Identification of a buried ß-strand as a novel disease-related motif in the human polysialyltransferases.

The polysialyltransferases ST8SIA2 and ST8SIA4 and their product, polysialic acid (polySia), are known to be related to cancers and mental disorders. ST8SIA2 and ST8SIA4 have conserved amino acid (AA) sequence motifs essential for the synthesis of the polySia structures on the neural cell adhesion molecule. To search for a new motif in the polysialyltransferases, we adopted the in silico Individual Meta Random Forest program that can predict disease-related AA substitutions. The Individual Meta Random Forest program predicted a new eight-amino-acids sequence motif consisting of highly pathogenic AA residues, thus designated as the pathogenic (P) motif. A series of alanine point mutation experiments in the pathogenic motif (P motif) showed that most P motif mutants lost the polysialylation activity without changing the proper enzyme expression levels or localization in the Golgi. In addition, we evaluated the enzyme stability of the P motif mutants using newly established calculations of mutation energy, demonstrating that the subtle change of the conformational energy regulates the activity. In the AlphaFold2 model, we found that the P motif was a buried ß-strand underneath the known surface motifs unique to ST8SIA2 and ST8SIA4. Taken together, the P motif is a novel buried ß-strand that regulates the full activity of polysialyltransferases from the inside of the molecule.

MDGA1 negatively regulates amyloid precursor protein-mediated synapse inhibition in the hippocampus.

Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.

Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing.

Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll ß-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.

Testosterone interrupts binding of Neurexin and Neuroligin that are expressed in a highly socialized rodent, Octodon degus.

Octodon degus is said to be one of the most human-like rodents because of its improved cognitive function. Focusing on its high sociality, we cloned and characterized some sociality-related genes of degus, in order to establish degus as a highly socialized animal model in molecular biology. We cloned degus Neurexin and Neuroligin as sociality-related genes, which are genetically related to autism spectrum disorder in human. According to our results, amino acid sequences of Neurexin and Neuroligin expressed in degus brain, are highly conserved to that of human sequences. Most notably, degus Neuroligin4 is highly similar to human Neuroligin4X, which is one of the most important autism-related genes, whereas mouse Neuroligin4 is known to be poorly similar to human Neuroligin4X. Furthermore, our work also indicated that testosterone directly binds to degus Neurexin and intercepts intercellular Neurexin-Neuroligin binding. Moreover, it is of high interest that testosterone is another key molecule of the higher incidence of autism in male. These results indicated that degus has the potential for animal model of sociality, and furthermore may promote understanding toward the pathogenic mechanism of autism.

Inhibitor screening assay for neurexin-LRRTM adhesion protein interaction involved in synaptic maintenance and neurological disorders.

Synaptic adhesion molecules, including presynaptic neurexins (NRXNs) and post-synaptic leucine-rich repeat transmembrane (LRRTM) proteins are important for development and maintenance of brain neuronal networks. NRXNs are probably the best characterized synaptic adhesion molecules, and one of the major presynaptic organizer proteins. The LRRTMs were found as ligands for NRXNs. Many of the synaptic adhesion proteins have been linked to neurological cognitive disorders, such as schizophrenia and autism spectrum disorders, making them targets of interest for both biological studies, and towards drug development. Therefore, we decided to develop a screening method to target the adhesion proteins, here the LRRTM-NRXN interaction, to find small molecule probes for further studies in cellular settings. To our knowledge, no potent small molecule compounds against the neuronal synaptic adhesion proteins are available. We utilized the AlphaScreen technology, and developed an assay targeting the NRXN-LRRTM2 interaction. We carried out screening of 2000 compounds and identified hits with moderate IC50-values. We also established an orthogonal in-cell Western blot assay to validate hits. This paves way for future development of specific high affinity compounds by further high throughput screening of larger compound libraries using the methods established here. The method could also be applied to screening other NRXN-ligand interactions.

The Polybasic Region of the Polysialyltransferase ST8Sia-IV Binds Directly to the Neural Cell Adhesion Molecule, NCAM.

Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers. Our laboratory has previously shown that the neural cell adhesion molecule, NCAM, has an acidic surface patch in its first fibronectin type III repeat (FN1) that is critical for the polysialylation of N-glycans on the adjacent immunoglobulin domain (Ig5). We have also identified a polysialyltransferase (polyST) polybasic region (PBR) that may mediate substrate recognition. However, a direct interaction between the NCAM FN1 acidic patch and the polyST PBR has yet to be demonstrated. Here, we have probed this interaction using isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. We observe direct and specific binding between FN1 and the PBR peptide that is dependent upon acidic residues in FN1 and basic residues of the PBR. NMR titration experiments verified the role of the FN1 acidic patch in the recognition of the PBR and suggest a conformational change of the Ig5-FN1 linker region following binding of the PBR to the acidic patch. Finally, mutation of residues identified by NMR titration experiments impacts NCAM polysialylation, supporting their mechanistic role in protein-specific polysialylation.

The N Terminus of the Prion Protein Mediates Functional Interactions with the Neuronal Cell Adhesion Molecule (NCAM) Fibronectin Domain.

The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrPC function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrPC protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrPC co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrPC N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrPC N terminus as a dynamic and functional element responsible for protein-protein interaction.

Increased Polysialylation of the Neural Cell Adhesion Molecule in a Transgenic Mouse Model of Sialuria.

Sialuria is a rare autosomal dominant disorder of mammalian metabolism, caused by defective feedback inhibition of the UDP-N-acetylglucosamine-2-epimerase N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. Sialuria is characterized by overproduction of free sialic acid in the cell cytoplasm. Patients exhibit vastly increased urinary excretion of sialic acid and show differently pronounced developmental delays. The physiopathology of sialuria is not well understood. Here we established a transgenic mouse line that expresses GNE containing the sialuria mutation R263L, in order to investigate the influence of an altered sialic acid concentration on the organism. The transgenic mice that expressed the mutated RNA excreted up to 400 times more N-acetylneuraminic acid than wild-type mice. Additionally, we found higher sialic acid concentration in the brain cytoplasm. Analyzing the (poly)sialylation of neural cell adhesion molecule (NCAM) revealed increased polysialylation in brains of transgenic mice compared to wild-type. However, we found only minor changes in membrane-bound sialylation in various organs but, surprisingly, a significant increase in surface sialylation on leukocytes. Our results suggest that the intracellular sialic acid concentration regulates polysialylation on NCAM in vivo; this could play a role in the manifestation of the developmental delays in sialuria patients.

Chondroitin sulfates and their binding molecules in the central nervous system.

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases.

CASK stabilizes neurexin and links it to liprin-α in a neuronal activity-dependent manner.

CASK, a MAGUK family protein, is an essential protein present in the presynaptic compartment. CASK's cellular role is unknown, but it interacts with multiple proteins important for synapse formation and function, including neurexin, liprin-α, and Mint1. CASK phosphorylates neurexin in a divalent ion-sensitive manner, although the functional relevance of this activity is unclear. Here we find that liprin-α and Mint1 compete for direct binding to CASK, but neurexin1ß eliminates this competition, and all four proteins form a complex. We describe a novel mode of interaction between liprin-α and CASK when CASK is bound to neurexin1ß. We show that CASK phosphorylates neurexin, modulating the interaction of liprin-α with the CASK-neurexin1ß-Mint1 complex. Thus, CASK creates a regulatory and structural link between the presynaptic adhesion molecule neurexin and active zone organizer, liprin-α. In neuronal culture, CASK appears to regulate the stability of neurexin by linking it with this multi-protein presynaptic active zone complex.

Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding.

Synaptic adhesion molecules are key components in development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of chemical synapses. Several families of neuronal adhesion molecules have been identified such as the neuronal cell adhesion molecules, neurexins and neuroligins, and in particular recently several leucine-rich repeat proteins, e.g., Netrin G-ligands, SLITRKs, and LRRTMs. The LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic synapse function and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have been found to be ligands for heparan sulfate proteoglycans, including glypican. We report here the crystal structure of a thermostabilized mouse LRRTM2, with a Tm 30 °C higher than that of the wild-type protein. We localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation, and prior information about the interaction of the ligand with neurexins, which allowed us to propose a tentative model for the LRRTM-neurexin interaction complex. We also determined affinities of the thermostabilized LRRTM2 and wild-type LRRTM1 and LRRTM2 for neurexin-ß1 with and without Ca(2+). Cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapselike contacts. The structural and functional data presented here provide the first structure of an LRRTM protein and allow us to propose a model for the molecular mechanism of LRRTM function in the synaptic adhesion.

Roles of the Laodelphax striatellus Down syndrome cell adhesion molecule in Rice stripe virus infection of its insect vector.

The arthropod Down syndrome cell adhesion molecule (Dscam) mediates pathogen-specific recognition via an extensive protein isoform repertoire produced by alternative splicing. To date, most studies have focused on the subsequent pathogen-specific immune response, and few have investigated the entry into cells of viruses or endosymbionts. In the present study, we cloned and characterized the cDNA of Laodelphax striatellus Dscam (LsDscam) and investigated the function of LsDscam in rice stripe virus (RSV) infection and the influence on the endosymbiont Wolbachia. LsDscam displayed a typical Dscam domain architecture, including 10 immunoglobulin (Ig) domains, six fibronectin type III domains, one transmembrane domain and a cytoplasmic tail. Alternative splicing occurred at the N-termini of the Ig2 and Ig3 domains, the complete Ig7 domain, the transmembrane domain and the C-terminus, comprising 10, 51, 35, two and two variable exons, respectively. Potentially LsDscam could encode at least 71 400 unique isoforms and 17 850 types of extracellular regions. LsDscam was expressed in various L. striatellus tissues. Knockdown of LsDscam mRNA via RNA interference decreased the titres of both RSV and Wolbachia, but did not change the numbers of the extracellular symbiotic bacterium Acinetobacter rhizosphaerae. Specific Dscam isoforms may play roles in enhancing the infection of vector-borne viruses or endosymbionts.

Investigating the potential immune role of fish NCAMs: Molecular cloning and expression analysis in mandarin fish.

The immune role of NCAMs has been revealed in mammals, yet there is no such report in fish. Hence, we analyzed the molecular characterizations and immune-associated expression patterns of NCAMs in mandarin fish. Three NCAM members, named mfNCAM1a, mfNCAM1b and mfNCAM2, were identified. Among the cDNA sequences of mfNCAMs, AU-rich elements in the 3' UTRs of mfNCAM1b and mfNCAM2 as well as VASE sequences in the fourth Ig-like domain-encoding regions of mfNCAM1a and mfNCAM1b were discovered. Moreover, the syntenic analysis suggested that the duplication of NCAM1 is fish-specific. At mRNA and protein levels, the expression analyses revealed that mfNCAMs existed in both systemic and mucosal immune tissues, and located within lymphoid cells. Upon stimulated either by LPS or poly I:C, the expression level of mfNCAM1a was significantly up-regulated in head kidney, spleen, liver, and gut, whereas mfNCAM1b only in head kidney and liver, and mfNCAM2 only in liver. Additionally, the cells coexpressed mfNCAM1 and mfNCCRP-1 might imply the equivalents to mammalian NK cells. Our finding firstly demonstrates the member-specific immune-related tissue expression pattern and immune activity for fish NCAMs. Current data indicate that mfNCAM2 has little immune activity, while the immune activity of mfNCAM1a exists in more tissues than mfNCAM1b, and mfNCAM1a may tend to respond more actively to viral while mfNCAM1b to bacterial stimulants. Additionally, NCAM1b should be a fish-specific member with unique immune function, judging from its different expression pattern, immune activity as well as phylogenetic relationship to mfNCAM1a.

Membrane-tethered monomeric neurexin LNS-domain triggers synapse formation.

Neurexins are presynaptic cell-adhesion molecules that bind to postsynaptic cell-adhesion molecules such as neuroligins and leucine-rich repeat transmembrane proteins (LRRTMs). When neuroligins or LRRTMs are expressed in a nonneuronal cell, cocultured neurons avidly form heterologous synapses onto that cell. Here we show that knockdown of all neurexins in cultured hippocampal mouse neurons did not impair synapse formation between neurons, but blocked heterologous synapse formation induced by neuroligin-1 or LRRTM2. Rescue experiments demonstrated that all neurexins tested restored heterologous synapse formation in neurexin-deficient neurons. Neurexin-deficient neurons exhibited a decrease in the levels of the PDZ-domain protein CASK (a calcium/calmodulin-activated serine/threonine kinase), which binds to neurexins, and mutation of the PDZ-domain binding sequence of neurexin-3ß blocked its transport to the neuronal surface and impaired heterologous synapse formation. However, replacement of the C-terminal neurexin sequence with an unrelated PDZ-domain binding sequence that does not bind to CASK fully restored surface transport and heterologous synapse formation in neurexin-deficient neurons, suggesting that no particular PDZ-domain protein is essential for neurexin surface transport or heterologous synapse formation. Further mutagenesis revealed, moreover, that the entire neurexin cytoplasmic tail was dispensable for heterologous synapse formation in neurexin-deficient neurons, as long as the neurexin protein was transported to the neuronal cell surface. Furthermore, the single LNS-domain (for laminin/neurexin/sex hormone-binding globulin-domain) of neurexin-1ß or neurexin-3ß, when tethered to the presynaptic plasma membrane by a glycosylinositolphosphate anchor, was sufficient for rescuing heterologous synapse formation in neurexin-deficient neurons. Our data suggest that neurexins mediate heterologous synapse formation via an extracellular interaction with presynaptic and postsynaptic ligands without the need for signal transduction by the neurexin cytoplasmic tail.

The polysialyltransferases interact with sequences in two domains of the neural cell adhesion molecule to allow its polysialylation.

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an "extra" N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons.

During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia-NCAMs) modulate cell-cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia-NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb's to polySia, NCAM, ß-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell-cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

A novel method for the simultaneous enrichment, identification, and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.

IGSF9 family proteins.

The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene, whereas vertebrates contain two to four genes. In cnidarians, the gene appears to encode a secreted protein, but transmembrane isoforms of the protein have also evolved, and in many species, alternative splicing facilitates the expression of both transmembrane and secreted isoforms. In most species, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle and mammalian IGSF9 proteins are contradictory.

Regulation of adhesion by flexible ectodomains of IgCAMs.

To perform their diverse biological functions the adhesion activities of the cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) might be regulated by local clustering, proteolytical shedding of their ectodomains or rapid recycling to and from the plasma membrane. Another form of regulation of adhesion might be obtained through flexible ectodomains of IgCAMs which adopt distinct conformations and which in turn modulate their adhesion activity. Here, we discuss variations in the conformation of the extracellular domains of CEACAM1 and CAR that might influence their binding and signaling activities. Furthermore, we concentrate on alternative splicing of single domains and short segments in the extracellular regions of L1 subfamily members that might affect the organization of the N-terminal located Ig-like domains. In particular, we discuss variations of the linker sequence between Ig-like domains 2 and 3 (D2 and D3) that is required for the horseshoe conformation.

Role of neurexin heparan sulfate in the molecular assembly of synapses - expanding the neurexin code?

Synapses are the minimal information processing units of the brain and come in many flavors across distinct circuits. The shape and properties of a synapse depend on its molecular organisation, which is thought to largely depend on interactions between cell adhesion molecules across the synaptic cleft. An established example is that of presynaptic neurexins and their interactions with structurally diverse postsynaptic ligands: the diversity of neurexin isoforms that arise from alternative promoters and alternative splicing specify synaptic properties by dictating ligand preference. The recent finding that a majority of neurexin isoforms exist as proteoglycans with a single heparan sulfate (HS) polysaccharide adds to this complexity. Sequence motifs within the HS polysaccharide may differ between neuronal cell types to contribute specificity to its interactions, thereby expanding the coding capacity of neurexin diversity. However, an expanding number of HS-binding proteins have been found capable to recruit neurexins via the HS chain, challenging the concept of a code provided by neurexin splice isoforms. Here we discuss the possible roles of the neurexin HS in light of what is known from other HS-protein interactions, and propose a model for how the neurexin HS polysaccharide may contribute to synaptic assembly. We also discuss how the neurexin HS may be regulated by co-secreted carbonic anhydrase-related and FAM19A proteins, and highlight some key issues that should be resolved to advance the field.