP1bC2 is as inactive as
unprocessed SemD it appears possible that dimerization may be required
for correct processing of pro-SemD to its active form. For example,
dimerization could be required for recognition of PCS3/4 by the
processing enzyme(s).
In summary, dimerization of SemD appears to be an essential part in its maturation process. Formation of dimeric molecules is not restricted to the class III semaphorins but can be found in at least one other class of these proteins. Although it may be a general characteristic of this family, different classes of semaphorins probably depend on different molecular mechanisms to accomplish it.
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ACKNOWLEDGEMENTS |
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We thank C. Hass and A. Trutzel for excellent technical assistance, Dr. H. Rohrer for nerve growth factor, Dr. S. Heller for the pBK-Fc plasmid, and Dr. H. Betz for a critical reading of the manuscript and constant support.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Pu102/4-1 and SFB 269.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.
Present address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg,
Germany.
§ To whom correspondence should be addressed. Fax: 49-69-96769-441; E-mail: pueschel{at}mpih-frankfurt.mpg.de.
1 The abbreviations used are: SemD, semaphorin D; SemB, semaphorin B; SemD(65k) and SemD(95k), 65-kDa and 95-kDa isoforms of SemD, respectively; CTD, carboxyl-terminal domain; Fc, constant part of human immunoglobulin G1; PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney.
2 A. W. Püschel, unpublished results.
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REFERENCES |
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