Sema Domain Research Articles

Cholinergic signaling via muscarinic M1 receptor confers resistance to docetaxel in prostate cancer

Docetaxel is the most commonly used chemotherapy for advanced prostate cancer (PC), including castration-resistant disease (CRPC), but the eventual development of docetaxel resistance constitutes a major clinical challenge. Here, we demonstrate activation of the cholinergic muscarinic M1 receptor (CHRM1) in CRPC cells upon acquiring resistance to docetaxel, which is manifested in tumor tissues from PC patients post- vs. pre-docetaxel. Genetic and pharmacological inactivation of CHRM1 restores the efficacy of docetaxel in resistant cells. Mechanistically, CHRM1, via its first and third extracellular loops, interacts with the SEMA domain of cMET and forms a heteroreceptor complex with cMET, stimulating a downstream mitogen-activated protein polykinase program to confer docetaxel resistance. Dicyclomine, a clinically available CHRM1-selective antagonist, reverts resistance and restricts the growth of multiple docetaxel-resistant CRPC cell lines and patient-derived xenografts. Our study reveals a CHRM1-dictated mechanism for docetaxel resistance and identifies a CHRM1-targeted combinatorial strategy for overcoming docetaxel resistance in PC.

Pinpointing the interaction site between semaphorin-3A and its inhibitory peptide.

Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A-NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l-phenylalanine or photo-l-leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A-NRP-1 interaction.

Identifying Function Determining Residues in Neuroimmune Semaphorin 4A.

Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.

N-glycosylation regulates MET processing and signaling.

MET, the receptor for the hepatocyte growth factor (HGF), is strongly associated with resistance to tyrosine kinase inhibitors, key drugs that are used in the therapy of non–small cell lung cancer. MET contains 11 potential N‐glycosylation sites, but the site‐specific roles of these N‐glycans have not been elucidated. We report herein that these N‐glycans regulate the proteolytic processing of MET and HGF‐induced MET signaling, and that this regulation is site specific. Inhibitors of N‐glycosylation were found to suppress the processing and trafficking of endogenous MET in H1975 and EBC‐1 lung cancer cells and exogenous MET in CHO‐K1 cells. We purified the recombinant extracellular domain of human MET and determined the site‐specific N‐glycan structures and occupancy using mass spectrometry. The results indicated that most sites were fully glycosylated and that the dominant population was the complex type. To examine the effects of the deletion of N‐glycans of MET, we prepared endogenous MET knockout Flp‐In CHO cells and transfected them with a series of N‐glycan–deletion mutants of MET. The results showed that several N‐glycans are implicated in the processing of MET. The findings also suggested that the N‐glycans of the SEMA domain of MET positively regulate HGF signaling, and the N‐glycans of the region other than the SEMA domain negatively regulate HGF signaling. Processing, cell surface expression, and signaling were significantly suppressed in the case of the all‐N‐glycan–deletion mutant. The overall findings suggest that N‐glycans of MET affect the status and the function of the receptor in a site‐specific manner.

Architecture of the Sema3A/PlexinA4/Neuropilin tripartite complex

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.

Novel Truncating and Missense Variants in SEMA6B in Patients With Early-Onset Epilepsy.

Progressive myoclonic epilepsy (PME) is a rare neurodegenerative disease, characterized by myoclonic seizures and tonic clonic seizures, with genetical and phenotypical heterogeneity. The semaphorin 6B (SEMA6B) gene has been recently reported a causal gene of PME. Independent studies are warranted to further support these findings. Here we report that one nonsense variant in NM_032108.3 exon17 c.2056C > T (p.Gln686∗) and one missense variant in exon14 c.1483G > T (p.Gly495Trp) of SEMA6B, both occurring de novo, underlie early-onset epilepsy with variable severity and different response to treatment in two patients. In vitro analyses have demonstrated that the nonsense variant, p.Gln686∗, results in a truncated protein with remarkably increased expression compared to that of the wild type. The truncated protein presented more homogeneous and failed to locate in the plasma membrane. The missense variant p.Gly495Trp affects evolutionarily conserved amino acid and is located in the sema domain, a key functional domain of SEMA6B. It was predicted to perturb the SEMA6B function by altering the tertiary structure of mutant protein, although neither change of protein length and expression nor difference of cellular distribution was observed. Co-immunoprecipitation studies have demonstrated that both variants influence protein binding of SEMA6B and PlxnA2 with varying degrees. Our results provide further evidence to support the initial findings of SEMA6B being causal to epilepsy and indicate that mediating Semaphorin/Plexin signaling is the potential mechanism of the SEMA6B-related disease.

Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

Crizotinib inhibits activation of MET pathway caused by MET extracellular SEMA domain duplication

ObjectiveAberrant MET activation, which promotes cell proliferation and tumor metastasis, occurs in many types of cancer and results from multiple mechanisms. A novel MET duplication mutation was found in a non-small cell lung cancer (NSCLC) patient. The clinical response to crizotinib was investigated and the functional relevance was characterized in cellular models. Materials and MethodsNext-generation sequencing (NGS) was performed on the tumor tissue and circulating tumor DNA (ctDNA) of a patient with advanced NSCLC. In vitro studies including western blot, proliferation assays and colony formation assays were used to confirm the clinical observations. ResultsThe patient was identified to harbor a duplication of the MET SEMA domain. After a month of treatment, the patient showed a marked response to crizotinib, a multikinase inhibitor with potent activity against MET. Functional in vitro studies demonstrated that expression of MET SEMA duplication in NIH-3T3 cells stimulated the activation of MET signaling. Crizotinib treatment obviously repressed cell proliferation, colony formation, and MET signaling pathway. ConclusionCrizotinib treatment resulted in a clinical response in a patient with MET SEMA duplication. Results of cellular analyses together with the clinical data suggest that this novel alteration may represent an actionable target in NSCLC patients.

Cryo-EM structure of the PlexinC1/A39R complex reveals inter-domain interactions critical for ligand-induced activation

Plexins are receptors for semaphorins that transduce signals for regulating neuronal development and other processes. Plexins are single-pass transmembrane proteins with multiple domains in both the extracellular and intracellular regions. Semaphorin activates plexin by binding to its extracellular N-terminal Sema domain, inducing the active dimer of the plexin intracellular region. The mechanism underlying this activation process of plexin is incompletely understood. We present cryo-electron microscopic structure of full-length human PlexinC1 in complex with the viral semaphorin mimic A39R. The structure shows that A39R induces a specific dimer of PlexinC1 where the membrane-proximal domains from the two PlexinC1 protomers are placed close to each other, poised to promote the active dimer of the intracellular region. This configuration is imposed by a distinct conformation of the PlexinC1 extracellular region, stabilized by inter-domain interactions among the Sema and membrane-proximal domains. Our mutational analyses support the critical role of this conformation in PlexinC1 activation.

MP09-11 LANDSCAPE OF SOMATIC AND GERMLINE VARIATIONS IN UNRESECTABLE CHINESE PROSTATE CANCER PATIENTS

INTRODUCTION AND OBJECTIVE: Then landscape of genomic variations in unresectable Chinese prostate cancer (PCa) patients was unclear, which was essential in guiding drug therapy and prognosis predicting. We performed next generation sequencing (NGS) in unresectable PCa patients in order to study the somatic and germline variations in ctDNA, especially the prevalence and types of variations within two novel tumor suppressor genes PLXNA1 and PCDH9, and co-occurrence between PLXNA1 variations and the variations within other PCa-relevant pathways. METHODS: Targeted NGS was performed on ctDNA obtained from 243 unresectable Chinese PCa patients using a validated 90-PCa-relevant-gene panel. The whole coding sequence (cds) regions and partial intron regions of the 90-gene were sequenced. Co-occurrence analysis was performed using R software, version 3.6.1 (R Foundation for Statistical Computing) and maftools package. RESULTS: In 243 blood samples collected, 233 (95.9%) patients carried somatic or germline variations, and 112 (46.1%) patients have at least one mutation classified as pathogenic or likely pathogenic. There were 671 somatic and 308 germline mutations detected, with an average of 4.0 mutations per patient. The top frequent somatic mutation genes were AR (12.8%), TP53 (8.6%), KMT2C (4.5%), BRAF (2.3%) and CDK12 (2.3%). And the top frequent germline mutation genes were BRCA2 (9.1%), APC (4.9%), ATR (4.9%), ATM (4.5%) and BARD1 (4.1%), most of which belong to DNA damage repair pathway. Notably, 12 patients (4.9%) harbored PLXNA1 alterations and 1 patient (0.4%) harbored PCDH9 alterations. Among 14 PLXNA1 variations, 11 were single nucleotide variations and 3 were copy number gains, which occurred mainly in Sema domain and IPT/TIG domain. Furthermore, co-occurrence of variations within PLXNA1 and two genes (CCND1 and RB1) in cell cycle pathway was significant (p<0.05, Fisher Exact test). CONCLUSIONS: This study shows the landscape of both somatic and germline variations in unresectable Chinese PCa patients, which might be valuable to assess potential druggable markers and prognostic predictors. Source of Funding: None.

Semaphorin 4A, 4C, and 4D: Function comparison in the autoimmunity, allergy, and cancer

Semaphorins are a group of proteins that are divided into eight subclasses and identified by a conserved Sema domain on their carboxyl terminus. Sema4A, 4C, and 4D are the members of the fourth class of semaphorin family, which are known as membrane semaphorins; however, these molecules can be altered to soluble semaphorins by proteolytic cleavage. Semaphorins have various roles in the immune, nervous, and metabolic systems. In the immune system, these molecules contribute to the formation of cellular, humoral, and innate immune responses, such as inflammation, leukocyte migration, immunological synapse formation, and germinal center events. Given the diverse roles of semaphorins in the immune system, in this review, we have tried to give a comprehensive look at the role of these molecules in autoimmunity, allergy, and cancer. Sema4D and 4A seem to play a critical role in the pathogenesis of some autoimmune diseases, such as multiple sclerosis. In contrast, it has been shown that Sema4A and 4C have beneficial effects on allergies, and their absence can exacerbate the severity of the disease. In the case of cancer, an increase in all three of these molecules has been reported. Sema4D and 4C can contribute to tumor progression in human patients or experimental models, while the role of Sema4A has not yet been fully understood. In conclusion, semaphorins seem to be a favorable therapeutic target for autoimmune diseases and allergies. However, in cancer, studies have not yet been able to identify the exact role of semaphorins, and further studies are needed.

The Role of c-Met as a Biomarker and Player in Innate and Acquired Resistance in Non-Small-Cell Lung Cancer: Two New Mutations Warrant Further Studies

The c-Met receptor is a therapeutically actionable target in non-small-cell lung cancer (NSCLC), with one approved drug and several agents in development. Most suitable biomarkers for patient selection include c-Met amplification and exon-14 skipping. Our retrospective study focused on the frequency of different c-Met aberrations (overexpression, amplification and mutations) in 153 primary, therapy-naïve resection samples and their paired metastases, from Biobank@UZA. Furthermore, we determined the correlation of c-Met expression with clinicopathological factors, Epidermal Growth Factor Receptor (EGFR)-status and TP53 mutations. Our results showed that c-Met expression levels in primary tumors were comparable to their respective metastases. Five different mutations were detected by deep sequencing: three (E168D, S203T, N375S) previously described and two never reported (I333T, G783E). I333T, a new mutation in the Sema(phorin) domain of c-Met, might influence the binding of antibodies targeting the HGF-binding domain, potentially causing innate resistance. E168D and S203T mutations showed a trend towards a correlation with high c-Met expression (p = 0.058). We found a significant correlation between c-MET expression, EGFR expression (p = 0.010) and EGFR mutations (p = 0.013), as well as a trend (p = 0.057) with regards to TP53 mutant activity. In conclusion this study demonstrated a strong correlation between EGFR mutations, TP53 and c-Met expression in therapy-naïve primary resection samples. Moreover, we found two new c-Met mutations that warrant further studies.

Molecular Signatures for Combined Targeted Treatments in Diffuse Malignant Peritoneal Mesothelioma.

Background—There are currently no effective therapies for diffuse malignant peritoneal mesothelioma (DMPM) patients with disease recurrence. In this study, we investigated the biology of DMPM by analyzing the EGFR family, Axl, and MET, in order to assess the presence of cross-talk between these receptors, suggesting the effectiveness of combined targeted treatments in DMPM. Method—We analyzed a series of 22 naïve epithelioid DMPM samples from a single institute, two of which showed higher-grade malignancy (“progressed”). EGFR, HER2, HER3, Axl, and MET activation and expression were investigated by biochemical analysis, real-time PCR immunofluorescence, immunohistochemistry, next-generation sequencing, miRNA, and mRNA in situ hybridization. Results—In most DMPMs, a strong EGFR activation was associated with HER2, HER3, Axl, and MET co-activation, mediated mainly by receptor heterodimerization and autocrine-paracrine loops induced by the expression of their cognate ligands. Axl expression was downregulated by miRNA34a. Mutations in MET Sema domain were exclusively found in two “progressed” DMPMs, and the combined Axl and MET inhibition reduced cellular motility in a DMPM cell line obtained from a “progressed” DMPM. Conclusion—The results indicate that the coordinated activity of multiple cross-talks between RTKs is directly involved in the biology of DMPM, suggesting the combined inhibition of PIK3 and mTOR as an effective strategy that may be easily implemented in clinical practice, and indicating that the combined inhibition of EGFR/HER2 and HER3 and of Axl and MET deserves further investigation.

1959P - HER2 inhibition in aggressive squamous cell carcinomas driven by a common MET sema domain polymorphism

BackgroundOncogenic MET signaling is often deployed by tumors for malignant transformation through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. We present biochemical and clinical findings on a polymorphism of MET, which is common in the East Asian population. MethodsGermline DNA (gDNA) of lung, nasopharyngeal, liver, breast, gastric and colon cancer cohorts among East Asians (n=1,076) were genotyped for MET (p.N375S) polymorphism using digital droplet PCR (ddPCR). Relapse free survival (RFS) with curative intent was compared using log-rank test. Screening for protein-protein interaction was performed with SILAC. Clinical study is now enrolling MET-N375S+ squamous cell (SCC) head and neck patients to be given 30mg afatinib daily. Treatment outcome was determined with F-FDG PET/CT scan. Baseline (BL) and post-treatment biopsies were analyzed with immunohistochemistry for response biomarkers and in situ hybridization-proximity ligation assay (ISH-PLA) for receptor interaction. ResultsFraction of the MET-N375S+ cases is comparable across cancer patients and cancer-free controls (9-18%). METN375S was associated with significantly shorter relapse-free survival (RFS) in SCC of the head and neck (HNSCC; P=0.005) and lung (LUSC; P=0.029). SILAC revealed interaction of MET-N375S with HER2 (P<0.05). Two patients with refractory HNSCC who completed 1 cycle of afatinib, a pan-HER inhibitor, showed radiological tumour shrinkage and reduction in metabolic activity, with partial response by RECIST 1.1 measurement observed in one patient. Evaluable BL biopsy showed strong MET-HER2 interaction (ISH-PLA) with strong staining of phosphorylated MET and HER2 (IHC), which were significantly reduced post-treatment, indicative of successive intervention of MET-N375S/HER2 signaling. ConclusionsWe established MET-N375S as a biologically-distinct molecular subset of SCC that drives malignancy through HER2 signaling, and could serve as a predictive biomarker to select refractory patients with dismal prognosis for afatinib treatment. Clinical trial identificationNCT03938012. Actual study start date: October 3, 2017; Estimated primary completion date: October 2021; Estimated study completion date: October 2022. Legal entity responsible for the studyNational University Cancer Institute, Singapore. FundingNational Medical Research Council, Singapore. DisclosureAll authors have declared no conflicts of interest.

Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning

Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.

Impaired ligand-dependent MET activation caused by an extracellular SEMA domain missense mutation in lung cancer.

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion‐metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET‐knockout cells were prepared and reconstituted with WT‐MET or V370D‐MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT‐MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D‐MET cells. The V370D mutation abrogated HGF‐dependent drug resistance of lung cancer cells to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI). Compared with WT‐MET cells, V370D‐MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth‐stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D‐MET and HGF was reduced compared with that for WT‐MET. Further analysis of the association between V370D‐MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D‐MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss‐of‐function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.

Functional divergence of Plexin B structural motifs in distinct steps of Drosophila olfactory circuit assembly.

Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the Drosophila olfactory circuit (Li et al., 2018b). Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.

A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma

Human semaphorin 4D (SEMA4D), a type I integral membrane glycoprotein, regulates key cellular functions (e.g. cell-cell communication, platelet activation). Its 120 kDa extracellular region can be shed from the membrane to release soluble SEMA4D (sSEMA4D). Studies on circulating sSEMA4D levels are mostly performed with poorly characterized assays and use serum and plasma as matrix.We developed and validated a sandwich ELISA utilizing two monoclonal antibodies with resolved epitopes and determined affinities. Human serum and plasma samples were analyzed, and the influence of protease activity on sSEMA4D concentration was tested by collecting samples in the presence of the protease inhibitor TAPI-1.Both antibodies recognize conformational epitopes in the sema domain. Validation for plasma (EDTA, citrate, heparin) showed valid specificity, precision, accuracy, dilution linearity, and robustness. The assay shows a calibration range from 62.5 to 2000 pmol/L with a quantification limit of 31 pmol/L. sSEMA4D was significantly higher in serum than in plasma, whereas serum and plasma levels from samples collected in the presence of TAPI-1 showed no statistical difference.This ELISA provides a reliable tool for the quantification of sSEMA4D in human plasma. Serum is not recommended as matrix due to the accumulation of shed SEMA4D during blood coagulation altering serum sSEMA4D levels.

Origin and evolution of plexins, semaphorins, and Met receptor tyrosine kinases

The transition from unicellular to multicellular organisms poses the question as to when genes that regulate cell-cell interactions emerged during evolution. The receptor and ligand pairing of plexins and semaphorins regulates cellular interactions in a wide range of developmental and physiological contexts. We surveyed here genomes of unicellular eukaryotes and of non-bilaterian and bilaterian Metazoa and performed phylogenetic analyses to gain insight into the evolution of plexin and semaphorin families. Remarkably, we detected plexins and semaphorins in unicellular choanoflagellates, indicating their evolutionary origin in a common ancestor of Choanoflagellida and Metazoa. The plexin domain structure is conserved throughout all clades; in contrast, semaphorins are structurally diverse. Choanoflagellate semaphorins are transmembrane proteins with multiple fibronectin type III domains following the N-terminal Sema domain (termed Sema-FN). Other previously not yet described semaphorin classes include semaphorins of Ctenophora with tandem immunoglobulin domains (Sema-IG) and secreted semaphorins of Echinoderamata (Sema-SP, Sema-SI). Our study also identified Met receptor tyrosine kinases (RTKs), which carry a truncated plexin extracellular domain, in several bilaterian clades, indicating evolutionary origin in a common ancestor of Bilateria. In addition, a novel type of Met-like RTK with a complete plexin extracellular domain was detected in Lophotrochozoa and Echinodermata (termed Met-LP RTK). Our findings are consistent with an ancient function of plexins and semaphorins in regulating cytoskeletal dynamics and cell adhesion that predates their role as axon guidance molecules.

Further Evidence for the Implication of the MET Gene in Non-Syndromic Autosomal Recessive Deafness

Mutations in the mesenchymal epithelial transition factor (MET) gene are frequently associated with multiple human cancers but can also lead to human non-syndromic autosomal recessive deafness (DFNB97). In the present study, we identified a novel homozygous missense mutation in the METgene causing a non-syndromic hearing impairment DFNB97 form. Whole-exome sequencing was performed to determine the genetic causes of hearing loss in a Moroccan consanguineous family with an affected daughter. The structural analysis of native and mutant in the SEMA domain of the MET receptor was investigated using a molecular dynamics simulation (MDS) approach. We identified a novel pathogenic homozygous c.948A>G (p.Ile316Met) mutation in the MET gene in one deaf Moroccan young girl carrying a total bilateral non-syndromic hearing impairment. The results of the MDS approach show that an Ile316Met mutation in the SEMA domain leads to protein flexibility loss. This may produce a major impact on the structural conformation of the MET receptor, which also affects the function and binding site of the receptor. This is the first time that a mutation in the MET gene is described in a Moroccan family. Moreover, this study reports the second family in the world associating deafness and mutation in the MET gene.