RESUMO
Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.
Assuntos
Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Neuropilina-2/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Substituição de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células COS , Chlorocebus aethiops , Células Dendríticas/citologia , Humanos , Camundongos , Camundongos Knockout , Neuropilina-2/genética , Estrutura Terciária de Proteína , Ácidos Siálicos/genética , Sialiltransferases/genética , Especificidade por SubstratoRESUMO
Polysialic acid is a unique carbohydrate polymer specifically attached to a limited number of glycoproteins. Among them is synaptic cell adhesion molecule 1 (SynCAM 1), a member of the immunoglobulin (Ig) superfamily composed of three extracellular Ig-like domains. Polysialylation of SynCAM 1 is cell type-specific and was exclusively found in NG2 cells, a class of multifunctional progenitor cells that form specialized synapses with neurons. Here, we studied the molecular requirements for SynCAM 1 polysialylation. Analysis of mice lacking one of the two polysialyltransferases, ST8SiaII or ST8SiaIV, revealed that polysialylation of SynCAM 1 is exclusively mediated by ST8SiaII throughout postnatal brain development. Alternative splicing of the three variable exons 8a, 8b, and 8c can theoretically give rise to eight transmembrane isoforms of SynCAM 1. We detected seven transcript variants in the developing mouse brain, including three variants containing exon 8c, which was so far regarded as a cryptic exon in mice. Polysialylation of SynCAM 1 was restricted to four isoforms in perinatal brain. However, cell culture experiments demonstrated that all transmembrane isoforms of SynCAM 1 can be polysialylated by ST8SiaII. Moreover, analysis of domain deletion constructs revealed that Ig1, which harbors the polysialylation site, is not sufficient as an acceptor for ST8SiaII. The minimal polypeptide required for polysialylation contained Ig1 and Ig2, suggesting an important role for Ig2 as a docking site for ST8SiaII.
Assuntos
Encéfalo/patologia , Moléculas de Adesão Celular/metabolismo , Imunoglobulinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Sialiltransferases/metabolismo , Sinapses/metabolismo , Processamento Alternativo/fisiologia , Animais , Sequência de Bases , Encéfalo/citologia , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Linhagem Celular , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Neurônios/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sialiltransferases/genética , Sinapses/genéticaRESUMO
The glycan polysialic acid is well-known as a unique posttranslational modification of the neural cell adhesion molecule NCAM. Despite remarkable acceptor specificity, however, a few other proteins can be targets of polysialylation. Here, we recapitulate the biosynthesis of polysialic acid by the two polysialyltransferases ST8SIA2 and ST8SIA4 and highlight the increasing evidence that variation in the human ST8SIA2 gene is linked to schizophrenia and possibly other neuropsychiatric disorders. Moreover, we summarize the knowledge on the role of NCAM polysialylation in brain development gained by the analysis of NCAM- and polysialyltransferase-deficient mouse models. The last part of this review is focused on recent advances in identifying SynCAM 1 and neuropilin-2 as novel acceptors of polysialic acid in NG2 cells of the perinatal brain and in dendritic cells of the immune system, respectively.
Assuntos
Moléculas de Adesão Celular/metabolismo , Imunoglobulinas/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neuropilina-2/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Animais , Encéfalo/enzimologia , Molécula 1 de Adesão Celular , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Células Dendríticas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Sialiltransferases/deficiência , Sialiltransferases/genética , Especificidade por SubstratoRESUMO
Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam(-/-) mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam(-/-) brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam(-/-) background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam(-/-) brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.