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Background and Purpose- Absent or diminished α-galactosidase A (GLA) and acid α-glucosidase (GAA) enzyme activity are core features of Fabry and Pompe disease, respectively. Patients with Fabry or Pompe disease may have dilated intracranial arteries but whether lower GLA or GAA enzyme activity relates to brain arterial dilatation in other populations is unknown. Methods- Participants included Parkinson disease patients and nonblood-related controls, whose GLA and GAA enzymatic activities were measured in dried blood spots. Independent readers measured the axial arterial diameter of the ascending portion of the cavernous internal carotid arteries and the most proximal segment of the basilar artery in T2 black voids. Linear regression models were built to investigate the relationship between brain arterial diameters and lysosomal enzymatic activities. Results- The cohort included 107 participants (mean age, 66.5±10.3; 67% men). In an adjusted linear regression model, lower GLA activity was associated with larger brain arterial diameters (B=0.50±0.23, P=0.03). The strength of association was the greatest for the basilar artery diameter (B=0.80±0.33, P=0.02). Similarly, lower GAA activity was associated with an increased basilar arterial diameter (B=0.73±0.35, P=0.04). Conclusions- Lower GLA and GAA enzymatic activities were associated with larger brain arterial diameters, particularly the basilar artery diameter. Lower lysosomal enzymatic function in patients without Fabry or Pompe disease may play a role in brain arterial dilatation.
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Arterias Cerebrales/diagnóstico por imagen , Arterias Cerebrales/enzimología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Lisosomas/enzimología , alfa-Galactosidasa/metabolismo , Anciano , Encéfalo/irrigación sanguínea , Encéfalo/diagnóstico por imagen , Encéfalo/enzimología , Estudios de Cohortes , Dilatación Patológica/enzimología , Activación Enzimática/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos Parkinsonianos/diagnóstico por imagen , Trastornos Parkinsonianos/enzimologíaRESUMEN
Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.
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Cilios/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/genética , Fallo Renal Crónico/tratamiento farmacológico , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Animales , Diferenciación Celular/efectos de los fármacos , Cilios/patología , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Fallo Renal Crónico/genética , Fallo Renal Crónico/patología , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Purinas/administración & dosificación , Roscovitina , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
The inherited deficiency of the lysosomal glucocerebrosidase (GBA) due to mutations in the GBA gene results in Gaucher disease (GD). A vast majority of patients present with nonneuronopathic, type 1 GD (GD1). GBA deficiency causes the accumulation of two key sphingolipids, glucosylceramide (GL-1) and glucosylsphingosine (LysoGL-1), classically noted within the lysosomes of mononuclear phagocytes. How metabolites of GL-1 or LysoGL-1 produced by extralysosomal glucocerebrosidase GBA2 contribute to the GD1 pathophysiology is not known. We recently recapitulated hepatosplenomegaly, cytopenia, hypercytokinemia, and the bone-formation defect of human GD1 through conditional deletion of Gba in Mx1-Cre(+):GD1 mice. Here we show that the deletion of Gba2 significantly rescues the GD1 clinical phenotype, despite enhanced elevations in GL-1 and LysoGL-1. Most notably, the reduced bone volume and bone formation rate are normalized. These results suggest that metabolism of GL-1 or LysoGL-1 into downstream bioactive lipids is a major contributor to the bone-formation defect. Direct testing revealed a strong inhibition of osteoblast viability by nanomolar concentrations of sphingosine, but not of ceramide. These findings are consistent with toxicity of high circulating sphingosine levels in GD1 patients, which decline upon enzyme-replacement therapy; serum ceramide levels remain unchanged. Together, complementary results from mice and humans affected with GD1 not only pinpoint sphingosine as being an osteoblast toxin, but also set forth Gba2 as a viable therapeutic target for the development of inhibitors to ameliorate certain disabling consequences of GD1.
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Enfermedad de Gaucher/genética , Enfermedad de Gaucher/terapia , Eliminación de Gen , beta-Glucosidasa/genética , Animales , Línea Celular , Enfermedad de Gaucher/enzimología , Humanos , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoblastos/patología , Fenotipo , Esfingolípidos/metabolismo , Esfingosina/metabolismoRESUMEN
Gaucher disease (GD) involves the accumulation of glucosylceramide (GL1) and its deacylated lysolipid, glucosylsphingosine (lyso-GL1) which is implicated in mediating immune dysregulation and skeletal disease. The aim of our study was to assess plasma Lyso-GL1 as a biomarker of GD and its response to therapy. Plasma lyso-GL1 in 169 patients with GD type 1 (GD1) was measured by LC-MS/MS. Significant predictors of plasma LGL1 were assessed by Pearson's correlation coefficient, Wilcoxon Mann Whitney test and multiple linear regression. Propensity scores were used to match patients on treatment mode: Enzyme Replacement Therapy (ERT) vs. Eliglustat Tartrate SRT (ELI-SRT). Plasma Lyso-GL1 levels in healthy controls averaged 1.5 ng/ml (1.3-1.7; 95% CI). In untreated GD patients, the levels were massively elevated (180.9 ng/ml: 95% CI, 145.4-216.5) and imiglucerase ERT resulted in marked reduction (89 ng/ml: 95% CI, 69.2-129.4) (P < 0.001). Lyso-GL1 correlated with chitotriosidase (r = 0.59 P < 0.001), CCL18 (r = 0.62 P <0.001), hepatomegaly (r = 0.28 P < 0.001), splenomegaly (r = 0.27 P = 0.003), splenectomy (P = 0.01) and treatment mode (P < 0.001). By multiple linear regression, the strongest predictors of lyso-GL1 were age (P < 0.001), splenectomy (P = 0.02), Chitotriosidase (P < 0.001) and CCL18 levels (P = 0.001). After propensity score matching to obtain comparable groups of patients on ERT vs ELI-SRT, lyso-GL1 levels were lower among patients receiving ELI-SRT by 113 ng/ml (95% CI: 136-90.3 ng/ml P < 0.001). Plasma lyso-GL1 is a key biomarker of GD. ERT reduced lyso-GL1 levels. By propensity scoring, ELI-SRT resulted in greater reduction of lyso-GL1 than ERT. Am. J. Hematol. 91:1082-1089, 2016. © 2016 Wiley Periodicals, Inc.
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Enfermedad de Gaucher/sangre , Psicosina/análogos & derivados , Adolescente , Adulto , Biomarcadores/sangre , Niño , Preescolar , Terapia de Reemplazo Enzimático , Femenino , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/terapia , Humanos , Lactante , Masculino , Persona de Mediana Edad , Puntaje de Propensión , Psicosina/sangre , Pirrolidinas/uso terapéutico , Adulto JovenRESUMEN
The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide-based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi-dimensional liquid-chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre-cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope-labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT-PCR.
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Reactores Biológicos/virología , Biotecnología/métodos , Técnicas de Cultivo de Célula/métodos , Vesivirus/aislamiento & purificación , Virión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Células CHO , Supervivencia Celular/fisiología , Cromatografía por Intercambio Iónico , Cricetinae , Cricetulus , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Viral/genética , ARN Viral/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Vesivirus/química , Vesivirus/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/químicaRESUMEN
In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.
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Enfermedad de Gaucher/patología , Eliminación de Gen , Glucosilceramidasa/deficiencia , Macrófagos/patología , Animales , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Osteoporosis/etiología , Fenotipo , Regiones Promotoras GenéticasRESUMEN
The system of oceanic flows constituting the Atlantic Meridional Overturning Circulation (AMOC) moves heat and other properties to the subpolar North Atlantic, controlling regional climate, weather, sea levels, and ecosystems. Climate models suggest a potential AMOC slowdown towards the end of this century due to anthropogenic forcing, accelerating coastal sea level rise along the western boundary and dramatically increasing flood risk. While direct observations of the AMOC are still too short to infer long-term trends, we show here that the AMOC-induced changes in gyre-scale heat content, superimposed on the global mean sea level rise, are already influencing the frequency of floods along the United States southeastern seaboard. We find that ocean heat convergence, being the primary driver for interannual sea level changes in the subtropical North Atlantic, has led to an exceptional gyre-scale warming and associated dynamic sea level rise since 2010, accounting for 30-50% of flood days in 2015-2020.
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OBJECTIVE: Identify the prevalence of food insecurity (FI) and compare sociodemographic, mental, physical, behavioral, and environmental risk factors for FI among students at a private university, community college, and historically black college or university (HBCU). PARTICIPANTS: Adult students attending a private university, community college, or HBCU (n = 4,140) located within the southeastern United States. METHODS: Using an online survey (2017-2019), FI, sociodemographic, mental, physical, behavioral, and environmental data were collected to understand their association with FI. RESULTS: Up to 37.1% of students experienced FI. Identifying as black, other/multi-racial, having poor sleep, federal loans, depressive symptoms, high stress, social isolation, or a chronic condition were associated with FI. These associations varied by institution. CONCLUSIONS: FI is prevalent within diverse post-secondary institutions that serve traditional and nontraditional students with risk factors varying between institutions. The prevalence of FI and risk factors can inform institutional policy responses to ameliorate the effects of FI.
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Dried blood spot (DBS) methods are currently available for identification of a range of lysosomal storage disorders (LSDs). These disorders are generally characterized by a deficiency of activity of a lysosomal enzyme and by a broad spectrum of phenotypes. Diagnosis of LSD patients is often delayed, which is of particular concern as therapeutic outcomes (e.g. enzyme replacement therapy) are generally more favorable in early disease stages. Experts in the field of LSDs diagnostics and screening programs convened and reviewed experiences with the use of DBS methods, and discuss the diagnostic challenges, possible applications and quality programs in this paper. Given the easy sampling and shipping and stability of samples, DBS has evident advantages over other laboratory methods and can be particularly helpful in the early identification of affected LSD patients through neonatal screening, high-risk population screening or family screening.
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Pruebas con Sangre Seca/métodos , Pruebas con Sangre Seca/tendencias , Enfermedades por Almacenamiento Lisosomal/sangre , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Pruebas con Sangre Seca/normas , Humanos , Control de Calidad , Estándares de Referencia , Informe de InvestigaciónRESUMEN
Objective. To examine pharmacy career engagement, interest, and confidence in Doctor of Pharmacy (PharmD) students identifying as underrepresented racial minorities (URMs).Methods. A 15-item survey about career engagement, confidence, and goals was administered at a business session of a national conference. The survey included demographic items and items about career exposure prior to and during school, career aspirations after graduation, frequency of engagement in various settings, career factors, and career confidence. Cronbach alpha was used to examine survey reliability. Descriptive statistics and nonparametric statistical tests were used to analyze survey responses.Results. Sixty-nine URM students completed the survey. Most indicated frequent engagement with community pharmacy prior to and during school; no engagement with hospital pharmacy prior to school, yet occasional or frequent engagement during school; and no engagement with the pharmaceutical industry prior to and during school. Most selected hospital pharmacy as their career aspiration, followed by community pharmacy and industry. Approximately half indicated an interest in completing a postgraduate fellowship. Items selected as important to career choice included patient care, job security, and level of stress. Group differences were found by gender and year in school.Conclusion. Despite calls for diversity in pharmacy, there is a paucity of research in this area. This study provides a first glimpse into the career engagement, confidence, and goals of students identifying as URMs, raising a number of critical issues for pharmacy education. Moving forward, schools, employers, and researchers must work to better understand the career development of URM students, including barriers and facilitators to access and success.
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Educación en Farmacia , Estudiantes de Farmacia , Selección de Profesión , Objetivos , Humanos , Grupos Minoritarios , Reproducibilidad de los ResultadosRESUMEN
CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.
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Linfocitos T CD4-Positivos/inmunología , Sistemas CRISPR-Cas , Proliferación Celular/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/genética , Péptidos/metabolismo , Secuencia de Aminoácidos , Proteína 9 Asociada a CRISPR/genética , Citocinas , Edición Génica , Humanos , Staphylococcus aureus/genética , Linfocitos T/inmunologíaRESUMEN
Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Better understanding of the underlying disease mechanism(s) is an urgent need for the development of disease-modifying therapeutics. Limited studies have been performed in large patient cohorts to identify protein alterations in cerebrospinal fluid (CSF), a proximal site to pathology. We set out to identify disease-relevant protein changes in CSF to gain insights into the etiology of Parkinson's disease and potentially assist in disease biomarker identification. In this study, we used liquid chromatography-tandem mass spectrometry in data-independent acquisition (DIA) mode to identify Parkinson's-relevant biomarkers in cerebrospinal fluid. We quantified 341 protein groups in two independent cohorts (n = 196) and a longitudinal cohort (n = 105 samples, representing 40 patients) consisting of Parkinson's disease and healthy control samples from three different sources. A first cohort of 53 Parkinson's disease and 72 control samples was analyzed, identifying 53 proteins with significant changes (p < 0.05) in Parkinson's disease relative to healthy control. We established a biomarker signature and multiple protein ratios that differentiate Parkinson's disease from healthy controls and validated these results in an independent cohort. The second cohort included 28 Parkinson's disease and 43 control samples. Independent analysis of these samples identified 41 proteins with significant changes. Evaluation of the overlapping changes between the two cohorts identified 13 proteins with consistent and significant changes (p < 0.05). Importantly, we found the extended granin family proteins as reduced in disease, suggesting a potential common mechanism for the biological reduction in monoamine neurotransmission in Parkinson's patients. Our study identifies several novel protein changes in Parkinson's disease cerebrospinal fluid that may be exploited for understanding etiology of disease and for biomarker development.
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Cromograninas/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , Anciano , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteómica , Espectrometría de Masas en TándemRESUMEN
Objective Pathology overutilisation is a significant issue affecting the quality and cost of health care. Because junior medical officers (JMOs) order most pathology tests in the hospital setting, the aim of the present study was to identify the main reasons for hospital pathology overutilisation from the perspective of the JMO. Methods A qualitative method, using focus group methodology, was undertaken. Sixteen JMOs from two hospitals participated in three focus groups. Data were analysed using thematic analysis. Results Three major themes contributed to overutilisation: the real and perceived expectations of senior colleagues, the level of JMO clinical experience and strategies to manage JMO workload around clinical systems. Within these themes, 12 subthemes were identified. Conclusions Overutilisation of hospital pathology testing occurs when there are high social costs to JMOs for underordering, with little cost for overordering. Interventions should restore this balance through reframing overutilisation as both a costly and potentially harmful activity, promoting a supportive culture with regular senior guidance, and addressing clinical systems in which missed tests create an excessive workload. What is known about the topic? Mean overutilisation rates of pathology testing are reported to be as high as 44%. Although numerous studies have reported successful efforts to decrease hospital pathology overutilisation, no primary research was identified that examined the JMO perspective on this subject. What does this paper add? Clinical need is not the primary factor guiding the pathology-ordering decisions of junior practitioners; rather, medical team culture, limited JMO experience and systems factors have a significant role. What are the implications for practitioners? The social and behavioural determinants of pathology ordering must be considered to achieve appropriate pathology test utilisation. These include senior medical officer engagement, the guidance of JMOs and clinical workflows.
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Actitud del Personal de Salud , Pruebas Diagnósticas de Rutina , Uso Excesivo de los Servicios de Salud , Servicio de Patología en Hospital , Médicos/psicología , Pruebas Diagnósticas de Rutina/economía , Pruebas Diagnósticas de Rutina/métodos , Grupos Focales , Hospitales , Humanos , Internado y Residencia , Uso Excesivo de los Servicios de Salud/economía , Cuerpo Médico de Hospitales , Servicio de Patología en Hospital/economía , Pautas de la Práctica en MedicinaRESUMEN
A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 µg/mL with bias <15 %. The intra-assay coefficient of variation is less than 10 % for concentrations from 1.2 to 9 µg/mL, and less than 25 % for concentrations below 1.2 µg/mL. For glucosylceramide and ceramide trihexoside the linear range is 0.05-3 µg/mL with biases <10 % and <20 %, respectively. The intra-assay coefficient of variation for these analytes is less than 10 % at concentrations from 0.4 to 3 µg/mL, and less than 25 % for concentrations below 0.4 µg/mL.
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Cromatografía Líquida de Alta Presión/métodos , Pruebas con Sangre Seca/métodos , Glucosilceramidas/sangre , Glicoesfingolípidos/sangre , Espectrometría de Masas en Tándem/métodos , Métodos Analíticos de la Preparación de la Muestra , Glucosilceramidas/aislamiento & purificación , Glicoesfingolípidos/aislamiento & purificación , HumanosRESUMEN
Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann-Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann-Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls.
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Enfermedad de Fabry/complicaciones , Enfermedades Renales/diagnóstico , Enfermedades Renales/etiología , Biopsia , Análisis Mutacional de ADN , Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/tratamiento farmacológico , Femenino , Humanos , Recién Nacido , Isoenzimas/uso terapéutico , Enfermedades Renales/fisiopatología , Masculino , Tamizaje Neonatal , Linaje , Proteínas Recombinantes/uso terapéutico , Diálisis Renal , Factores de Riesgo , Factores de Tiempo , Trihexosilceramidas/orina , alfa-Galactosidasa/sangre , alfa-Galactosidasa/genética , alfa-Galactosidasa/uso terapéuticoRESUMEN
Globotriaosylceramide (GL3) is a heterogeneous glycosphingolipid that is elevated in the blood plasma of patients diagnosed with Fabry disease. GL3 consists of numerous isoforms, some of which are distinctly specific to human plasma. An electrospray-ionization LC/MS/MS method has been developed that has the capacity to monitor the GL3 isoform profiles in plasma extracts. Total GL3 is extracted from human plasma via chloroform/methanol liquid-liquid extraction, purified by C(18) solid-phase extraction and analyzed by multiple reaction monitoring LC/MS/MS. The relative responses of eight selected isoforms are calculated on the basis of the total GL3 response and the isoform responses are subsequently utilized to construct isoform profile plots.
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Cromatografía Liquida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Trihexosilceramidas/sangre , Humanos , IsomerismoRESUMEN
Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.
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Leche/química , Polisacáridos/química , Polisacáridos/metabolismo , alfa-Glucosidasas/química , alfa-Glucosidasas/metabolismo , Animales , Animales Modificados Genéticamente , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Femenino , Glicosilación , Humanos , Glándulas Mamarias Animales/metabolismo , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/farmacología , Polisacáridos/aislamiento & purificación , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , alfa-Glucosidasas/genéticaRESUMEN
Fabry disease is a lysosomal storage disease arising from deficiency of the enzyme alpha-galactosidase A. Two recombinant protein therapeutics, Fabrazyme (agalsidase beta) and Replagal (agalsidase alfa), have been approved in Europe as enzyme replacement therapies for Fabry disease. Both contain the same human enzyme, alpha-galactosidase A, but they are produced using different protein expression systems and have been approved for administration at different doses. To determine if there is recognizable biochemical basis for the different doses, we performed a comparison of the two drugs, focusing on factors that are likely to influence biological activity and availability. The two drugs have similar glycosylation, both in the type and location of the oligosaccharide structures present. Differences in glycosylation were mainly limited to the levels of sialic acid and mannose-6-phosphate present, with Fabrazyme having a higher percentage of fully sialylated oligosaccharides and a higher level of phosphorylation. The higher levels of phosphorylated oligomannose residues correlated with increased binding to mannose-6-phosphate receptors and uptake into Fabry fibroblasts in vitro. Biodistribution studies in a mouse model of Fabry disease showed similar organ uptake. Likewise, antigenicity studies using antisera from Fabry patients demonstrated that both drugs were indistinguishable in terms of antibody cross-reactivity. Based on these studies and present knowledge regarding the influence of glycosylation on protein biodistribution and cellular uptake, the two protein preparations appear to be functionally indistinguishable. Therefore, the data from these studies provide no rationale for the use of these proteins at different therapeutic doses.