The Chemorepulsive Activity of the Axonal Guidance Signal Semaphorin D Requires Dimerization*

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 car-boxyl-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 sug-gest that dimerization is a general feature of semaphorins which depends on class-specific sequences and is important for their function. (FlagSemD D CTD) it migrated at a size similar to that of FlagSemD. The molecular mass of native FlagSemDP1b as determined by gel filtration chromatography was 190 kDa, consistent with the expected size for homodimeric SemD(95k). Little immunoreactivity was detected at higher molecular masses (data not shown), indicating that no additional oligomeric complexes were formed and that the SemD(95k) homodimer represents the majority of recombinant protein secreted by HEK 293 cells.

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 semaphorins which depends on class-specific sequences and is important for their function.
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)(3)(4)(5)(6)(7)(8)(9)(10)(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 SemD 1 and its chick ortholog collapsin-1 (3,6,(12)(13)(14)(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 pro-proteins 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 nonneural 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.

EXPERIMENTAL PROCEDURES
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 Ser 580 -Val 772 were deleted to generate FlagSemD⌬CTD. 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 Ser 580 -Val 772 by oligonucleotides (5Ј-GGAACAGGTAAGTGGATCC-3Ј) 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 His 682 -Ala 760 were replaced by DYKD-DDDKRS 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 coprecipitation (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 l 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 g/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 -Cu 2ϩ 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 crosslinking, 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 l) 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).

RESULTS
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 COOHterminal 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 SemB⌬TCFlag migrated as a monomer of 94 kDa after deletion of its carboxyl-terminal end (amino acids His 682 -Ala 760 ; Fig. 2, lanes 1 and 4).
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 Cys 567 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, muta- tion of Cys 723 prevented the formation of dimers completely (Fig. 3, lanes 4 and 7).
To substantiate these observations further, cross-linking studies were performed with a sulfhydryl oxidizing agent. Samples of recombinant semaphorins were reacted with iodoacetamide directly after harvesting the conditioned media to acetylate free thiols and avoid unspecific disulfide formation. As expected, FlagSemD, FlagSemD⌬CTD, and FlagSemDP1bC2 migrated as monomeric proteins with apparent molecular masses of 65, 65, and 95 kDa, respectively, whereas FlagSemDP1b formed dimers of 190 kDa (Fig. 4, lanes 1 and 2). Incubation of acetylated dimeric FlagSemDP1b with dithiothreitol resulted in the reduction of disulfide bonds as indicated by a shift in the molecular mass of FlagSemDP1b to 95 kDa (Fig. 4, lane 3). Cross-linking of the reduced proteins by incu-bation with increasing concentrations of the sulfhydryl oxidizing agent o-phenanthroline) 2 -Cu 2ϩ resulted in the re-formation of the 190-kDa complex only for FlagSemDP1b but not the other proteins analyzed (Fig. 4, lanes 5 and 6). Thus Cys 723 is responsible for the formation of an intermolecular disulfide bond, and its mutation blocks the dimerization of SemD completely.
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 COOHterminal 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 (FlagSemD⌬CTD) it migrated at a size similar to that of FlagSemD.
The molecular mass of native FlagSemDP1b as determined by gel filtration chromatography was 190 kDa, consistent with the expected size for homodimeric SemD(95k). Little immunoreactivity was detected at higher molecular masses (data not shown), indicating that no additional oligomeric complexes were formed and that the SemD(95k) homodimer represents the majority of recombinant protein secreted by HEK 293 cells. Introduction of the C723A mutation into FlagSemDP1b (FlagSemDP1bC2) prevented dimerization as indicated by a reduction of the molecular mass to approximately 100 kDa. These experiments confirm that Cys 723 in the COOH-terminal domain is essential for dimerization of SemD and that processing at PCS1 results not only in the removal of the COOHterminal domain but also in the conversion of SemD to a monomeric form. These results also revealed that SemD(65k) and the dimerized COOH-terminal domain do not appear to associate as they run at a molecular mass of 70 and 50 kDa, respectively, which is inconsistent with the formation of aggregates including both fragments.
Cys 723 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 Cys 560 resulted in a 7-fold reduction of repulsion compared with FlagSemD (Fig. 6A). In contrast, mutation of Cys 723 almost completely abolished the repulsive activity of SemD; FlagSemDP1bC2 was almost 100-fold less active than Flag-SemD (Fig. 6A). Similar results were obtained with constructs that did not contain a mutation in PCS1 (data not shown).
Thus, Cys 723 appears to be essential for the repulsive activity of SemD.
The COOH-terminal domain of class III semaphorins might function exclusively by promoting the formation of SemD dimers, or it may have additional functions. To distinguish between these possibilities, the COOH-terminal domain was replaced by the constant part of the human ␥-immunoglobulin, and the activity of the resulting hybrid protein was analyzed in a co-culture assay. These chimeric proteins displayed almost no detectable repulsive activity irrespective of processing at PCS1 (Fig. 6C). Similarly, FlagSemD⌬CTD did not show any repulsive effects despite a significantly higher expression compared with that of the other tested SemD variants (Fig. 6, B and C; note that five times less medium was loaded in lane 4). Western blot analysis confirmed that with the exception of FlagSemD⌬CTD all three proteins were expressed at comparable levels (Fig. 6B). DISCUSSION 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 Cys 723 . 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 Cys 723 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 Cys 567 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 COOHterminal 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 FlagSemD⌬CTD 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 COOHterminal 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.