RESUMO
Human natural killer-1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.
Assuntos
Antígenos CD57/biossíntese , Glucuronosiltransferase/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Epitopos/metabolismo , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Polissacarídeos/metabolismoRESUMO
In the mammalian nervous system, protein N-glycosylation plays an important role in neuronal physiology. In this study, we performed a comprehensive N-glycosylation analysis of mouse GluA1, one of the major subunits of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate type glutamate receptor, which possesses six potential N-glycosylation sites in the N-terminal domain. By mass spectrometry-based analysis, we identified the N-glycoforms and semiquantitatively determined the site-specific N-glycosylation occupancy of GluA1. In addition, only the N401-glycosylation site demonstrated incomplete N-glycosylation occupancy. Therefore, we generated a peptide antibody that specifically detects the N401-glycan-free form to precisely quantify N401-glycosylation occupancy. Using this antibody, we clarified that N401 occupancy varies between cell types and increases in an age-dependent manner in mouse forebrains. To address the regulatory mechanism of N401-glycosylation, binding proteins of GluA1 around the N401 site were screened. HSP70 family proteins, including Bip, were identified as candidates. Bip has been known as a molecular chaperone that plays a key role in protein folding in the ER (endoplasmic reticulum). To examine the involvement of Bip in N401-glycosylation, the effect of Bip over-expression on N401 occupancy was evaluated in HEK293T cells, and the results demonstrated Bip increases the N401 glycan-free form by mediating selective prolongation of its protein half-life. Taken together, we propose that the N401-glycosite of GluA1 receives a unique control of modification, and we also propose a novel N-glycosylation occupancy regulatory mechanism by Bip that might be associated with α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors function in the brain.
Assuntos
Anticorpos/genética , Anticorpos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Animais , Sítios de Ligação/fisiologia , Feminino , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , GravidezRESUMO
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
The AMPA-type glutamate receptor (AMPA-R) plays a primary role in principal excitatory synaptic transmission and many neuronal functions including synaptic plasticity that underlie learning and memory. N-glycosylation is one of the major post-translational modifications of membrane proteins, but its specific roles in neurons remain largely unknown. AMPA-R subunits are N-glycosylated at their extracellular domains during their biosynthesis in the lumen of the endoplasmic reticulum and Golgi system. Six N-glycosylation sites are presumed to exist in the extracellular domain of GluA1, which is a member of the AMPA-R subunits. We observed that the intracellular trafficking and cell surface expression were strongly suppressed in the GluA1 mutants lacking N-glycans at N63/N363 in HEK293T cells. Multimer analysis using Blue Native-PAGE displayed the impaired tetramer formation in the glycosylation mutants (N63S and N363S), indicating that the mis-transport was caused by impaired tetramer formation. N63S and N363S mutants were primarily degraded via the lysosomal pathway. Flag-tagged N363S GluA1, but not N63S GluA1, expressed in primary cortical neuron cultures prepared from GluA1 knockout mice was observed to localize at the cell surface. Co-expression of GluA2 partially rescued tetramer formation and the cell surface expression of N363S GluA1 but not N63S GluA1, in HEK293T cells. Electrophysiological analysis also demonstrated functional heteromers of N363S GluA1 with GluA2. These data suggest that site-specific N-glycans on GluA1 subunit regulates tetramer formation, intracellular trafficking, and cell surface expression of AMPA-R. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
Assuntos
Glicosilação , Canais Iônicos/fisiologia , Proteínas de Membrana/biossíntese , Receptores de AMPA/fisiologia , Animais , Fenômenos Eletrofisiológicos/genética , Células HEK293 , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Neurônios/metabolismo , Cultura Primária de Células , Receptores de AMPA/biossíntese , Receptores de AMPA/genética , Receptores de AMPA/metabolismoRESUMO
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
Core fucosylation is catalyzed by α1,6-fucosyltransferase (FUT8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8-knock-out (Fut8(-/-)) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early form long term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8(-/-) mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by postsynaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in postsynapses, were greatly increased in Fut8(-/-) mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8(-/-) mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which increase sensitivity for postsynaptic depolarization and persistently activate N-methyl-d-aspartate receptors as well as Ca(2+) influx and CaMKII and then impair LTP.
Assuntos
Fucosiltransferases/deficiência , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Animais , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Feminino , Fucose/metabolismo , Fucosiltransferases/genética , Glicosilação , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Camundongos Knockout , Multimerização Proteica , Transdução de Sinais , Transmissão SinápticaRESUMO
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
Immunogenicity is a major caveat of protein therapeutics. In particular, the long-term administration of protein therapeutic agents leads to the generation of antidrug antibodies (ADAs), which reduce drug efficacy while eliciting adverse events. One promising solution to this issue is the use of mirror-image proteins consisting of d-amino acids, which are resistant to proteolytic degradation in immune cells. We have recently reported the chemical synthesis of the enantiomeric form of the variable domain of the antibody heavy chain (d-VHH). However, identifying mirror-image antibodies capable of binding to natural ligands remains challenging. In this study, we developed a novel screening platform to identify a d-VHH specific for vascular endothelial growth factor A (VEGF-A). We performed mirror-image screening of two newly constructed synthetic VHH libraries displayed on T7 phage and identified VHH sequences that effectively bound to the mirror-image VEGF-A target (d-VEGF-A). We subsequently synthesized a d-VHH candidate that preferentially bound the native VEGF-A (l-VEGF-A) with submicromolar affinity. Furthermore, immunization studies in mice demonstrated that this d-VHH elicited no ADAs, unlike its corresponding l-VHH. Our findings highlight the utility of this novel d-VHH screening platform in the development of protein therapeutics exhibiting both reduced immunogenicity and improved efficacy.
Assuntos
Fator A de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Camundongos , Humanos , Engenharia de Proteínas/métodos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Biblioteca de PeptídeosRESUMO
Retinoic acid (RA) is a well established anti-tumor agent inducing differentiation in various cancer cells. Recently, a robust up-regulation of human natural killer-1 sulfotransferase (HNK-1ST) was found in several subsets of melanoma cells during RA-mediated differentiation. However, the molecular mechanism underlying the tumor suppression mediated by HNK-1ST remains unclear. Here, we show that HNK-1ST changed the glycosylation state and reduced the ligand binding activity of α-dystroglycan (α-DG) in RA-treated S91 melanoma cells, which contributed to an attenuation of cell migration. Knockdown of HNK-1ST restored the glycosylation of α-DG and the migration of RA-treated S91 cells, indicating that HNK-1ST functions through glycans on α-DG. Using CHO-K1 cells, we provide direct evidence that HNK-1ST but not other homologous sulfotransferases (C4ST1 and GalNAc4ST1) suppresses the glycosylation of α-DG. The activity-abolished mutant of HNK-1ST did not show the α-DG-modulating function, indicating that the sulfotransferase activity of HNK-1ST is essential. Finally, the HNK-1ST-dependent incorporation of [(35)S]sulfate groups was detected on α-DG. These findings suggest a novel role for HNK-1ST as a tumor suppressor controlling the functional glycans on α-DG and the importance of sulfate transfer in the glycosylation of α-DG.
Assuntos
Distroglicanas/metabolismo , Laminina/metabolismo , Polissacarídeos/metabolismo , Sulfotransferases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células CHO , Movimento Celular/genética , Cricetinae , Cricetulus , Distroglicanas/genética , Glicosilação , Humanos , Laminina/genética , Mutação , Polissacarídeos/genética , Sulfotransferases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
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
Human natural killer-1 (HNK-1) carbohydrate, comprising a unique trisaccharide HSO(3)-3GlcAß1-3Galß1-4GlcNAc, shows well-regulated expression and unique functions in the nervous system. Recent studies have revealed sophisticated and complicated expression mechanisms for HNK-1 glycan. Activities of biosynthetic enzymes are controlled through the formation of enzyme-complexes and regulation of subcellular localization. Functional aspects of HNK-1 carbohydrate were examined by overexpression, knockdown, and knockout studies of these enzymes. HNK-1 is involved in several neural functions such as synaptic plasticity, learning and memory, and the underlying molecular mechanisms have been illustrated upon identification of the target carrier glycoproteins of HNK-1 such as the glutamate receptor subunit GluA2 or tenascin-R. In this review, we describe recent findings about HNK-1 carbohydrate that provide further insights into the mechanism of its expression and function in the nervous system.
Assuntos
Antígenos CD57/fisiologia , Sistema Nervoso/metabolismo , Animais , Antígenos CD57/biossíntese , Antígenos CD57/química , Embrião de Galinha , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/fisiologia , Epitopos/biossíntese , Epitopos/química , Regulação da Expressão Gênica no Desenvolvimento , Glucuronosiltransferase/genética , Glucuronosiltransferase/fisiologia , Humanos , Camundongos , Modelos Biológicos , Sistema Nervoso/crescimento & desenvolvimento , Doenças do Sistema Nervoso/genética , Plasticidade Neuronal , Polissacarídeos/biossíntese , Polissacarídeos/química , Polissacarídeos/fisiologia , RatosRESUMO
T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.
Assuntos
Bacteriófago T7 , Biblioteca de Peptídeos , Sequência de Aminoácidos , Bacteriófago T7/genética , Bacteriófago T7/metabolismo , Peptídeos/química , DNA/metabolismo , Epitopos/química , Clonagem MolecularRESUMO
Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAß1-3Galß1-4GlcNAc-), is biosynthesized by the successive actions of ß-1,4-galactosyltransferase (ß4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking ß4GalT-II, one of seven ß4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although ß4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of ß4GalT-II is not likely due to a general loss of the ß1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that ß4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of ß4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of ß4GalT-II was increased about 2.5-fold compared with that in the absence of ß4GalT-II. Finally, we showed that co-expression of ß4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of ß4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.
Assuntos
Antígenos CD57/biossíntese , Galactosiltransferases/metabolismo , Glucuronosiltransferase/metabolismo , Animais , Linhagem Celular , Galactosiltransferases/deficiência , Regulação da Expressão Gênica , Glucuronosiltransferase/deficiência , Humanos , Cinética , Redes e Vias Metabólicas , Camundongos , Camundongos KnockoutRESUMO
Ischemia/reperfusion (I/R) is an important cause of acute renal failure. Recent studies have shown that the complement system mediated by the mannan-binding protein (MBP), which is a C-type serum lectin recognizing mannose, fucose and N-acetylglucosamine residues, plays a critical role in the pathogenesis of ischemic acute renal failure. MBP causes complement activation through the MBP lectin pathway and a resulting complement component, C3b, is accumulated on the brush borders of kidney proximal tubules in a renal I/R-operated mouse kidney. However, the initial step of the complement activation has not been studied extensively. We previously identified both meprins α and ß, highly glycosylated zinc metalloproteases, localized on kidney proximal tubules as endogenous MBP ligands. In the present study, we demonstrated that serum-type MBP (S-MBP) and C3b were co-localized with meprins on both the cortex and the medulla in the renal I/R-operated mouse kidney. S-MBP was indicated to interact with meprins in vivo in the I/R-operated mouse kidney and was shown to initiate the complement activation through the interaction with meprins in vitro. Taken together, the present study strongly suggested that the binding of S-MBP to meprins triggers the complement activation through the lectin pathway and may cause the acute renal failure due to I/R on kidney transplantation and hemorrhagic shock.
Assuntos
Lectina de Ligação a Manose da Via do Complemento , Rim/metabolismo , Lectina de Ligação a Manose/metabolismo , Metaloendopeptidases/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Complemento C3b/metabolismo , Expressão Gênica , Humanos , Rim/imunologia , Rim/patologia , Masculino , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/genética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Ligação Proteica , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologiaRESUMO
HNK-1 (human natural killer-1) carbohydrate epitope (HSO(3)-3GlcAß1-3Galß1-4GlcNAc-) recognized by a HNK-1 monoclonal antibody is highly expressed in the nervous system and biosynthesized by a glucuronyltransferase (GlcAT-P or GlcAT-S), and sulfotransferase (HNK-1ST). A similar oligosaccharide (HSO(3)-3GlcAß1-3Galß1-3Galß1-4Xyl) also recognized by the HNK-1 antibody had been found in a glycosaminoglycan (GAG)-protein linkage region of α-thrombomodulin (TM) from human urine. However, which sulfotransferase is involved in sulfation of the terminal GlcA in the GAG-protein linkage region remains unclear. In this study, using CHO-K1 cells in which neither GlcAT-P nor GlcAT-S is endogenously expressed, we found that HNK-1ST has the ability to produce HNK-1 immunoreactivity on α-TM. We also demonstrated that HNK-1ST caused the suppression of chondroitin sulfate (CS) synthesis on TM and a reduction of its anti-coagulant activity. Moreover, using an in vitro enzyme assay system, the HNK-1-positive TM was found not to be utilized as a substrate for CS-polymerizing enzymes (chondroitin synthase (ChSy) and chondroitin polymerizing factor (ChPF)). These results suggest that HNK-1ST is involved in 3-O-sulfation of the terminal GlcA of the linkage tetrasaccharide which acts as an inhibitory signal for the initiation of CS biosynthesis on TM.
Assuntos
Sulfatos de Condroitina/biossíntese , Ácido Glucurônico/metabolismo , Oligossacarídeos/metabolismo , Sulfotransferases/metabolismo , Trombomodulina/metabolismo , Animais , Sequência de Carboidratos , Linhagem Celular , Sulfatos de Condroitina/química , Cricetinae , Cricetulus , Humanos , Dados de Sequência Molecular , Oligossacarídeos/química , Sulfatos , Trombomodulina/químicaRESUMO
HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an alpha-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.
Assuntos
Antígenos CD57/fisiologia , Caderinas/metabolismo , Neurônios/química , Receptores de AMPA/química , Animais , Epitopos , Hipocampo/citologia , Camundongos , Estabilidade Proteica , Transporte Proteico , Receptores de AMPA/metabolismo , Receptores de Glutamato/químicaRESUMO
Anti-myelin-associated glycoprotein (MAG) neuropathy is mediated by the binding of IgM M-proteins to the human natural killer-1 epitope of several glycoconjugates, including MAG and phosphacan. We recently reported that IgM M-proteins with a higher ratio of anti-phosphacan titer to anti-MAG titer (P/M ratio) were associated with a progressive clinical course. Herein, we investigated the temporal variability of the P/M ratio. The results showed that P/M ratios in worsened cases were significantly increased relative to stable or improved cases. Thus, temporal variability in the specificity of IgM M-proteins may be related to the disease course of anti-MAG neuropathy.