RESUMEN
OBJECTIVE: The Dutch Congenital hypothyroidism (CH) Newborn Screening (NBS) algorithm for thyroidal and central congenital hypothyroidism (CH-T and CH-C, respectively) is primarily based on determination of thyroxine (T4) concentrations in dried blood spots, followed by thyroid-stimulating hormone (TSH) and thyroxine-binding globulin (TBG) measurements enabling detection of both CH-T and CH-C, with a positive predictive value (PPV) of 21%. A calculated T4/TBG ratio serves as an indirect measure for free T4. The aim of this study is to investigate whether machine learning techniques can help to improve the PPV of the algorithm without missing the positive cases that should have been detected with the current algorithm. DESIGN & METHODS: NBS data and parameters of CH patients and false-positive referrals in the period 2007-2017 and of a healthy reference population were included in the study. A random forest model was trained and tested using a stratified split and improved using synthetic minority oversampling technique (SMOTE). NBS data of 4668 newborns were included, containing 458 CH-T and 82 CH-C patients, 2332 false-positive referrals and 1670 healthy newborns. RESULTS: Variables determining identification of CH were (in order of importance) TSH, T4/TBG ratio, gestational age, TBG, T4 and age at NBS sampling. In a Receiver-Operating Characteristic (ROC) analysis on the test set, current sensitivity could be maintained, while increasing the PPV to 26%. CONCLUSIONS: Machine learning techniques have the potential to improve the PPV of the Dutch CH NBS. However, improved detection of currently missed cases is only possible with new, better predictors of especially CH-C and a better registration and inclusion of these cases in future models.
Asunto(s)
Hipotiroidismo Congénito , Aprendizaje Automático , Tamizaje Neonatal , Bosques Aleatorios , Humanos , Hipotiroidismo Congénito/diagnóstico , Tiroxina/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Globulina de Unión a Tiroxina/análisis , Reacciones Falso Positivas , Algoritmos , Edad Gestacional , Recién NacidoRESUMEN
The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGß, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGß glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGß glycoforms, which allowed for the first time the detection of 33 hCGß glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.
Asunto(s)
Gonadotropina Coriónica Humana de Subunidad beta/análisis , Cromatografía Liquida/métodos , Hormonas Glicoproteicas de Subunidad alfa/análisis , Espectrometría de Masas/métodos , Nanotecnología/métodos , Animales , Células CHO , Gonadotropina Coriónica Humana de Subunidad beta/química , Cricetulus , Glicosilación , HumanosRESUMEN
In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGß), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 µm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 µL at 1 mg mL-1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.
Asunto(s)
Gonadotropina Coriónica/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Gonadotropina Coriónica/orina , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Femenino , Hormonas Glicoproteicas de Subunidad alfa/orina , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas/métodos , Embarazo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/orinaRESUMEN
Our aims were to investigate the presence of pituitary glycoprotein hormones in preterm and donor milk, and to examine the effects of Holder pasteurization and refrigeration on the levels of these hormones. We measured follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) in milk samples from mothers who delivered prematurely (n = 27) and in samples of mothers who delivered at term and donated milk to the Mother's Milk Bank of Iowa (n = 30). The gonadotropins and TSH were present in similar amounts within human milk produced for preterm and term infants. FSH increased 21% after refrigeration (p < 0.05), while LH declined by 39% (p < 0.05). Holder pasteurization decreased LH by 24% (p < 0.05) and increased TSH by 17% (p < 0.05). Holder pasteurization followed by refrigeration resulted in a 21% increase in FSH and a 41% decrease in LH (both p < 0.05), resulting in more than a 3-fold increase in donor milk FSH:LH ratios (p < 0.05 versus fresh donor milk). Despite structural similarities, the gonadotropins are differentially impacted by Holder pasteurization and refrigeration, and this results in marked alterations in the relative amount of FSH and LH that may be administered to preterm infants, potentially swinging hormonal balance towards ovarian hyperstimulation in females and hypogonadism in males.
Asunto(s)
Análisis de los Alimentos , Hormonas Glicoproteicas de Subunidad alfa/análisis , Leche Humana/química , Pasteurización , Hormonas Hipofisarias/análisis , Refrigeración , HumanosRESUMEN
Objective: To investigate the clinicopathological features of gangliocytic paraganglioma(GP). Methods: Clinical data and pathological diagnosis of the 4 cases of GP were obtained through the medical record inquiry from January 2011 to December 2017 at the First Affiliated Hospital of Zhengzhou University. Routine HE staining and immunohistochemistry of CKpan, Syn, CgA, CD56, NSE and NF were performed. Clinical follow-up of the patients was obtained through telephone communication. Results: All 4 patients, including 2 male and 2 female patients, presented with intermittent abdominal pain and distention. The median age was 56 years. Preoperative CT showed local thickening of the duodenum wall with slight enhancement in all four cases. Endoscopic ultrasonography showed low level echo in the mucous layer and submucosa involved by the tumor in 3 of 4 cases. The maximal diameter of the tumor ranged from 0.6 to 1.8 cm with an average of 1.2 cm. Microscopically, the tumors consisted of epithelioid, spindle and ganglion-like cells, and the proportion of the three cell types was different among cases. Epithelioid cells expressed CKpan, Syn, CgA and CD56. Spindle cells expressed S-100 protein and SOX-10 and ganglion-like cells expressed NF, Syn, CgA and CD56.All tumour cells expressed NSE. All 4 patients had no recurrence a post-surgery follow-up period of 3 to 30 months. Conclusions: GP of the duodenum is a benign tumor with excellent prognosis after endoscopic excision. Although its incidence is very low, its diagnosis should be considered for any mass lesion of the duodenum, especially involving mucosa and submucosa of the second dudenal segment.
Asunto(s)
Neoplasias Duodenales/química , Neoplasias Duodenales/patología , Paraganglioma/química , Paraganglioma/patología , Antígeno CD56/análisis , Proteínas Portadoras/análisis , Creatina Quinasa/análisis , Neoplasias Duodenales/diagnóstico por imagen , Femenino , Hormonas Glicoproteicas de Subunidad alfa/análisis , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Oligodesoxirribonucleótidos , Paraganglioma/diagnóstico por imagen , Pronóstico , Proteínas S100 , Sinapsinas/análisisRESUMEN
OBJECTIVE: The term "null cell" adenoma was first proposed in 1980 to designate pituitary adenomas lacking clinical, biochemical and morphological markers to disclose their cell origin. DESIGN: The aim of this study was to investigate the presence of α- and ß-gonadotropin subunits in clinically nonfunctioning pituitary tumors, which were initially immunonegative and thus diagnosed as null cell adenomas. For this reason, we reapplied immunohistochemistry using a more sensitive method comprising a tyramide signal amplification technique, combined with a polymer antibody immunohistochemical detection system. RESULTS: With this approach, all these previously negative tumors became positive for α- and ß-gonadotropin hormone subunits. CONCLUSIONS: Our results prove that so-called "null cell" adenomas produce α-SU or/and ß-FSH or ß-LH and therefore are gonadotrph adenomas in origin.
Asunto(s)
Adenoma/química , Biomarcadores de Tumor/análisis , Linaje de la Célula , Hormona Folículo Estimulante de Subunidad beta/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Hormona Luteinizante de Subunidad beta/análisis , Neoplasias Hipofisarias/química , Adenoma/clasificación , Adenoma/ultraestructura , Humanos , Inmunohistoquímica , Microscopía Electrónica , Fenotipo , Neoplasias Hipofisarias/clasificación , Neoplasias Hipofisarias/ultraestructura , Terminología como AsuntoRESUMEN
Abstract : To investigate whether schistosomiasis can contribute to appendiceal goblet cell carcinoid, appendix samples were obtained from 3 patients with combined appendiceal schistosomiasis and goblet cell carcinoid (CSG), 6 patients with goblet cell carcinoid only (GCC), 12 patients with appendiceal schistosomiasis only (ASO), and 12 cases with normal appendix (NA), all of similar gender ratio and age distributions. Hematoxylin and eosin-(H&E) stained sections were studied in 3 CSGs and 12 ASOs to diagnose schistosomiasis by detecting schistosome eggs. H&E and alcian blue/PAS-stained sections and immunohistochemistry of CgA and CEA were employed to establish the diagnosis of GCC in the 3 CSGs and 6 GCCs. Then, to determine whether schistosomiasis can contribute to GCC, immunostaining patterns of CgA and Ki67 in mucosal crypt epithelia were investigated and compared among all 33 cases. Our results revealed typical histological and immunohistochemical phenotypes of GCC in the 3 CSGs and 6 GCCs and schistosome egg deposits in 3 CSGs and 12 ASOs. We found that the expression levels of both CgA and Ki67 in mucosal crypt epithelia were significantly higher in CSG than in GCC (P < 0.05 â=â 0.013 and P â=â 0.004, respectively). Moreover, high expression levels of both CgA and Ki67 in mucosal crypt epithelia favor ASO as compared to NA (P < 0.001 â=â 3.4 × 10(-6) and 3.1 × 10(-5), respectively). Our findings suggest that appendiceal schistosomiasis was associated with increased proliferation and neuroendocrine differentiation of mucosal pluripotent crypt cells and that it may contribute to GCC, which is documented to originate from mucosal pluripotent crypt cells in mucosal crypt epithelia.
Asunto(s)
Neoplasias del Apéndice/parasitología , Tumor Carcinoide/parasitología , Esquistosomiasis mansoni/complicaciones , Neoplasias del Apéndice/diagnóstico , Neoplasias del Apéndice/cirugía , Apendicitis/diagnóstico , Apéndice/parasitología , Biomarcadores de Tumor/análisis , Tumor Carcinoide/diagnóstico , Tumor Carcinoide/cirugía , China/epidemiología , Diagnóstico Diferencial , Femenino , Hormonas Glicoproteicas de Subunidad alfa/análisis , Humanos , Antígeno Ki-67/análisis , Masculino , Persona de Mediana Edad , Prevalencia , Esquistosomiasis mansoni/diagnóstico , Esquistosomiasis mansoni/epidemiologíaRESUMEN
PURPOSE: Assessment of first-trimester combined screening for trisomy 18 and 13 with the combined use of the risk algorithms for trisomy 21, 18 and 13. MATERIALS AND METHODS: First-trimester combined screening based on maternal and gestational age, fetal NT, PAPP-A and free ß-hCG was assessed in 39â,004 pregnancies. Patient-specific risks for trisomy 21, 18, 13 were computed based on the current FMF London algorithm. RESULTS: The study population consisted of 38â,751 singleton pregnancies including 39 cases with trisomy 18 or 13.âIn the aneuploid group, median delta NT was 0.72âmm, PAPP-A was 0.21 MoM and free ß-hCG was 0.33 MoM. Although only 41â% of the NT measurements of fetuses with trisomy 18 or 13 were above the 95th percentile, the detection rates for trisomy 18 or 13 were 82â% with the trisomy 18/13 algorithm and 56.4â% with the trisomy 21 algorithm. The respective false-positive rates were 0.7â% and 4.7â%. The combination of the trisomy 18/13 and the trisomy 21 algorithm with the same cut-offs led to a detection rate of 94.9â% at an overall false-positive rate of 5.0â%. CONCLUSION: Despite a substantial underestimation of the fetal NT, the combined use of the trisomy 18/13 and the trisomy 21 algorithm of the FMF London leads to a detection rate for trisomy 18/13 of about 95â% for a false-positive rate of 5.0â%.
Asunto(s)
Anomalías Múltiples/diagnóstico , Algoritmos , Trastornos de los Cromosomas/diagnóstico , Síndrome de Down/diagnóstico , Primer Trimestre del Embarazo , Diagnóstico Prenatal/métodos , Trisomía/diagnóstico , Anomalías Múltiples/genética , Adulto , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 18/genética , Síndrome de Down/genética , Femenino , Alemania , Edad Gestacional , Hormonas Glicoproteicas de Subunidad alfa/análisis , Humanos , Recién Nacido , Edad Materna , Medida de Translucencia Nucal , Valor Predictivo de las Pruebas , Embarazo , Proteína Plasmática A Asociada al Embarazo/análisis , Trisomía/genética , Síndrome de la Trisomía 13 , Ultrasonografía PrenatalRESUMEN
Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCGAsunto(s)
Gonadotropina Coriónica/análisis
, Cromatografía Líquida de Alta Presión/métodos
, Cromatografía de Fase Inversa/métodos
, Hormona Luteinizante/análisis
, Preparaciones Farmacéuticas/análisis
, Gonadotropina Coriónica Humana de Subunidad beta/análisis
, Hormonas Glicoproteicas de Subunidad alfa/análisis
, Humanos
, Proteínas Recombinantes/análisis
RESUMEN
With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.
Asunto(s)
Electroforesis Capilar/métodos , Hormonas Glicoproteicas de Subunidad alfa/análisis , Espectrometría de Masas/métodos , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Análisis de Fourier , Glicopéptidos/análisis , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Humanos , Ratones , Polisacáridos/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMEN
OBJECTIVE: To understand the properties of each available gonadotropin preparation, especially in terms of the differences between urinary-derived and recombinant preparations. STUDY DESIGN: Human menopausal gonadotropin (hMG), highly purified urinary-derived follicle-stimulating hormone (uFSH-HP) and recombinant FSH (rFSH) were subjected to 2-dimensional gel electrophoresis (2-DE), and protein spots were visualized by silver-staining procedures. Major spots were analyzed by mass spectrometry. Fluorescent-labeled preparations were also subjected to 2-DE to evaluate the quantities of FSH isohormones contained in each preparation. RESULTS: 2-DE and mass spectrometry analyses of hMG identified many extracellular proteins as major impurities and several plasma membrane proteins including prion proteins. Both uFSH-HP and rFSH demonstrated slight impurities and showed several alpha and beta subunit isohormones. rFSH contained higher amounts of the basic isohormones of the alpha subunit than uFSH-HP, whereas the predominance of the basic isohormones was less significant in the beta subunit. CONCLUSION: Proteomic analyses demonstrated the detailed protein profiles of each preparation. Differences in the quantities of alpha subunit isohormones may contribute to the variations in FSH activity observed between recombinant and urinary-derived FSH preparations.
Asunto(s)
Hormona Folículo Estimulante de Subunidad beta/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Menotropinas/química , Urofolitropina/química , Electroforesis en Gel Bidimensional , Femenino , Humanos , Espectrometría de Masas , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: DNA aptamers are single-stranded nucleotide sequences that bind specifically to target molecules. By combining the advantages of PCR for amplifying specific DNA sequences and aptamer technology, we have developed a new strategy to detect target molecules such as proteins. METHODS: Ovine follicle-stimulating hormone alpha subunit (oFSHalpha) was used as the model protein to generate a specific DNA aptamer via an in vitro evolutionary process. A targeted regional-mapping approach and a target-capturing assay were used to identify the binding region on the aptamer molecule. In the detection assay, referred to as "aptamer-based regionally protected PCR" (ARP-PCR), the aptamer was allowed to bind to the target protein in solution before digestion with DNase I. The region of the aptamer bound to the target was protected from DNase I cleavage. The target-binding region of the aptamer protected from the enzymatic treatment was then amplified by the PCR. RESULTS: Aptamers against oFSHalpha were generated. Six sequences of 20 selected aptamer clones were identical. This aptamer sequence was divided into 4 regions according to the aptamer's secondary structure. From examination of the target-binding ability of each region, we determined the specific binding region, for which primers were designed. With the aptamer and primers to detect oFSHalpha by means of the ARP-PCR method, we were able to detect the target protein at concentrations as low as 10(-14) mol/L. CONCLUSIONS: Combining the use of a DNA aptamer with the PCR is a potentially useful analytic tool for detection of proteins at low concentrations. .
Asunto(s)
Aptámeros de Nucleótidos/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Reacción en Cadena de la Polimerasa/métodos , Animales , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Hormonas Glicoproteicas de Subunidad alfa/química , Hormonas Glicoproteicas de Subunidad alfa/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , OvinosRESUMEN
Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSHbeta (AsFSHbeta) and LHbeta (AsLHbeta) subunit cDNAs are 345, 363, 387 and 414bp in length, and encode mature peptides of 115, 121, 129 and 138aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSHbeta and AsLHbeta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSHbeta and AsLHbeta) in mature sturgeon pituitaries, but only AsFSHbeta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSHbeta was expressed in 4-year-old individuals, whereas in females, AsFSHbeta was just expressed in 5-year-old individuals.
Asunto(s)
Gonadotropinas/análisis , Hipófisis/química , Factores de Edad , Animales , Clonación Molecular , ADN Complementario , Femenino , Peces , Hormona Folículo Estimulante/análisis , Hormona Folículo Estimulante/genética , Hormonas Glicoproteicas de Subunidad alfa/análisis , Hormonas Glicoproteicas de Subunidad alfa/genética , Gonadotropinas/genética , Hormona Luteinizante/análisis , Hormona Luteinizante/genética , Masculino , Factores Sexuales , Distribución TisularRESUMEN
To identify genes that are most responsive to a sustained activation of a G(s) protein-coupled receptor, HEK293 cells were stably transfected with the beta(2)-adrenergic receptor and stimulated with agonist isoproterenol (1 mum). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common alpha-subunit of human glycoprotein hormones (GPHalpha). Induction of GPHalpha transcription in response to cAMP elevations resulted in a dramatic increase (600-fold) of protein secretion as shown by RT-PCR and a highly specific time-resolved immunofluorometric assay. Cloning and sequencing of the GPHalpha cDNA and mass spectrometric analysis of HPLC-purified GPHalpha derived from serum-free HEK293-beta(2)-adrenergic receptor-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent Western blots revealed that this was a large hyperglycosylated form of GPHalpha that had not been associated with a beta-subunit previously. This uncombined variant is known to be either cosecreted with GPHs from the pituitary, the placenta, and a variety of tumors or secreted without GPHs from APUD cells and rare tumors. Moreover, it is similar to GPHalpha found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G protein-coupled receptors in HEK293 cells, the expression of large GPHalpha was controlled by G(s)- and G(q)- but not G(i)-dependent receptors and mediated via cAMP and Ca(++) release. This suggests that Gq- or G(s)-coupled receptors other than the classical GnRH receptor may play a role in the regulation of nonpituitary, nonplacental GPHalpha secretion under physiological and pathological conditions.
Asunto(s)
Hormonas Glicoproteicas de Subunidad alfa/genética , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Riñón/metabolismo , Transcripción Genética , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/análisis , Humanos , Isoproterenol/farmacología , Riñón/citología , Riñón/embriología , Datos de Secuencia Molecular , Receptores Adrenérgicos beta 2/metabolismo , Receptores LHRH/metabolismo , TransfecciónRESUMEN
Follicle stimulating hormone (FSH) is secreted from the pituitary gland to regulate reproduction in vertebrates. FSH signals through a G-protein coupled receptor (FSHR) on the target cell surface. We describe here the strategy to produce a soluble FSH-FSHR complex that involves the co-secretion of a truncated FSHR ectodomain (FSHR(HB)) and a covalently linked FSHalphabeta heterodimer from baculovirus-infected insect cells. FSH binds to FSHR(HB) with a high affinity comparable to that for the full-length receptor. The crystal structure of the FSH-FSHR(HB) complex provides explanations for the high affinity and specificity of FSH interaction with FSHR, and it shows an unexpected dimerization of these complexes. Here we also compare the crystal structure with theoretical models of the FSH-FSHR-binding mode. We conclude that the FSH-FSHR(HB) structure gives an authentic representation of FSH binding to intact FSHR.
Asunto(s)
Hormona Folículo Estimulante de Subunidad beta/química , Hormona Folículo Estimulante de Subunidad beta/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/química , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Receptores de HFE/química , Receptores de HFE/metabolismo , Animales , Baculoviridae , Gonadotropina Coriónica/química , Cromatografía en Gel , Cristalización , Dimerización , Hormona Folículo Estimulante de Subunidad beta/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Glicosilación , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de HFE/análisis , Solubilidad , Relación Estructura-ActividadRESUMEN
The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the alpha and beta subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGalphabeta) was used to investigate roles of critical residues of the alpha subunit in hormone receptor interaction and biological activity. The alpha subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type beta subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGalpha P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGalpha T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the alpha subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.
Asunto(s)
Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Mutación/genética , Anticuerpos Monoclonales , Gonadotropina Coriónica Humana de Subunidad beta/análisis , Fermentación , Hormonas Glicoproteicas de Subunidad alfa/análisis , Hormonas Glicoproteicas de Subunidad alfa/genética , Humanos , Sueros Inmunes , Proteínas Mutantes/aislamiento & purificación , Radioinmunoensayo , Receptores de Gonadotropina/metabolismo , Relación Estructura-ActividadRESUMEN
The revolutionary development of biotechnology-derived therapeutic proteins has provided the expected improvements in quality, purity and consistency, as demonstrated in recombinant human FSH (rhFSH). However, the development of urine-derived gonadotrophins has not always shown comparable improvements. More recently, highly purified urine-derived FSH (uFSH-HP) products have become widely available. The relative purity, level of urine-derived contaminants, and consistency of one such highly purified human uFSH (uhFSH) (urofollitropin) has been assessed and directly compared with rhFSH (follitropin alpha). It has been demonstrated that the highly purified urofollitropin contains variable levels of urine-derived contaminant proteins and demonstrates a variable level of FSH purity, FSH isoforms, and delivered dose. These variable factors may contribute to the control of ovarian stimulation. The relative purity, variable consistency and the presence of contaminants indicates that the urofollitropin is, at best, a partially purified uFSH that is not able to meet the quality attributes of follitropin alpha (rhFSH).
Asunto(s)
Fármacos para la Fertilidad Femenina/normas , Hormona Folículo Estimulante/normas , Hormonas Glicoproteicas de Subunidad alfa/normas , Urofolitropina/normas , Western Blotting , Cromatografía Líquida de Alta Presión , Densitometría , Contaminación de Medicamentos , Electroforesis en Gel de Poliacrilamida , Femenino , Fármacos para la Fertilidad Femenina/análisis , Hormona Folículo Estimulante/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Humanos , Inmunohistoquímica , Isoformas de Proteínas/análisis , Control de Calidad , Proteínas Recombinantes/normas , Urofolitropina/análisis , Urofolitropina/químicaRESUMEN
The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.
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Gatos/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Tirotropina/biosíntesis , Tirotropina/aislamiento & purificación , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva , Western Blotting/veterinaria , Células CHO , Cromatografía de Afinidad/veterinaria , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Hormonas Glicoproteicas de Subunidad alfa/biosíntesis , Hormonas Glicoproteicas de Subunidad alfa/genética , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Humanos , Inmunoensayo/métodos , Proteínas Recombinantes/genética , Tirotropina/genética , TransfecciónRESUMEN
The use of gonadotrophins for the treatment of infertility began in the 1930s following early work on the pituitary-ovarian axis and the discovery of FSH and LH. The technological development of pharmaceutical gonadotrophins over the last 40 years has shown improvements in specific activity, purity, degradation and impurities. Throughout these pharmaceutical developments the gonadotrophin content of both urinary and recombinant preparations has been assessed using an animal in-vivo bioassay. This paper reflects upon the manufacturing history of recombinant human FSH (r-hFSH) and follitropin alfa filled by mass (FbM), and evaluates the impact of introducing a pharmaceutical product that is formulated and assayed by a physicochemical method for r-hFSH protein content. It also compares the analytical consistency of follitropin alfa FbM with another commercially available r-hFSH, follitropin beta.
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Hormona Folículo Estimulante/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Infertilidad Femenina/tratamiento farmacológico , Bioensayo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Hormona Folículo Estimulante/historia , Hormona Folículo Estimulante/uso terapéutico , Hormona Folículo Estimulante de Subunidad beta/análisis , Hormonas Glicoproteicas de Subunidad alfa/historia , Hormonas Glicoproteicas de Subunidad alfa/uso terapéutico , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Polisacáridos/metabolismo , Isoformas de Proteínas/análisis , Control de CalidadRESUMEN
A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSHbeta and LHbeta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSHbeta and LHbeta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSHbeta and LHbeta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSHbeta and LHbeta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSHbeta and LHbeta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSHbeta cells mainly distributed in the middle area of PPD, while the LHbeta cells distributed more widely, including in the area similar to the FSHbeta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSHbeta and LHbeta cells. Double immunofluoresence localization further demonstrated FSHbeta and LHbeta expression in distinct cells in the PPD area, although the FSHbeta and LHbeta cells were detected in the identical area of PPD.