A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent.

Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis.

View Point: Semaphorin-3E: An Emerging Modulator of Natural Killer Cell Functions?

Semaphorin-3E (Sema-3E) is a member of a large family of proteins originally identified as axon guidance cues in neural development. It is expressed in different cell types, such as immune cells, cancer cells, neural cells, and epithelial cells. Subsequently, dys-regulation of Sema-3E expression has been reported in various biological processes that range from cancers to autoimmune and allergic diseases. Recent work in our laboratories revealed a critical immunoregulatory role of Sema-3E in experimental allergic asthma. We further speculate possible immune modulatory function(s) of Sema-3E on natural killer (NK) cells.

[Nrp-1 expression in healing process of traumatic brain injury combined with tibial fracture].

OBJECTIVE: To examine the expression of neuropilin-1 (Nrp-1), semaphorin 3A (Sema3A) and vascular endothelial growth factor (VEGF) in the healing process of tibial fracture after traumatic brain injury and to explore the mechanism of Nrp-1 in the formation of pathological callus after nerve injury.
 Methods: A total of 192 Wister female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into a control group (Group C), a tibia fracture group (Group F), a traumatic brain injury group (Group TBI), a traumatic brain injury combined with the tibia fracture group (Group TBI+F) (n=48 in each group). Tissue samples were collected at 3, 5, 7, 14, 21 and 28 days, respectively (n=8 for each time point). The expression of Nrp-1 in callus tissues were examined by immunohistochemistry and Western blot, while the expression of Sema3A and VEGF were detected by Western blot.
 Results: Compared with the Group F , the expression of Nrp-1 in chondrocytes of bone formation area and cartilage area was higher in the Group TBI+F, particularly at the 7th , 14th and 21st day (P<0.05), while the expression of Nrp-1 in osteoblast cells of fresh bone trabecula region was lower in the Group TBI+F, particularly at the 14th and 21st day (both P<0.05). Western blot showed that the expression of Nrp-1, Sema 3A and VEGF had the same trends in the Group TBI+F and the Group F. However, at each time point, the expression of Nrp-1 in the Group TBI+F was significantly higher and slowly decreased, particularly at the 14th, 21st and 28th day (all P<0.05). Meanwhile, the ratio of Sema 3A/VEGF in the Group TBI+F was significantly higher than that in the Group F, with statistical difference (P<0.05).
 Conclusion: The Nrp-1 is expressed abnormally in the process of fracture healing after nerve injury. It may play a role in the formation of pathological callus after nerve injury by promoting the preliminary and proliferation of chondrocytes, and inhibiting the growth of nerve fibers in the soft callus as well as the differentiation of osteoblast cell.

SEMA3A suspended in matrigel improves titanium implant fixation in ovariectomized rats.

The aim of this study was to evaluate the effect of SEMA3A released from matrigel on implant fixation in ovariectomized (OVX) rats. Sixty female rats were subjected to bilateral ovariectomy. Twelve weeks later, rats were randomly divided into three groups according to implants they accepted: (1) Control, implants with distilled water; (2) Matrigel, implants with matrigel coating; (3) Matrigel + SEMA3A, implants with coating of SEMA3A suspended in matrigel. Implants were inserted in metaphysis of proximal tibiae in all animas bilaterally. In vitro release of SEMA3A was tested using enzyme linked immunosorbent assay. In vitro release of SEMA3A was detectable during the first 10 days, and a burst release of was observed during the first 3 days. No significant difference was observed between Control and Matrigel group. The protective effects of SEMA3A in matrigel on peri-implant bone, implant osseointegration and fixation was confirmed. Compared to matrigel alone, SEMA3A suspended in matrigel increased percent bone volume by 88.7% and 83.3% (p < 0.01), bone-to-implant contact ratio by 148.9% (p < 0.01), and 24.8% (p < 0.05), the maximal push-out force by 149.3% and 209.2% (p < 0.01) at 4 and 8 weeks after implant insertion, respectively. Surface modification with SEMA3A suspended in matrigel improved implant osseointegration and fixation in the proximal tibiae of OVX rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2060-2065, 2017.

Structure of the Plexin Ectodomain Bound by Semaphorin-Mimicking Antibodies.

Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these "semaphorin-mimicking" antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved "C" shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies.

Cationic Peptides and Peptidomimetics Bind Glycosaminoglycans as Potential Sema3A Pathway Inhibitors.

Semaphorin3A (Sema3A) is a vertebrate-secreted protein that was initially characterized as a repulsive-guidance cue. Semaphorins have crucial roles in several diseases; therefore, the development of Sema3A inhibitors is of therapeutic interest. Sema3A interacts with glycosaminoglycans (GAGs), presumably through its C-terminal basic region. We used different biophysical techniques (i.e., NMR, surface plasmon resonance, isothermal titration calorimetry, fluorescence, and UV-visible spectroscopy) to characterize the binding of two Sema3A C-terminus-derived basic peptides (FS2 and NFS3) to heparin and chondroitin sulfate A. We found that these peptides bind to both GAGs with affinities in the low-micromolar range. On the other hand, a peptoid named SICHI (semaphorin-induced chemorepulsion inhibitor), which is positively charged at physiological pH, was first identified by our group as being able to block Sema3A chemorepulsion and growth-cone collapse in axons at the extracellular level. To elucidate the direct target for the reported SICHI inhibitory effect in the Sema3A signaling pathway, we looked first to the protein-protein interaction between secreted Sema3A and the Nrp1 receptor. However, our results show that SICHI does not bind directly to the Sema3A sema domain or to Nrp1 extracellular domains. We evaluated a new, to our knowledge, hypothesis, according to which SICHI binds to GAGs, thereby perturbing the Sema3A-GAG interaction. By using the above-mentioned techniques, we observed that SICHI binds to GAGs and competes with Sema3A C-terminus-derived basic peptides for binding to GAGs. These data support the ability of SICHI to block the biologically relevant interaction between Sema3A and GAGs, thus revealing SICHI as a new, to our knowledge, class of inhibitors that target the GAG-protein interaction.

Immobilization of chitosan film containing semaphorin 3A onto a microarc oxidized titanium implant surface via silane reaction to improve MG63 osteogenic differentiation.

Improving osseointegration of extensively used titanium (Ti) implants still remains a main theme in implantology. Recently, grafting biomolecules onto a Ti surface has attracted more attention due to their direct participation in the osseointegration process around the implant. Semaphorin 3A (Sema3A) is a new proven osteoprotection molecule and is considered to be a promising therapeutic agent in bone diseases, but how to immobilize the protein onto a Ti surface to acquire a long-term effect is poorly defined. In our study, we tried to use chitosan to wrap Sema3A (CS/Sema) and connect to the microarc oxidized Ti surface via silane glutaraldehyde coupling. The microarc oxidization could formulate porous topography on a Ti surface, and the covalently bonded coating was homogeneously covered on the ridges between the pores without significant influence on the original topography. A burst release of Sema3A was observed in the first few days in phosphate-buffered saline and could be maintained for >2 weeks. Coating in phosphate-buffered saline containing lysozyme was similar, but the release rate was much more rapid. The coating did not significantly affect cellular adhesion, viability, or cytoskeleton arrangement, but the osteogenic-related gene expression was dramatically increased and calcium deposition was also abundantly detected. In conclusion, covalent bonding of CS/Sema could strongly improve osteogenic differentiation of osteoblasts and might be applied for Ti implant surface biofunctionalization.

Biallelic SEMA3A defects cause a novel type of syndromic short stature.

Chromosomal microarray testing is commonly used to identify disease causing de novo copy number variants in patients with developmental delay and multiple congenital anomalies. In such a patient we now observed an 150 kb deletion on chromosome 7q21.11 affecting the first exon of the axon guidance molecule gene SEMA3A (sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A). This deletion was inherited from the healthy father, but considering the function of SEMA3A and phenotypic similarity to the knock-out mice, we still assumed a pathogenic relevance and tested for a recessive second defect. Sequencing of SEMA3A in the patient indeed revealed the de novo in-frame mutation p.Phe316_Lys317delinsThrSerSerAsnGlu. Cloning of the mutated allele in combination with two informative SNPs confirmed compound heterozygosity in the patient. While the altered protein structure was predicted to be benign, aberrant splicing resulting in a premature stop codon was proven by RT-PCR to occur in about half of the transcripts from this allele. Expression profiling in human fetal and adult cDNA panels, confirmed a high expression of SEMA3A in all brain regions as well as in adult and fetal heart and fetal skeletal muscle. Normal intellectual development in the patient was surprising but may be explained by the remaining 20% of SEMA3A expression level demonstrated by quantitative RT-PCR. We therefore report a novel autosomal recessive syndrome characterized by postnatal short stature with relative macrocephaly, camptodactyly, septal heart defect and several minor anomalies caused by biallelic mutations in SEMA3A.

Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate type E motifs in rodent brains.

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186-200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.

Specific immobilization of biotinylated fusion proteins NGF and Sema3A utilizing a photo-cross-linkable diazirine compound for controlling neurite extension.

In this study we report the successful synthesis of N-(2-mercaptoethyl)-3-(3-methyl-3H-diazirine-3-yl) propanamide (N-MCEP-diazirine), with sulfhydryl and amine photoreactive ends to allow recombinant protein tethering to chitosan films. This regimen allows mimicry of the physiological endeavor of axon pathfinding in the nervous system where neurons rely on cues for guidance during development and regeneration. Our strategy incorporates strong covalent and noncovalent interactions, utilizing N-MCEP-diazirine, maleimide-streptavidin complex, and two custom biotinylated-fusion proteins, nerve growth factor (bNGF), and semaphorin3A (bSema3A). Synthetic yield of N-MCEP-diazirine was 87.3 ± 1.9%. Characteristic absorbance decrease at 348 nm after N-MCEP-diazirine exposure to UV validated the photochemical properties of the diazirine moiety, and the attachment of cross-linker to chitosan films was verified with Fourier transform infrared spectroscopy (FTIR). Fluorescence techniques showed no significant difference in the detection of immobilized proteins compared to absorbing the proteins to films (p < 0.05); however, in vitro outgrowth of dorsal root ganglia (DRG) was more responsive to immobilized bNGF and bSema3A compared to adsorbed bNGF and bSema3A over a 5 day period. Immobilized bNGF significantly increased DRG length over time (p < 0.0001), but adsorbed bNGF did not increase in axon extension from day 1 to day 5 (p = 0.4476). Immobilized bSema3A showed a significant decrease in neurite length (524.42 ± 57.31 µm) at day 5 compared to adsorbed bSema3A (969.13 ± 57.31 µm). These results demonstrate the superiority of our immobilization approach to protein adsorption because biotinylated-fusion proteins maintain their active confirmation and their tethering can be spatially controlled via a UV activated N-MCEP-diazirine cross-linker.

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Algorithms for the design of maximum hydropathic complementarity molecules.

In this article, we address the problem of designing a string with optimal complementarity properties with respect to another given string according to a given criterion. The motivation comes from a drug design application, in which the complementarity between two sequences (proteins) is measured according to the values of the hydropathic coefficients associated with the sequence elements (amino acids). We present heuristic and exact optimization algorithms, and we report on some computational experiments on amino peptides taken from Semaphorin and human Interleukin-1ß, which have already been investigated in the literature using heuristic algorithms. With our techniques, we proved the optimality of a known solution for Semaphorin-3A, and we discovered several other optimal and near-optimal solutions in a short computing time; we also found in fractions of a second an optimal solution for human interleukin-1ß, whose complementary value is one order of magnitude better than previously known ones. The source code of a prototype C++ implementation of our algorithms is freely available for noncommercial use on the web. As a main result, we showed that in this context mathematical programming methods are more successful than heuristics, such as simulated annealing. Our algorithm unfolds its potential, especially when different measures could be used for scoring peptides, and is able to provide not only a single optimal solution, but a ranking of provable good ones; this ranking can then be used by biologists as a starting basis for further refinements, simulations, or in vitro experiments.

Neuropilin 1 directly interacts with Fer kinase to mediate semaphorin 3A-induced death of cortical neurons.

Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons.

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding.

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

The CRMP family of proteins and their role in Sema3A signaling.

The CRMP proteins were originally identified as mediators of Sema3A signaling and neuronal differentiation. Much has been learned about the mechanism by which CRMPs regulate cellular responses to Sema3A. In this review, the evidence for CRMP as a component of the Sema3A signaling cascade and the modulation of CRMP by plexin and phosphorylation are considered. In addition, current knowledge of the function of CRMP in a variety of cellular processes, including regulation of the cytoskeleton and endocytosis, is discussed in relationship to the mechanisms of axonal growth cone Sema3A response. The secreted protein Sema3A (collapsin-1) was the first identified vertebrate semaphorin. Sema3A acts primarily as a repulsive axon guidance cue, and can cause a dramatic collapse of the growth cone lamellipodium. This process results from the redistribution of the F-actin cytoskeleton and endocytosis of the growth cone cell membrane. Neuropilin-1 (NP1) and members of the class A plexins (PlexA) form a Sema3A receptor complex, with NP1 serving as a high-affinity ligand binding partner, and PlexA transducing the signal into the cell via its large intracellular domain. Although the effect of Sema3A on growth cones was first described nearly 15 years ago, the intracellular signaling pathways that lead to the cellular effects have only recently begun to be understood. Monomeric G-proteins, various kinases, the redox protein, MICAL, and protein turnover have all been implicated in PlexA transduction. In addition, the collapsin-response-mediator protein (CRMP) family of cytosolic phosphoproteins plays a crucial role in Sema3A/NP1/PlexA signal transduction. Current knowledge regarding CRMP functions are reviewed here.

Negative regulation of platelet function by a secreted cell repulsive protein, semaphorin 3A.

Semaphorin 3A (Sema3A) is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and inhibits integrin function in an autocrine fashion. In this study, we investigated the effects of Sema3A on platelet function by using 2 distinct human Sema3A chimera proteins. We detected expression of functional Sema3A receptors in platelets and dose-dependent and saturable binding of Sema3A to platelets. Sema3A dose-dependently inhibited activation of integrin alphaIIbbeta3 by all agonists examined including adenosine diphosphate (ADP), thrombin, convulxin, phorbol 12-myristate 13-acetate, and A23187. Sema3A inhibited not only platelet aggregation induced by thrombin or collagen but also platelet adhesion and spreading on immobilized fibrinogen. Moreover, Sema3A impaired alphaIIbbeta3-independent spreading on glass coverslips and aggregation-independent granular secretion. Sema3A inhibited agonist-induced elevation of filamentous action (F-actin) contents, phosphorylation of cofilin, and Rac1 activation. In contrast, Sema3A did not affect the levels of cyclic nucleotides or agonist-induced increase of intracellular Ca2+ concentrations. Thus, the extensive inhibition of platelet function by Sema3A appears to be mediated, at least in part, through impairment of agonist-induced Rac1-dependent actin rearrangement.

A complementary peptide approach applied to the design of novel semaphorin/neuropilin antagonists.

Semaphorin 3A can inhibit axonal growth and induce neuronal apoptosis following binding to neuropilin-1, with the membrane proximal MAM (meprin, A5, mu) domain in neuropilin-1 playing a key role in the formation of a higher order receptor complex. If functional motifs on semaphorin 3A and/or the MAM domain can be identified, then small-constrained peptides might be developed as antagonists. We have scored peptide pairs for complementary hydropathy and antisense homology to identify a candidate functional motif in the Ig domain of semaphorin 3A, and in the MAM domain of neuropilin-1. Synthetic peptides corresponding to these sequences fully inhibit growth cone collapse induced by semaphorin 3A. A number of smaller peptides derived from the parental sequence also inhibited the response, particularly after they were constrained by a disulfide bond. Finally, we have used an algorithm to design a peptide that is a near-perfect hydropathic complement of the candidate functional site in the MAM domain; this also inhibits the semaphorin 3A response. Thus, an algorithm-driven methodology has led to the identification of three independent semaphorin 3A antagonists. Semaphorin 3F stimulates growth cone collapse following binding to the closest relative to neuropilin-1 in the genome, neuropilin-2. Where tested, the peptides that antagonise semaphorin 3A failed to inhibit the semaphorin 3F response.

Structure of the semaphorin-3A receptor binding module.

The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.