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1.
J Neurochem ; 163(5): 375-390, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36227633

RESUMEN

Phosphacan, a chondroitin sulfate proteoglycan, is a repulsive cue of cerebellar granule cells. This study aims to explore the molecular mechanism. The glycosylphosphatidylinositol-anchored neural adhesion molecule TAG-1 is a binding partner of phosphacan, suggesting that the repulsive effect of phosphacan is possibly because of its interaction with TAG-1. The repulsive effect was greatly reduced on primary cerebellar granule cells of TAG-1-deficient mice. Surface plasmon resonance analysis confirmed the direct interaction of TAG-1 with chondroitin sulfate C. On postnatal days 1, 4, 7, 11, 15, and 20 and in adulthood, phosphacan was present in the molecular layer and internal granular layer, but not in the external granular layer. In contrast, transient TAG-1 expression was observed exclusively within the premigratory zone of the external granular layer on postnatal days 1, 4, 7, and 11. Boyden chamber cell migration assay demonstrated that phosphacan exerted its repulsive effect on the spontaneous and brain-derived neurotrophic factor (BDNF)-induced migration of cerebellar granule cells. The BDNF-induced migration was inhibited by MK-2206, an Akt inhibitor. The pre-treatment with a raft-disrupting agent, methyl-ß-cyclodextrin, also inhibited the BDNF-induced migration, suggesting that lipid rafts are involved in the migration of cerebellar granule cells. In primary cerebellar granule cells obtained on postnatal day 7 and cultured for 7 days, the ganglioside GD3 and TAG-1 preferentially localized in the cell body, whereas the ganglioside GD1b and NB-3 localized in not only the cell body but also neurites. Pre-treatment with the anti-GD3 antibody R24, but not the anti-GD1b antibody GGR12, inhibited the spontaneous and BDNF-induced migration, and attenuated BDNF-induced Akt activation. These findings suggest that phosphacan is responsible for the repulsion of TAG-1-expressing cerebellar granule cells via GD3 rafts to attenuate BDNF-induced migration signaling.


Asunto(s)
Moléculas de Adhesión Celular Neuronal , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Animales , Ratones , Ratas , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Cerebelo/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo
2.
J Biol Chem ; 294(17): 6659-6669, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-30833330

RESUMEN

IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)-treated IgE cannot access its binding site on bone marrow-derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)-treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies.


Asunto(s)
Anafilaxia/prevención & control , Enzimas/metabolismo , Inmunoglobulina E/inmunología , Vibrio cholerae/enzimología , Anafilaxia/inmunología , Animales , Sitios de Unión , Células de la Médula Ósea/inmunología , Inmunoglobulina E/química , Inmunoglobulina E/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Mastocitos/inmunología , Ratones , Polisacáridos/metabolismo , Inhibidores de Proteasas/farmacología , Conformación Proteica , Tripsina/metabolismo
3.
Int Immunol ; 31(8): 515-530, 2019 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-30859183

RESUMEN

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.


Asunto(s)
Células Asesinas Naturales/citología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Línea Celular , Humanos , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C
4.
Glycoconj J ; 35(5): 477-491, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30173355

RESUMEN

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-ß-GalNAc derivatives and 4-azido-ß-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-ß-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC50 value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.


Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Galactosamina/síntesis química , Galactosamina/farmacología , Sulfotransferasas/antagonistas & inhibidores , Amidas/química , Animales , Inhibidores Enzimáticos/química , Galactosamina/química , Humanos , Sulfotransferasas/metabolismo
5.
Connect Tissue Res ; 59(2): 178-190, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28488903

RESUMEN

Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using VcanΔ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, VcanΔ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and VcanΔ3/Δ3 mice died at the neonatal period (P0). At P0, VcanΔ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in VcanΔ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor ß (TGFß) signaling mediated through the Smad2/3-dependent pathway was down-regulated in VcanΔ3/Δ3 fibroblasts and a reduced TGFß storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFß signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFß signaling.


Asunto(s)
Dermis/crecimiento & desarrollo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Transducción de Señal , Versicanos/metabolismo , Animales , Células Cultivadas , Dermis/citología , Matriz Extracelular/genética , Fibroblastos/citología , Ratones , Mutación , Dominios Proteicos , Versicanos/genética , Versicanos/farmacología
6.
Glycobiology ; 26(6): 592-606, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26791444

RESUMEN

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains.


Asunto(s)
Proteínas Bacterianas/química , Sulfatos de Condroitina/análisis , Disacáridos/análisis , Escherichia coli/enzimología , Oligosacáridos/análisis , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Aminopiridinas/química , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Secuencia de Carbohidratos , Condroitín Liasas/química , Condroitín Liasas/metabolismo , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/síntesis química , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Disacáridos/química , Escherichia coli/genética , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Hialuronoglucosaminidasa/química , Hialuronoglucosaminidasa/metabolismo , Oligosacáridos/biosíntesis , Oligosacáridos/síntesis química , Análisis de Secuencia , Coloración y Etiquetado/métodos , Sulfotransferasas/química , Sulfotransferasas/metabolismo , ortoaminobenzoatos/química
7.
Glycoconj J ; 33(6): 985-994, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27287227

RESUMEN

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.


Asunto(s)
Antígenos de Protozoos/química , Sulfatos de Condroitina/química , Plasmodium falciparum/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Sitios de Unión , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Femenino , Humanos , Malaria Falciparum/genética , Malaria Falciparum/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Embarazo , Complicaciones Parasitarias del Embarazo/genética , Complicaciones Parasitarias del Embarazo/metabolismo
8.
Cell Signal ; 124: 111456, 2024 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-39384005

RESUMEN

Leptin, a hormone mainly secreted by adipocytes, has attracted significant attention since its discovery in 1994. Initially known for its role in appetite suppression and energy regulation, leptin is now recognized for its influence on various physiological processes, including immune response, bone formation, and reproduction. It exerts its effects by binding to receptors and initiating an intracellular signaling cascade. Heparan sulfate (HS) is known to regulate the intracellular signaling of various ligands. HS is present as the glycan portion of HSPGs on cell surfaces and in intercellular spaces, with diverse structures due to extensive sulfation and epimerization. Although HS chains on HSPGs are involved in many physiological processes, the detailed effects of HS chains on leptin signaling are not well understood. This study examined the role of HS chains on HSPGs in leptin signaling using Neuro2A cells expressing the full-length leptin receptor (LepR). We showed that cell surface HS was essential for efficient leptin signaling. Enzymatic degradation of HS significantly reduced leptin-induced phosphorylation of downstream molecules, such as signal transducer and activator of transcription 3 and p44/p42 Mitogen-activated protein kinase. In addition, HS regulated LepR expression and internalization, as treatment with HS-degrading enzymes decreased cell surface LepR. HS was also found to exhibit a weak interaction with LepR. Enzymatic removal of HS enhanced the interaction between LepR and low-density lipoprotein receptor-related protein 1, suggesting that HS negatively regulates this interaction. In conclusion, HS plays a significant role in modulating LepR availability on the cell surface, thereby influencing leptin signaling. These findings provide new insights into the complex regulation of leptin signaling and highlight potential therapeutic targets for metabolic disorders and obesity.

9.
J Biochem ; 176(3): 229-236, 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-38861406

RESUMEN

Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively, in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids and generated CSA and CSE liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.


Asunto(s)
Sulfatos de Condroitina , Liposomas , Liposomas/metabolismo , Liposomas/química , Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/química , Humanos , Células HEK293 , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/química
10.
J Biol Chem ; 287(43): 36022-8, 2012 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-22936799

RESUMEN

Bifunctional chondroitin synthase K4CP catalyzes glucuronic acid and N-acetylgalactosamine transfer activities and polymerizes a chondroitin chain. Here we have determined that an N-terminal region (residues 58-134) coordinates two transfer reactions and enables K4CP to catalyze polymerization. When residues 58-107 are deleted, K4CP loses polymerase activity while retaining both transfer activities. Peptide (113)DWPSDL(118) within this N-terminal region interacts with C-terminal peptide (677)YTWEKI(682). The deletion of either sequence abolishes glucuronic acid but not N-acetylgalactosamine transfer activity in K4CP. Both donor bindings and transfer activities are lost by mutating (677)YTWEKI(682) to (677)DAWEDI(682). On the other hand, acceptor substrates retain their binding to K4CP mutants. The characteristics of these K4CP mutants highlight different states of the enzyme reaction, providing an underlying structural basis for how these peptides play essential roles in coordinating the two glycosyltransferase activities for K4CP to elongate the chondroitin chain.


Asunto(s)
Condroitín/química , Escherichia coli/enzimología , Hexosiltransferasas/química , Péptidos/química , Secuencias de Aminoácidos , Catálisis , Condroitín/biosíntesis , Condroitín/genética , Escherichia coli/genética , Glicosilación , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Mutación , Péptidos/genética , Péptidos/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad
11.
J Biol Chem ; 287(52): 43390-400, 2012 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-23129769

RESUMEN

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.


Asunto(s)
Sulfatos de Condroitina , Biblioteca de Genes , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/genética , Escherichia coli/enzimología , Escherichia coli/genética , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferasas/genética , Sulfotransferasas/metabolismo
12.
Glycobiology ; 23(12): 1520-30, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24052236

RESUMEN

Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.


Asunto(s)
Baculoviridae/enzimología , Bombyx/química , Sulfatos de Condroitina/metabolismo , Condroitinasas y Condroitín Liasas/metabolismo , Animales , Sulfatos de Condroitina/química
13.
J Neurosci ; 31(47): 17091-102, 2011 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-22114278

RESUMEN

Chondroitin sulfate (CS) proteoglycans are strong inhibitors of structural rearrangement after injuries of the adult CNS. In addition to CS chains, keratan sulfate (KS) chains are also covalently attached to some proteoglycans. CS and KS sometimes share the same core protein, but exist as independent sugar chains. However, the biological significance of KS remains elusive. Here, we addressed the question of whether KS is involved in plasticity after spinal cord injury. Keratanase II (K-II) specifically degraded KS, i.e., not CS, in vivo. This enzyme digestion promoted the recovery of motor and sensory function after spinal cord injury in rats. Consistent with this, axonal regeneration/sprouting was enhanced in K-II-treated rats. K-II and the CS-degrading enzyme chondroitinase ABC exerted comparable effects in vivo and in vitro. However, these two enzymes worked neither additively nor synergistically. These data and further in vitro studies involving artificial proteoglycans (KS/CS-albumin) and heat-denatured or reduced/alkylated proteoglycans suggested that all three components of the proteoglycan moiety, i.e., the core protein, CS chains, and KS chains, were required for the inhibitory activity of proteoglycans. We conclude that KS is essential for, and has an impact comparable to that of CS on, postinjury plasticity. Our study also established that KS and CS are independent requirements for the proteoglycan-mediated inhibition of axonal regeneration/sprouting.


Asunto(s)
Sulfato de Queratano/fisiología , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Traumatismos de la Médula Espinal/metabolismo , Acetilglucosaminidasa/farmacología , Animales , Femenino , Regeneración Nerviosa/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Vértebras Torácicas
14.
J Biol Chem ; 286(33): 29026-29034, 2011 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-21715327

RESUMEN

Chondroitin sulfate is a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4-9) and temperature (30-60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.


Asunto(s)
Proteínas de la Cápside/metabolismo , Condroitinasas y Condroitín Liasas/metabolismo , Ácido Hialurónico/metabolismo , Nucleopoliedrovirus/enzimología , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Condroitinasas y Condroitín Liasas/química , Condroitinasas y Condroitín Liasas/genética , Ácido Hialurónico/química , Concentración de Iones de Hidrógeno , Nucleopoliedrovirus/genética , Spodoptera , Especificidad por Sustrato/fisiología
15.
Chemistry ; 18(24): 7388-93, 2012 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-22615170

RESUMEN

Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 µM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.


Asunto(s)
Hexosiltransferasas/metabolismo , Polisacáridos/metabolismo , Catálisis , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Glicosilación , Cinética , Modelos Químicos , Polisacáridos/química , Cuarzo/metabolismo
16.
Artículo en Inglés | MEDLINE | ID: mdl-22297996

RESUMEN

Baculovirus envelope protein ODV-E66 (67-704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67-704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P6(2) or P6(4), with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å(3) Da(-1).


Asunto(s)
Baculoviridae/enzimología , Condroitín Liasas/química , Productos del Gen env/química , Cristalización , Difracción de Rayos X
17.
J Biol Chem ; 285(3): 1597-606, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19915003

RESUMEN

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA beta1-4 and GlcNAc alpha1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Glucuronosiltransferasa/metabolismo , Glicosaminoglicanos/biosíntesis , Glicosiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Antígenos Bacterianos/biosíntesis , Bioingeniería , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glicosiltransferasas/biosíntesis , Glicosiltransferasas/química , Glicosiltransferasas/genética , Heparitina Sulfato/metabolismo , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/biosíntesis , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Mutación Puntual , Polímeros/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
J Biol Chem ; 285(44): 34155-67, 2010 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-20729547

RESUMEN

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.


Asunto(s)
Hexosiltransferasas/fisiología , N-Acetilgalactosaminiltransferasas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Glicosaminoglicanos/química , Hexosiltransferasas/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Células 3T3 NIH , Homología de Secuencia de Aminoácido
19.
J Biol Chem ; 285(47): 36645-55, 2010 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-20843788

RESUMEN

Activin A, a member of the transforming growth factor-ß family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys(259)-Lys(263) and Lys(270)-Lys(272), in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys(259)-Gly(277) containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.


Asunto(s)
Activinas/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Proteínas Recombinantes/metabolismo , Activinas/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Técnicas para Inmunoenzimas , Laminina/metabolismo , Luciferasas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Homología de Secuencia de Aminoácido
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