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INTRODUCTION: Along with recent advances in analytical technologies, tricarboxylic acid-cycle intermediates are increasingly identified as promising makers for cellular ischemia and mitochondrial dysfunction during hemorrhagic shock. For traumatized patients, the knowledge of the role of lipid oxidation substrates is sparse. In this study, we aimed to analyze the dynamics of systemic acylcarnitine (AcCa) release in a standardized polytrauma model with hemorrhagic shock. METHODS: Fifty-two male pigs (50 ± 5 kg) were randomized into two groups: group isolated fracture was subject to a standardized femur shaft fracture, and group polytrauma was subject to a femur fracture, followed by blunt chest trauma, liver laceration, and a pressure-controlled hemorrhagic shock for 60 minutes. Resuscitation was performed with crystalloids. Fractures were stabilized by intramedullary nailing. Venous samples were collected at six time points (baseline, trauma, resuscitation, 2 hours, 4 hours, and 6 hours). Lipidomic analysis was performed via liquid chromatography coupled mass spectrometry. Measurements were collated with clinical markers and near-infrared spectrometry measurements of tissue perfusion. Longitudinal analyses were performed with linear mixed models, and Spearman's correlations were calculated. A p value of 0.05 was defined as threshold for statistical significance. RESULTS: From a total of 303 distinct lipids, we identified two species of long-chain AcCas. Both showed a highly significant ( p < 0.001) twofold increase after hemorrhagic shock in group polytrauma that promptly normalized after resuscitation. This increase was associated with a significant decrease of the base excess ( p = 0.005), but recovery after resuscitation was faster. For both AcCas, there were significant correlations with decreased muscle tissue oxygen delivery ( p = 0.008, p = 0.003) and significant time-lagged correlations with the increase of creatine kinase ( p < 0.001, p < 0.001). CONCLUSION: Our results point to plasma AcCas as a possible indicator for mitochondrial dysfunction and cellular ischemia in hemorrhagic shock. The more rapid normalization after resuscitation in comparison with acid base changes may warrant further investigation.
Assuntos
Carnitina , Modelos Animais de Doenças , Lipidômica , Traumatismo Múltiplo , Choque Hemorrágico , Animais , Choque Hemorrágico/sangue , Choque Hemorrágico/terapia , Traumatismo Múltiplo/sangue , Traumatismo Múltiplo/complicações , Masculino , Carnitina/análogos & derivados , Carnitina/sangue , Suínos , Lipidômica/métodos , Ressuscitação/métodos , Fraturas do Fêmur/sangue , Fraturas do Fêmur/cirurgia , Biomarcadores/sangue , Biomarcadores/metabolismoRESUMO
BACKGROUND: Metabolic cardiomyopathy (MCM), characterized by intramyocardial lipid accumulation, drives the progression to heart failure with preserved ejection fraction (HFpEF). Although evidence suggests that the mammalian silent information regulator 1 (Sirt1) orchestrates myocardial lipid metabolism, it is unknown whether its exogenous administration could avoid MCM onset. We investigated whether chronic treatment with recombinant Sirt1 (rSirt1) could halt MCM progression. METHODS: db/db mice, an established model of MCM, were supplemented with intraperitoneal rSirt1 or vehicle for 4 weeks and compared with their db/ + heterozygous littermates. At the end of treatment, cardiac function was assessed by cardiac ultrasound and left ventricular samples were collected and processed for molecular analysis. Transcriptional changes were evaluated using a custom PCR array. Lipidomic analysis was performed by mass spectrometry. H9c2 cardiomyocytes exposed to hyperglycaemia and treated with rSirt1 were used as in vitro model of MCM to investigate the ability of rSirt1 to directly target cardiomyocytes and modulate malondialdehyde levels and caspase 3 activity. Myocardial samples from diabetic and nondiabetic patients were analysed to explore Sirt1 expression levels and signaling pathways. RESULTS: rSirt1 treatment restored cardiac Sirt1 levels and preserved cardiac performance by improving left ventricular ejection fraction, fractional shortening and diastolic function (E/A ratio). In left ventricular samples from rSirt1-treated db/db mice, rSirt1 modulated the cardiac lipidome: medium and long-chain triacylglycerols, long-chain triacylglycerols, and triacylglycerols containing only saturated fatty acids were reduced, while those containing docosahexaenoic acid were increased. Mechanistically, several genes involved in lipid trafficking, metabolism and inflammation, such as Cd36, Acox3, Pparg, Ncoa3, and Ppara were downregulated by rSirt1 both in vitro and in vivo. In humans, reduced cardiac expression levels of Sirt1 were associated with higher intramyocardial triacylglycerols and PPARG-related genes. CONCLUSIONS: In the db/db mouse model of MCM, chronic exogenous rSirt1 supplementation rescued cardiac function. This was associated with a modulation of the myocardial lipidome and a downregulation of genes involved in lipid metabolism, trafficking, inflammation, and PPARG signaling. These findings were confirmed in the human diabetic myocardium. Treatments that increase Sirt1 levels may represent a promising strategy to prevent myocardial lipid abnormalities and MCM development.
Assuntos
Diabetes Mellitus , Cardiomiopatias Diabéticas , Insuficiência Cardíaca , Animais , Humanos , Camundongos , Diabetes Mellitus/metabolismo , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/prevenção & controle , Insuficiência Cardíaca/metabolismo , Inflamação/metabolismo , Lipidômica , Lipídeos , Miócitos Cardíacos/metabolismo , PPAR gama/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Volume Sistólico , Triglicerídeos/metabolismo , Função Ventricular EsquerdaRESUMO
Sphingolipids (SL) represent a structurally diverse class of lipids that are central to cellular physiology and neuronal development and function. Defects in the sphingolipid metabolism are typically associated with nervous system disorders. The C4-dihydroceramide desaturase (DEGS1) catalyzes the conversion of dihydroceramide to ceramide, the final step in the SL de-novo synthesis. Loss of function mutations in DEGS1 cause a hypomyelinating leukodystrophy, which is associated with increased plasma dihydrosphingolipids (dhSL) and with the formation of an atypical SPB 18:1(14Z);O2 metabolite. Here, we characterize two novel DEGS1 variants of unknown significance (VUS), provide a structural model with a predicted substrate binding site, and propose a regulatory link between DEGS1 and fatty acid desaturase 3 (FADS3). Both VUS involve single amino acid substitutions near the C-terminus within conserved regions of the enzyme. Patient 1 (p.R311K variant) shows severe progressive tetraspasticity, intellectual disability, and epilepsy in combination with brain magnetic resonance imaging (MRI) findings, typical for DEGS1-related leukodystrophy. Patient 2 (p.G270E variant) presents with delayed psychomotor development, oculomotor apraxia, and a normal brain MRI. Plasma from the p.R311K carrier showed a significantly elevated dhSL species and the presence of SPB 18:1(14Z);O2, while the plasma SL profile for the p.G270E variant was not altered. This suggests the p.R331K variant is pathogenic, while the p.G270E appears benign. As an increase in dihydroSL species is also seen in other pathological disorders of the SL metabolism, the SPB 18:1(14Z);O2 seems to be a more specific biomarker to discriminate between pathogenic and benign DEGS1 variants.
Assuntos
Síndrome de Cogan , Doenças do Sistema Nervoso , Humanos , Substituição de Aminoácidos , Biomarcadores , Esfingolipídeos/metabolismoRESUMO
BACKGROUND: Occult hypoperfusion describes the absence of sufficient microcirculation despite normal vital signs. It is known to be associated with prolonged elevation of serum lactate and later complications in severely injured patients. We hypothesized that changes in circulating lipids are related to responsiveness to resuscitation. The purpose of this study is investigating the relation between responsiveness to resuscitation and lipidomic course after poly trauma. METHODS: Twenty-five male pigs were exposed a combined injury of blunt chest trauma, liver laceration, controlled haemorrhagic shock, and femoral shaft fracture. After 1 h, animals received resuscitation and fracture stabilization. Venous blood was taken regularly and 233 specific lipids were analysed. Animals were divided into two groups based on serum lactate level at the end point as an indicator of responsiveness to resuscitation (<2 mmol/L: responder group (R group), â§2 mmol/L: occult hypoperfusion group (OH group)). RESULTS: Eighteen animals met criteria for the R group, four animals for the OH group, and three animals died. Acylcarnitines showed a significant increase at 1 h compared to baseline in both groups. Six lipid subgroups showed a significant increase only in R group at 2 h. There was no significant change at other time points. CONCLUSIONS: Six lipid groups increased significantly only in the R group at 2 h, which may support the idea that they could serve as potential biomarkers to help us to detect the presence of occult hypoperfusion and insufficient resuscitation. We feel that further study is required to confirm the role and mechanism of lipid changes after trauma.
RESUMO
Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on samples enriched for isolated OSs show that Bbs1 is required for BBSome-complex stability and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a role for Bbs1/BBSome in OS lipid homeostasis, suggesting a pathomechanism underlying retinal degeneration in BBS.
Assuntos
Síndrome de Bardet-Biedl , Animais , Síndrome de Bardet-Biedl/genética , Cílios/metabolismo , Lipídeos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Peixe-Zebra/metabolismoRESUMO
Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.
Assuntos
Apolipoproteínas M/metabolismo , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Lipoproteínas HDL/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Transporte Biológico , Bovinos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Permeabilidade , Placa Aterosclerótica , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Receptores de Esfingosina-1-Fosfato/genéticaRESUMO
OBJECTIVE: CNS damage can increase the susceptibility of the blood-brain barrier (BBB) to changes induced by systemic inflammation. The aim of this study is to better understand BBB permeability in patients with MS and to examine whether compromised BBB integrity in some of these patients is associated with CNS damage and systemic inflammation. METHODS: Routine CSF measurements of 121 patients with MS were analyzed including number and type of infiltrating cells, total protein, lactate, and oligoclonal bands, as well as intrathecal production of immunoglobulins and CSF/serum quotients for albumin, immunoglobulins, and glucose. In addition, in a subcohort of these patients, we performed ex vivo immunophenotyping of CSF-infiltrating and paired circulating lymphocytes using a panel of 13 monoclonal antibodies, we quantified intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1), and we performed intrathecal lipidomic analysis. RESULTS: Patients with MS with abnormal high levels of albumin in the CSF showed a distinct CSF cell infiltrate and markers of CNS damage such as increased intrathecal levels of NF-L and CHI3L1 as well as a distinct CSF lipidomic profile. In addition, these patients showed higher numbers of circulating proinflammatory Th1 and Th1* cells compatible with systemic inflammation. Of interest, the abnormally high levels of albumin in the CSF of those patients were preserved over time. CONCLUSIONS: Our results support the hypothesis that CNS damage may increase BBB vulnerability to systemic inflammation in a subset of patients and thus contribute to disease heterogeneity.
Assuntos
Albuminas/líquido cefalorraquidiano , Lesões Encefálicas/líquido cefalorraquidiano , Inflamação/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Adulto , Biomarcadores/líquido cefalorraquidiano , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/imunologia , Feminino , Humanos , Imunoglobulina G/líquido cefalorraquidiano , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologiaRESUMO
Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.
Assuntos
Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Camundongos , Serina C-Palmitoiltransferase/genética , Especificidade por SubstratoRESUMO
OBJECTIVE: Debate continues regarding the influence of dietary fats and sugars on the risk of developing metabolic diseases, including insulin resistance and nonalcoholic fatty liver disease (NAFLD). We investigated the effect of two eucaloric diets, one enriched with saturated fat (SFA) and the other enriched with free sugars (SUGAR), on intrahepatic triacylglycerol (IHTAG) content, hepatic de novo lipogenesis (DNL), and whole-body postprandial metabolism in overweight males. RESEARCH DESIGN AND METHODS: Sixteen overweight males were randomized to consume the SFA or SUGAR diet for 4 weeks before consuming the alternate diet after a 7-week washout period. The metabolic effects of the respective diets on IHTAG content, hepatic DNL, and whole-body metabolism were investigated using imaging techniques and metabolic substrates labeled with stable-isotope tracers. RESULTS: Consumption of the SFA diet significantly increased IHTAG by mean ± SEM 39.0 ± 10.0%, while after the SUGAR diet IHTAG was virtually unchanged. Consumption of the SFA diet induced an exaggerated postprandial glucose and insulin response to a standardized test meal compared with SUGAR. Although whole-body fat oxidation, lipolysis, and DNL were similar following the two diets, consumption of the SUGAR diet resulted in significant (P < 0.05) decreases in plasma total, HDL, and non-HDL cholesterol and fasting ß-hydroxybutyrate plasma concentrations. CONCLUSIONS: Consumption of an SFA diet had a potent effect, increasing IHTAG together with exaggerating postprandial glycemia. The SUGAR diet did not influence IHTAG and induced minor metabolic changes. Our findings indicate that a diet enriched in SFA is more harmful to metabolic health than a diet enriched in free sugars.
Assuntos
Glicemia/efeitos dos fármacos , Carboidratos da Dieta/farmacologia , Gorduras na Dieta/farmacologia , Ácidos Graxos , Fígado/efeitos dos fármacos , Período Pós-Prandial/efeitos dos fármacos , Adulto , Glicemia/metabolismo , Estudos Cross-Over , Dieta , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Humanos , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Fígado/metabolismo , Masculino , Refeições , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fatores de RiscoRESUMO
Glycosylation is an integral part in health and disease, as emphasized by the growing number of identified glycosylation defects. In humans, proteins are modified with a diverse range of glycoforms synthesized in complex biosynthetic pathways. Glycosylation disorders have been described in congenital disorders of glycosylation (CDG) as well as in acquired disease conditions such and non-alcoholic fatty liver disease (NAFLD). A hallmark in a subset of CDG cases is the reduced glycosylation site occupancy of asparagine-linked glycans. Using an optimized method protocol, we determined the glycosylation site occupancy from four proteins of hepatic and lymphatic origin from CDG and NAFLD patients. We found variable degrees of site occupancy, depending on the tissue of origin and the disease condition. In CDG glycosylation sites of IgG2 and IgA1 were occupied to normal levels. In NAFLD haptoglobin and transferrin glycosylation sites were hyper-glycosylated, a property qualifying for its use as a potential biomarker. Furthermore, we observed, that glycosylation sites of liver-originating transferrin and haptoglobin are differentially occupied under physiological conditions, a further instance not noticed in serum proteins to date. Our findings suggest the use of serum protein hyperglycosylation as a biomarker for early stages of NAFLD.
Assuntos
Defeitos Congênitos da Glicosilação/sangue , Haptoglobinas/metabolismo , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Biomarcadores/sangue , Defeitos Congênitos da Glicosilação/complicações , Feminino , Glicosilação , Humanos , Masculino , Hepatopatia Gordurosa não Alcoólica/etiologiaRESUMO
Acanthamoeba polyphaga mimivirus is a giant virus encoding 1262 genes among which many were previously thought to be exclusive to cellular life. For example, mimivirus genes encode enzymes involved in the biosynthesis of nucleotide sugars and putative glycosyltransferases. We identified in mimivirus a glycogenin-1 homologous gene encoded by the open reading frame R707. The R707 protein was found to be active as a polymerizing glucosyltransferase enzyme. Like glycogenin-1, R707 activity was divalent-metal-ion-dependent and relied on an intact DXD motif. In contrast with glycogenin-1, R707 was, however, not self-glucosylating. Interestingly, the product of R707 catalysis featured α1-6, ß1-6 and α1-4 glycosidic linkages. Mimivirus R707 is the first reported glycosyltransferase able to catalyse the formation of both α and ß linkages. Mimivirus-encoded glycans play a role in the infection of host amoebae. Co-infection of Acanthamoeba with mimivirus and amylose and chitin hydrolysate reduced the number of infected amoebae, thus supporting the importance of polysaccharide chains in the uptake of mimivirus by amoebae. The identification of a glycosyltransferase capable of forming α and ß linkages underlines the peculiarity of mimivirus and enforces the concept of a host-independent glycosylation machinery in mimivirus.
Assuntos
Acanthamoeba/virologia , Glucose/metabolismo , Glucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Mimiviridae/metabolismo , Mimiviridae/patogenicidade , Proteínas Virais/metabolismo , Glucose/química , Glucosiltransferases/química , Glicoproteínas/química , Glicosídeos/química , Glicosídeos/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Proteínas Virais/químicaRESUMO
Acanthamoeba polyphaga mimivirus is a member of the giant nucleocytoplasmic large DNA viruses, infecting various Acanthamoeba spp. The genomes of giant viruses encode components previously thought to be exclusive to cellular life, such as proteins involved in nucleic acid and protein synthesis. Recent work on enzymes involved in carbohydrate biosynthesis and metabolism show that instead of utilizing host cell resources, Mimivirus produces its own glycosylation machinery. To obtain a more detailed view of glycosylation in Mimivirus, we developed a periodate oxidation-based method to selectively enrich Mimivirus surface glycoproteins. O-Glycosylation in Mimivirus glycoproteins was identified by permethylation and matrix-assisted laser desorption/ionization-mass spectrometry analyses of beta-eliminated glycans. We sequenced 26 previously undescribed O-glycans, most of which contain glucose as their reducing end saccharide. These data will facilitate future studies on the functional significance of glycosylation in Mimivirus.
Assuntos
Glicoproteínas/metabolismo , Mimiviridae/metabolismo , Proteínas Virais/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicosilação , Oxirredução , Proteínas Virais/biossíntese , Proteínas Virais/químicaRESUMO
Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens.
Assuntos
Artrite/virologia , Colágeno/imunologia , Mimiviridae/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Artrite/imunologia , Bovinos , Colágeno/genética , Modelos Animais de Doenças , Humanos , Imunização , Camundongos , Camundongos Endogâmicos DBA , Mimiviridae/genética , Proteínas Virais/genéticaRESUMO
Deficiency in N-linked protein glycosylation is a long-known characteristic of alcoholic liver disease and congenital disorders of glycosylation. Previous investigations of ethanol-induced glycosylation deficiency demonstrated perturbations in the early steps of substrate synthesis and in the final steps of capping N-linked glycans in the Golgi. The significance of the biosynthesis of N-glycan precursors in the endoplasmic reticulum, however, has not yet been addressed in alcoholic liver disease. Ethanol-metabolizing hepatoma cells were treated with increasing concentrations of ethanol. Transcript analysis of genes involved in the biosynthesis of N-glycans, activity assays of related enzymes, dolichol-phosphate quantification, and analysis of dolichol-linked oligosaccharides were performed. Upon treatment of cells with ethanol, we found a decrease in the final N-glycan precursor Dol-PP-GlcNAc(2) Man(9) Glc(3) and in C95- and C100-dolichol-phosphate levels. Transcript analysis of genes involved in N-glycosylation showed a 17% decrease in expression levels of DPM1, a subunit of the dolichol-phosphate-mannose synthase, and an 8% increase in RPN2, a subunit of the oligosaccharyl transferase. Ethanol treatment decreases the biosynthesis of dolichol-phosphate. Consequently, the formation of N-glycan precursors is affected, resulting in an aberrant precursor assembly. Messenger RNA levels of genes involved in N-glycan biosynthesis are slightly affected by ethanol treatment, indicating that the assembly of N-glycan precursors is not regulated at the transcriptional level. This study confirms that ethanol impairs N-linked glycosylation by affecting dolichol biosynthesis leading to impaired dolichol-linked oligosaccharide assembly. Together our data help to explain the underglycosylation phenotype observed in alcoholic liver disease and congenital disorders of glycosylation.
Assuntos
Dolicóis/biossíntese , Etanol/farmacologia , Glicosilação/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Configuração de Carboidratos , Células Cultivadas , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Fosfatos de Dolicol/metabolismo , Dolicóis/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hexosiltransferases , Humanos , Inativação Metabólica , Manosiltransferases/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transferrina/metabolismoRESUMO
Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.
Assuntos
Colágeno/metabolismo , Glicosiltransferases/química , Mimiviridae/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Acanthamoeba/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/metabolismo , Clonagem Molecular , Colágeno/química , Vetores Genéticos , Glicosilação , Humanos , Hidroxilisina/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycosylation site occupancy and oligosaccharide structure must be measured quantitatively. In this chapter, we describe current methods enabling the determination of N-glycosylation by assessment of cellular dolichol phosphate levels, dolichol-linked oligosaccharides, and the occupancy of N-glycosylation sites. We also provide detailed methods for the analysis of O-glycosylation, whose role in intracellular protein maturation is often overlooked.
Assuntos
Dolicóis/química , Glicoproteínas/química , Oligossacarídeos/análise , Resposta a Proteínas não Dobradas , Animais , Cromatografia Líquida de Alta Pressão/métodos , Fosfatos de Dolicol/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Oligossacarídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Defeitos Congênitos da Glicosilação/metabolismo , Dolicóis/metabolismo , Inibidores Enzimáticos/farmacologia , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Oligossacarídeos/biossíntese , Ácidos Tricarboxílicos/farmacologia , Antígenos CD59/biossíntese , Antígenos CD59/genética , Células Cultivadas , Colesterol/biossíntese , Colesterol/genética , Defeitos Congênitos da Glicosilação/genética , Dolicóis/genética , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Oligossacarídeos/genéticaRESUMO
Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchors of mono- and oligosaccharide precursors involved in protein glycosylation. The biological importance of Dol phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency. For characterization of inherited human diseases and evaluation of therapeutic trials, cultured cells often serve as a sole possible source for experimentation. Limited amounts of cell culture material render the quantitative analysis of Dol a challenging task. Here, we present HPLC- and mass spectrometry-based approaches to analyze and quantitate Dol-P from cultured human cells. The composition of naturally occurring Dol-P and the saturation state of the alpha-isoprene units was identified by negative-ion electrospray ionization mass spectrometry. Furthermore, fluorescently labeled Dol-P were separated by HPLC and quantified by comparison to known amounts of the internal standard polyprenol-P. The effect of pravastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase inhibitor, on the formation of Dol-P in HeLa cells was investigated. As expected, this treatment led to a decrease of Dol-P down to 35% of normal levels.
Assuntos
Fosfatos de Dolicol/análise , Espectrometria de Massas/métodos , Antracenos/metabolismo , Cromatografia Líquida de Alta Pressão , Fosfatos de Dolicol/química , Flavonoides/análise , Flavonoides/química , Células HeLa , Humanos , Fenóis/análise , Fenóis/química , Polifenóis , Pravastatina/farmacologia , Padrões de ReferênciaRESUMO
A major surface constituent of Echinococcus multilocularis adult worms, referred to as an EmA9 antigen, was immunoaffinity purified and identified as a high-molecular-weight glycoconjugate. Labeling studies using the monoclonal antibody MAbEmA9 indicated that this antigen undergoes a regulated expression during the development from the larval to the adult parasite. Chemical modification of carbohydrate by periodate oxidation resulted in a reduced reactivity with antigen-specific antibodies. Non-reductive beta-elimination of the purified molecule indicated the presence of O-linked glycans attached to threonine residues. Carbohydrate compositional analyses indicated the presence of N- and O-glycans with the ratio of carbohydrate to protein being 1.5:1 (w/w). N- and O-linked glycans were released by hydrazinolysis and analyzed as 2-aminobenzamide derivatized glycans by mass spectrometry together with HPLC and enzymatic sequencing. Novel linear O-linked saccharides with multiple beta-HexNAc extensions of reducing end Gal were identified. N-Linked glycans were also detected with oligomannose and mono-, bi-, tri- and tetra-antennary-type structures, most of which were found to be core-fucosylated. Taken together, the results indicate that the EmA9 antigen is a glycoprotein located at the outer surface of the adult E. multilocularis. The observation that the EmA9 antigen expression is developmentally regulated suggests an involvement of this glycoprotein in the establishment of the parasite in its canine host.
Assuntos
Antígenos de Helmintos/imunologia , Antígenos/química , Echinococcus multilocularis/imunologia , Glicosilação , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Antígenos de Helmintos/química , Carboidratos/química , Cromatografia Líquida de Alta Pressão/métodos , Echinococcus multilocularis/metabolismo , Hidrazinas/química , Manose/química , Espectrometria de Massas/métodos , Peso Molecular , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , ortoaminobenzoatos/químicaRESUMO
Collagen is a trimer of three left-handed alpha chains representing repeats of the motif Gly-X-Y, where (hydroxy)proline and (hydroxy)lysine residues are often found at positions X and Y. Selected hydroxylysines are further modified by the addition of galactose and glucose-galactose units. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation and is mediated by beta(1-O)galactosyl- and alpha(1-2)glucosyltransferase enzymes. We have identified two collagen galactosyltransferases using affinity chromatography and tandem mass spectrometry protein sequencing. The two collagen beta(1-O)galactosyltransferases corresponded to the GLT25D1 and GLT25D2 proteins. Recombinant GLT25D1 and GLT25D2 enzymes showed a strong galactosyltransferase activity toward various types of collagen and toward the serum mannose-binding lectin MBL, which contains a collagen domain. Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. The GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene is expressed only at low levels in the nervous system. The GLT25D1 and GLT25D2 enzymes are similar to CEECAM1, to which we could not attribute any collagen galactosyltransferase activity. The GLT25D1 and GLT25D2 genes now allow addressing of the biological significance of collagen glycosylation and the importance of this posttranslational modification in the etiology of connective tissue disorders.