RESUMO
CD22 (also known as Siglec-2) is an inhibitory receptor expressed in B cells. CD22 specifically recognizes α2,6 sialic acid, and interacts with α2,6 sialylated membrane proteins expressed on the same cell (cis-ligands) and those derived from outside of the cell (trans-ligands). Previously, CD22 cis-ligands were shown to regulate the activity of CD22, thereby regulating both BCR ligation-induced signaling and low level "tonic" signaling in the absence of BCR ligation that regulates survival and differentiation of B cells. Mouse CD22 prefers Neu5Gc to Neu5Ac thereby binding to α2,6-linked Neu5Gc with high affinity. Although human CD22 binds to a distinct α2,6 sialylated glycan with high affinity, expression of high affinity ligands is regulated in a conserved and stringent manner. However, how high vs. low affinity CD22 ligands regulate B cells is poorly understood. Here we demonstrate that interaction of CD22 with the endogenous ligands enhances BCR ligation-induced signaling but reduces tonic signaling in Cmah-/- mouse B cells deficient in Neu5Gc as well as wild-type B cells. Moreover, Cmah-/- B cells do not show alterations in the phenotypes correlated to tonic signaling. These results indicate that low affinity interaction of the CD22 cis-ligands with CD22 is sufficient for the regulation of B cell signaling, and suggest that expression of high affinity CD22 ligands might be involved in the regulation of B cells by competing the binding of CD22 with exogenous trans-ligands of CD22.
RESUMO
Spermatid production is a complex regulatory process in which coordination between hormonal control and apoptosis plays a pivotal role in maintaining a balanced number of sperm cells. Apoptosis in spermatogenesis is controlled by pro-apoptotic and anti-apoptotic molecules. Hormones involved in the apoptotic process during spermatogenesis include gonadotrophins, sex hormones, and glucocorticoid (GC). GC acts broadly as an apoptosis inducer by binding to its receptor (glucocorticoid receptor: GR) during organ development processes, such as spermatogenesis. However, the downstream pathway induced in GC-GR signaling and the apoptotic process during spermatogenesis remains poorly understood. We reported previously that GC induces full-length glucocorticoid-induced transcript 1 (GLCCI1-long), which functions as an anti-apoptotic mediator in thymic T cell development. Here, we demonstrate that mature murine testis expresses a novel isoform of GLCCI1 protein (GLCCI1-short) in addition to GLCCI1-long. We demonstrate that GLCCI1-long is expressed in spermatocytes along with GR. In contrast, GLCCI1-short is primarily expressed in spermatids where GR is absent; instead, the estrogen receptor is expressed. GLCCI1-short also binds to LC8, which is a known mediator of the anti-apoptotic effect of GLCCI1-long. A luciferase reporter assay revealed that ß-estradiol treatment synergistically increased Glcci1-short promotor-driven luciferase activity in Erα-overexpressing cells. Together with the evidence that the conversion of testosterone to estrogen is preceded by aromatase expression in spermatids, we hypothesize that estrogen induces GLCCI1-short, which, in turn, may function as a novel anti-apoptotic mediator in mature murine testis.
Assuntos
Glucocorticoides , Sêmen , Masculino , Camundongos , Animais , Espermatogênese , Espermátides , EstrogêniosRESUMO
CD22, one of the sialic acid-binding immunoglobulin-like lectins (Siglecs), regulates B lymphocyte signaling via its interaction with glycan ligands bearing the sequence Neu5Ac/Gcα(2â6)Gal. We have developed the synthetic sialoside GSC-718 as a ligand mimic for CD22 and identified it as a potent CD22 inhibitor. Although the synthesis of CD22-binding sialosides including GSC-718 has been reported by our group, the synthetic route was unfortunately not suitable for large-scale synthesis. In this study, we developed an improved scalable synthetic procedure for sialosides which utilized 1,5-lactam formation as a key step. The improved procedure yielded sialosides incorporating a series of aglycones at the C2 position. Several derivatives with substituted benzyl residues as aglycones were found to bind to mouse CD22 with affinity comparable to that of GSC-718. The new procedure developed in this study affords sialosides in sufficient quantities for cell-based assays, and will facilitate the search for promising CD22 inhibitors that have therapeutic potential.
Assuntos
Linfócitos B , Transdução de Sinais , Animais , Camundongos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos B/metabolismo , LigantesRESUMO
CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45-/- as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1-mediated dephosphorylation of CD22 in CD22 clusters.
Assuntos
Linfócitos B/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Linhagem Celular , Humanos , Antígenos Comuns de Leucócito/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.
Assuntos
Nefrose Lipoide , Síndrome Nefrótica , Podócitos , Camundongos , Humanos , Animais , Podócitos/metabolismo , Síndrome Nefrótica/metabolismo , Nefrose Lipoide/patologia , Camundongos Endogâmicos C3H , Proteinúria/metabolismo , Modelos Animais de Doenças , Imunização , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.
Assuntos
Glomerulosclerose Segmentar e Focal , Proteínas do Tecido Nervoso , Nestina , Podócitos , Proteína Supressora de Tumor p53 , Proteases Específicas de Ubiquitina , Animais , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Hipertrofia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina/genética , Nestina/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Podócitos/fisiologia , Proteína Quinase C/antagonistas & inibidores , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Regulação para CimaRESUMO
Guillain-Barré syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.
Assuntos
Gangliosídeos/imunologia , Predisposição Genética para Doença , Síndrome de Guillain-Barré/imunologia , Lectinas/imunologia , Mutação de Sentido Incorreto/imunologia , Polimorfismo de Nucleotídeo Único/imunologia , Receptores de Superfície Celular/imunologia , Alelos , Sequência de Aminoácidos , Autoanticorpos/imunologia , Sítios de Ligação/genética , Feminino , Gangliosídeos/metabolismo , Frequência do Gene , Genótipo , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Síndrome de Miller Fisher/genética , Síndrome de Miller Fisher/imunologia , Síndrome de Miller Fisher/metabolismo , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.
Assuntos
Apoptose/fisiologia , Glucocorticoides/fisiologia , Receptores de Glucocorticoides/fisiologia , Linfócitos T/citologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Linhagem Celular , Dineínas do Citoplasma/metabolismo , Dimerização , Regulação para Baixo , Técnicas de Silenciamento de Genes , Glucocorticoides/farmacologia , Humanos , Hipertrofia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/metabolismo , Fosforilação , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Receptores de Glucocorticoides/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Timo/patologia , Quinases Ativadas por p21/metabolismoRESUMO
The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1-4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits' ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.
Assuntos
Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , Membrana Celular/metabolismo , Expressão Gênica , Glicosilação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação , Estrutura Quaternária de Proteína , Receptores de Glutamato/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.
Assuntos
Evolução Biológica , Encéfalo/metabolismo , Ácidos Neuramínicos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/metabolismo , Locomoção , Espectrometria de Massas , Transtornos da Memória/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI-/- B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI-/- B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah-/- B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins.
Assuntos
Linfócitos B/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Mapeamento de Interação de Proteínas/métodos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Células Cultivadas , Lectinas/metabolismo , Camundongos , Ligação Proteica , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: Cells have evolved the mechanisms to survive nutritional shortages in the environment. In Saccharomyces cerevisiae, α-mannosidase Ams1 is known to play a role in catabolism of N-linked free oligosaccharides in the cytosol. Although, this enzyme is also known to be transported selectively from the cytosol to the vacuoles by autophagy, the physiological significance of this transport has not been clarified. METHODS: To elucidate the regulatory mechanism of the activity of Ams1, we assessed the enzymatic activity of the cell free extract of the wild-type and various gene disruptants under different nutritional conditions. In addition, the regulation of Ams1 at both transcription and post-translation was examined. RESULTS: The activity of Ams1 was significantly increased upon the depletion of glucose in the medium. Interestingly, the activity of the enzyme was also stimulated by nitrogen starvation. Our data showed that the activity of Ams1 is regulated by the stress responsive transcriptional factors Msn2/4 through the protein kinase A and the target of rapamycin complex 1 pathways. In addition, Ams1 is post-translationally activated by Pep4-dependent processing in the vacuoles. CONCLUSION: Yeast cells monitor extracellular nutrients to regulate mannoside catabolism via the cellular signaling pathway. GENERAL SIGNIFICANCE: This study revealed that intracellular Ams1 activity is exquisitely upregulated in response to nutrient starvation by induced expression as well as by Pep4-dependent enhanced activity in the vacuoles. The signaling molecules responsible for regulation of Ams1 were also clarified.
Assuntos
Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/fisiologia , alfa-Manosidase/metabolismo , Ácido Aspártico Endopeptidases/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Glucose/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/fisiologia , Nitrogênio/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Regulação para CimaRESUMO
Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.
Assuntos
Taxa de Filtração Glomerular , Glomérulos Renais/enzimologia , Ubiquitina Tiolesterase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genótipo , Células HEK293 , Humanos , Glomérulos Renais/embriologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Camundongos , Nefrose Lipoide/enzimologia , Nefrose Lipoide/genética , Nefrose Lipoide/fisiopatologia , Nestina/metabolismo , Permeabilidade , Fenótipo , Podócitos/enzimologia , Interferência de RNA , Ratos , Transfecção , Ubiquitina Tiolesterase/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The human natural killer-1 (HNK-1) carbohydrate, a unique trisaccharide possessing sulfated glucuronic acid in a non-reducing terminus (HSO3-3GlcAß1-3Galß1-4GlcNAc-), is highly expressed in the nervous system and its spatiotemporal expression is strictly regulated. Mice deficient in the gene encoding a key enzyme, GlcAT-P, of the HNK-1 biosynthetic pathway exhibit almost complete disappearance of the HNK-1 epitope in the brain, significant reduction of long-term potentiation, and aberration of spatial learning and memory formation. In addition to its physiological roles in higher brain function, the HNK-1 carbohydrate has attracted considerable attention as an autoantigen associated with peripheral demyelinative neuropathy, which relates to IgM paraproteinemia, because of high immunogenicity. It has been suggested, however, that serum autoantibodies in IgM anti-myelin-associated glycoprotein (MAG) antibody-associated neuropathy patients show heterogeneous reactivity to the HNK-1 epitope. SCOPE OF REVIEW: We have found that structurally distinct HNK-1 epitopes are expressed in specific proteins in the nervous system. Here, we overview the current knowledge of the involvement of these HNK-1 epitopes in the regulation of neural plasticity and discuss the impact of different HNK-1 antigens of anti-MAG neuropathy patients. MAJOR CONCLUSIONS: We identified the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor subunit GluA2 and aggrecan as HNK-1 carrier proteins. The HNK-1 epitope on GluA2 and aggrecan regulates neural plasticity in different ways. Furthermore, we found the clinical relationship between reactivity of autoantibodies to the different HNK-1 epitopes and progression of anti-MAG neuropathy. GENERAL SIGNIFICANCE: The HNK-1 epitope is indispensable for the acquisition of normal neuronal function and can be a good target for the establishment of diagnostic criteria for anti-MAG neuropathy.
Assuntos
Antígenos CD57/química , Epitopos/química , Glicoproteína Associada a Mielina/imunologia , Plasticidade Neuronal , Paraproteinemias/imunologia , Doenças do Sistema Nervoso Periférico/imunologia , Agrecanas/metabolismo , Animais , Autoanticorpos/biossíntese , Antígenos CD57/genética , Antígenos CD57/imunologia , Epitopos/genética , Epitopos/imunologia , Glucuronosiltransferase/deficiência , Glucuronosiltransferase/genética , Humanos , Imunoglobulina M/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Knockout , Glicoproteína Associada a Mielina/genética , Paraproteinemias/genética , Paraproteinemias/patologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Receptores de AMPA/genética , Receptores de AMPA/imunologiaRESUMO
Sialic acids (Sias) are often conjugated to the termini of cellular glycans and are key mediators of cellular recognition. Sias are nine-carbon acidic sugars, and, in vertebrates, the major species are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), differing in structure at the C5 position. Previously, we described a positive feedback loop involving regulation of Neu5Gc expression in mouse B cells. In this context, Neu5Gc negatively regulated B-cell proliferation, and Neu5Gc expression was suppressed upon activation. Similarly, resting mouse T cells expressed principally Neu5Gc, and Neu5Ac was induced upon activation. In the present work, we used various probes to examine sialoglycan expression by activated T cells in terms of the Sia species expressed and the linkages of Sias to glycans. Upon T-cell activation, sialoglycan expression shifted from Neu5Gc to Neu5Ac, and the linkage shifted from α2,6 to α2,3. These changes altered the expression levels of sialic acid-binding immunoglobulin-like lectin (siglec) ligands. Expression of sialoadhesin and Siglec-F ligands increased, and that of CD22 ligands decreased. Neu5Gc exerted a negative effect on T-cell activation, both in terms of the proliferative response and in the context of activation marker expression. Suppression of Neu5Gc expression in mouse T and B cells prevented the development of nonspecific CD22-mediated T cell-B cell interactions. Our results suggest that an activation-dependent shift from Neu5Gc to Neu5Ac and replacement of α2,6 by α2,3 linkages may regulate immune cell interactions at several levels.
Assuntos
Linfócitos B/metabolismo , Comunicação Celular/fisiologia , Ativação Linfocitária/fisiologia , Ácidos Siálicos/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/biossíntese , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Ácidos Siálicos/genética , Ácidos Siálicos/imunologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Glycosylation is a major protein modification. Although proteins are glycosylated/further modulated by several glycosyltransferases during trafficking from the endoplasmic reticulum to the Golgi apparatus, a certain glycan epitope has only been detected on a limited number of proteins. Of these glycan epitopes, Lewis X is highly expressed in the early stage of a developing brain and plays important roles in cell-cell interaction. The Lewis X epitope is comprised of a trisaccharide (Galß1-4 (Fucα1-3) GlcNAc), and a key enzyme for the expression of this epitope is α1,3-fucosyltransferase 9. However, the scaffolding glycan structure responsible for the formation of the Lewis X epitope as well as its major carrier protein has not been fully characterized in the nervous system. Here we showed that the Lewis X epitope was mainly expressed on phosphacan/receptor protein tyrosine phosphatase ß (RPTPß) in the developing mouse brain. Expression of the Lewis X epitope was markedly reduced in ß1,4-galactosyltransferase 2 (ß4GalT2) gene-deficient mice, which indicated that ß4GalT2 is a major galactosyltransferase required for the Lewis X epitope. We also showed that the Lewis X epitope almost disappeared due to the knockout of protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for the synthesis of O-mannosylated glycans, which indicated that the O-mannosylated glycan is responsible for presenting the Lewis X epitope. Since O-mannosylated glycans on phosphacan/RPTPß could also present human natural killer-1, another glycan epitope specifically expressed in the nervous system, our results revealed the importance of O-mannosylated glycan chains in the presentation of functional glycan epitopes in the brain.
Assuntos
Encéfalo/enzimologia , Antígenos CD15/metabolismo , Manose/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Animais , Vias Biossintéticas , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Células COS , Configuração de Carboidratos , Chlorocebus aethiops , Glicosilação , Mananas/metabolismo , Camundongos Knockout , Processamento de Proteína Pós-TraducionalRESUMO
The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAß1-3Galß1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.
Assuntos
Encéfalo/metabolismo , Antígenos CD57/metabolismo , Manose/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Antígenos CD57/química , Células COS , Configuração de Carboidratos , Chlorocebus aethiops , Glucuronosiltransferase/metabolismo , Glicosilação , Células HEK293 , Humanos , Manose/química , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/químicaRESUMO
Dystroglycan (DG) is a cell surface glycoprotein that connects extracellular matrix molecules to the intracellular cytoskeleton, functioning as mechanical and signaling axes in various physiological events. Since the ligand-binding activity of DG strictly depends on O-mannosyl glycans attached to its extracellular α-DG subunit, aberrant glycosylation causes dystroglycanopathy, a subclass of congenital muscular dystrophy. Accumulating evidence shows that like-acetylglucosaminyltransferase (LARGE), a glycosyltransferase involved in the biosynthesis of a phosphodiester-linked modification on O-mannose, is essential for α-DG to gain the ligand-binding activity. We previously reported that human natural killer-1 sulfotransferase (HNK-1ST), which was originally reported as one of the enzymes responsible for HNK-1 glycoepitope, had an ability to suppress the glycosylation and the function of α-DG. In this study, we investigated how HNK-1ST regulates the glycosylation of α-DG using deletion and mutation analyses. We generated an α-DG mutant which has only one threonine residue capable of being modified by LARGE. Focusing on the single post-phosphoryl modification site, we found that HNK-1ST showed an almost complete inhibition of the LARGE-dependent modification and transferred a sulfate group to the phosphodiester-linked moiety on O-mannose. Furthermore, using an in vitro enzymatic assay system, we demonstrated that the sulfated α-DG by HNK-1ST is no longer glycosylated by LARGE. These results illustrate one possible glycosylation pathway where α-DG function is regulated by opposing actions of HNK-1ST and LARGE.
Assuntos
Distroglicanas/metabolismo , Laminina/metabolismo , Polissacarídeos/metabolismo , Sulfotransferases/metabolismo , Sítios de Ligação , HumanosRESUMO
CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB).
Assuntos
Linfócitos B/imunologia , Imunoglobulina M/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Linfócitos B/metabolismo , Western Blotting , Linhagem Celular Tumoral , Retroalimentação , Citometria de Fluxo , Imunoglobulina M/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismoRESUMO
Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.