RESUMEN
The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100-120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis.
Asunto(s)
Antígenos de Neoplasias/química , Leucosialina/química , Acetilgalactosamina/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Electroforesis en Gel Bidimensional , Epítopos , Femenino , Glicosilación , Humanos , Leucosialina/genética , Leucosialina/inmunología , Leucosialina/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Interferencia de ARN , Espectrometría de Masas en TándemRESUMEN
Permanent neonatal diabetes mellitus (PNDM) is a rare disorder usually presenting within 6 months of birth. Although several genes have been linked to this disorder, in almost half the cases documented in Italy, the genetic cause remains unknown. Because the Akita mouse bearing a mutation in the Ins2 gene exhibits PNDM associated with pancreatic beta cell apoptosis, we sequenced the human insulin gene in PNDM subjects with unidentified mutations. We discovered 7 heterozygous mutations in 10 unrelated probands. In 8 of these patients, insulin secretion was detectable at diabetes onset, but rapidly declined over time. When these mutant proinsulins were expressed in HEK293 cells, we observed defects in insulin protein folding and secretion. In these experiments, expression of the mutant proinsulins was also associated with increased Grp78 protein expression and XBP1 mRNA splicing, 2 markers of endoplasmic reticulum stress, and with increased apoptosis. Similarly transfected INS-1E insulinoma cells had diminished viability compared with those expressing WT proinsulin. In conclusion, we find that mutations in the insulin gene that promote proinsulin misfolding may cause PNDM.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Diabetes Mellitus/genética , Proteínas de Choque Térmico/biosíntesis , Insulina/genética , Insulina/fisiología , Chaperonas Moleculares/biosíntesis , Mutación , Proteínas Nucleares/biosíntesis , Proinsulina/biosíntesis , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Chaperón BiP del Retículo Endoplásmico , Femenino , Heterocigoto , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Linaje , Factores de Transcripción del Factor Regulador X , Factores de Transcripción , Proteína 1 de Unión a la X-BoxRESUMEN
Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.
Asunto(s)
Diabetes Mellitus/genética , Mutación , Canales de Potasio de Rectificación Interna/genética , Análisis Mutacional de ADN , Diabetes Mellitus Tipo 1/genética , Femenino , Humanos , Lactante , Recién Nacido , Italia , MasculinoRESUMEN
In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m(2), and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening for mutations in candidate genes revealed a heterozygous glucokinase mutation in exon 10, substituting valine for alanine at codon 456 (A456V) in the proband and his mother. The purified recombinant glutathionyl S-transferase fusion protein of the A456V glucokinase revealed a decreased glucose S(0.5) (the concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k(cat) was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol/l. The theoretical and practical implications are manifold and significant.
Asunto(s)
Diabetes Mellitus/genética , Glucoquinasa/genética , Glucosa/metabolismo , Mutación Missense , Adolescente , Regulación Alostérica , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Diazóxido/uso terapéutico , Activación Enzimática , Glutatión Transferasa/genética , Homeostasis , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Cinética , Masculino , FosforilaciónRESUMEN
Transglutaminase 2 (TGase 2) is a Ca+2-dependent enzyme that catalyzes both intracellular and extracellular cross-linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane-mediated events required for glucose-stimulated insulin release from the pancreatic beta cells. Here we show that targeted disruption of TGase 2 impairs glucose-stimulated insulin secretion. TGase 2-/- mice show glucose intolerance after intraperitoneal glucose loading. TGase 2-/- mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS-2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2-/- mouse phenotype resembles that of the maturity-onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.
Asunto(s)
Glucemia/análisis , Diabetes Mellitus Tipo 2/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Mutación Missense , Transglutaminasas/genética , Transglutaminasas/fisiología , Adolescente , Secuencia de Aminoácidos , Animales , Células Cultivadas , Niño , Proteínas de Unión al GTP/química , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Islotes Pancreáticos/enzimología , Cinética , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Fosforilación , Proteína Glutamina Gamma Glutamiltransferasa 2 , Receptor de Insulina/metabolismo , Homología de Secuencia de Aminoácido , Transglutaminasas/químicaRESUMEN
BACKGROUND: Mutations in the hepatocyte nuclear factor (HNF)-1beta gene (TCF2) are responsible for a syndrome characterized by maturity-onset diabetes of the young, a nondiabetic renal disease, genital malformations, and liver dysfunction. METHODS: The HNF-1beta gene was screened for mutations in four members of an Italian family with early-onset, nonketotic diabetes or a familiar, nondiabetic renal disease and nonprogressive liver disorder. RESULTS: The genetic analysis revealed an already described nonsense mutation in codon 177 of HNF-1beta gene (R177X) in the four related subjects. Clinical features included diabetes in three of four patients, monolateral renal hypoplasia with cysts in the controlateral kidney in two patients, and bilaterally small hyperechoic kidneys without cysts in the other two patients. Renal function impairment was severe in one patient, requiring dialysis treatment, and mild in three. Three patients had nonprogressive liver dysfunction, with long-lasting enzyme alterations but no liver insufficiency or jaundice. CONCLUSION: HNF-1beta gene mutations are associated with a wide variability in severity and pattern of clinical symptoms within the same kindred regarding diabetes and renal impairment. Moderate liver dysfunction may be a so far overlooked component of the syndrome.
Asunto(s)
Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/genética , Enfermedades Renales Quísticas/genética , Hepatopatías/genética , Factores de Transcripción/genética , Adulto , Codón/genética , Femenino , Genitales/anomalías , Factor Nuclear 1-beta del Hepatocito , Humanos , Hepatopatías/fisiopatología , Masculino , Persona de Mediana Edad , Mutación Missense/genética , Linaje , SíndromeRESUMEN
Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously we reported that the role of TG2 in insulin secretion may involve cytoplasmic actin remodeling and a regulative action on other proteins during granule movement. The aim of this study was to gain a better insight into the role of TG2 transamidating activity in mitochondria and in the nucleus of INS-1E rat insulinoma cell line (INS-1E) during insulin secretion. To this end we labeled INS-1E with an artificial donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8min. Biotinylated proteins of the nuclear/mitochondrial-enriched fraction were analyzed using two-dimensional electrophoresis and mass spectrometry. Many mitochondrial proteins involved in Ca(2+) homeostasis (e.g. voltage-dependent anion-selective channel protein, prohibitin and different ATP synthase subunits) and many nuclear proteins involved in gene regulation (e.g. histone H3, barrier to autointegration factor and various heterogeneous nuclear ribonucleoprotein) were identified among a number of transamidating substrates of TG2 in INS-1E. The combined results provide evidence that a temporal link exists between glucose-stimulation, first phase insulin secretion and the action of TG on histone H3 both in INS-1E and human pancreatic islets. BIOLOGICAL SIGNIFICANCE: Research into the role of transglutaminase 2 during insulin secretion in INS-1E rat insulinoma cellular model is depicting a complex role for this enzyme. Transglutaminase 2 acts in the different INS-1E compartments in the same way: catalyzing a post-translational modification event of its substrates. In this work we identify some mitochondrial and nuclear substrates of INS-1E during first phase insulin secretion. The finding that TG2 interacts with nuclear proteins that include BAF and histone H3 immediately after (2-5min) glucose stimulus of INS-1E suggests that TG2 may be involved not only in insulin secretion, as suggested by our previous studies in cytoplasmic INS-1E fraction, but also in the regulation of glucose-induced gene transcription.
Asunto(s)
Núcleo Celular/enzimología , Células Secretoras de Insulina/enzimología , Transglutaminasas/metabolismo , Animales , Calcio/metabolismo , Línea Celular Tumoral , Proteínas de Unión al GTP , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ratas , Edulcorantes/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Transglutaminasas/genéticaRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease characterized by the presence of circulating autoantibodies directed against proteins of islet beta-cell. Autoantibody testing is used for diagnostic purposes; however, up to 2-5% of patients who are clinically diagnosed with T1D are found negative for known antibodies, suggesting that the T1D autoantigen panel is incomplete. With the aim of identifying new T1D autoantigen(s), we used sera from subjects clinically diagnosed with T1D, but who tested negative for the four T1D autoantibodies currently used in clinical practice and for genes responsible for sporadic cases of diabetes. Sera from these patients were challenged by Western blot against the proteome from human pancreatic beta-cells resolved by 2DE. Eleven proteins were identified by MS. A radiobinding assay (RBA) was developed to test the reactivity to Rab GDP dissociation inhibitor beta (GDIß) of T1D sera using an independent method. Depending on the construct used (open reading frame or COOH-terminus) 22% to 32% of fifty T1D sera showed increased binding to GDIß by RBA. In addition, 15% of patients with celiac disease had raised binding to the COOH-terminus GDIß. These results indicate that immunoproteomics is a feasible strategy for the identification of candidate T1D autoantigens. BIOLOGICAL SIGNIFICANCE: Several approaches have been previously used to look for new type 1 diabetes autoantigens. With the present work we show that carefully selected sera from rare patients with diabetes both negative for the 5 autoantibodies currently used in clinical practice and for genes responsible for sporadic cases of diabetes, may be exploited in experiments utilizing human pancreatic islets extracts as a target for SERPA to identify novel candidate T1D autoantigens.
Asunto(s)
Autoantígenos/sangre , Diabetes Mellitus Tipo 1/sangre , Células Secretoras de Insulina/metabolismo , Proteoma/metabolismo , Adolescente , Autoantígenos/inmunología , Células Cultivadas , Niño , Preescolar , Diabetes Mellitus Tipo 1/inmunología , Femenino , Humanos , Lactante , Células Secretoras de Insulina/patología , Masculino , Proteoma/inmunología , Suero/inmunología , Suero/metabolismoRESUMEN
Biallelic insulin receptor (INSR) gene mutations cause congenital syndromes of severe insulin resistance (SIR) known as Donohue syndrome (DS) and Rabson-Mendenhall syndrome (RMS). At presentation, DS and RMS are difficult to differentiate since they share many clinical features; however, while patients with DS usually die within 1 year of birth, individuals classified as RMS can reach adult age. INSR mutations can be also found in pubertal females with hyperinsulinism, hyperandrogenism, and acanthosis nigricans (type A SIR). We studied the INSR gene in five subjects with congenital SIR and in a patient with type A SIR. Nine biallelic INSR gene mutations (eight novels, including an in-frame deletion of INSR signal peptide) were identified in patients with congenital SIR; a heterozygous, spontaneous INSR mutation was detected in the patient with type A SIR. Two probands, presenting severe hirsutism at birth, died at the age of 3 months and were classified as DS, while other 2, currently 2 and 3 years old, were diagnosed with RMS (patients 3 and 4). The fifth patient with congenital SIR died when 14 months old. Nephrocalcinosis, hyperaldosteronism, hyperreninemia, and hypokalemia, in the absence of hypertension, were discovered in patients 3 and 5 when 24 and 4 months old, respectively. Patient 3, now 3 years/3 months old, still shows hyperreninemic hyperaldosteronism requiring potassium supplementation. We conclude that renal abnormalities resembling antenatal Bartter's syndrome type II, recently reported also by others, is a common observation in patients with congenital SIR.
Asunto(s)
Síndrome de Bartter/genética , Síndrome de Donohue/genética , Resistencia a la Insulina/genética , Mutación , Receptor de Insulina/genética , Acantosis Nigricans/complicaciones , Acantosis Nigricans/diagnóstico , Acantosis Nigricans/genética , Adolescente , Síndrome de Bartter/diagnóstico , Preescolar , Síndrome de Donohue/diagnóstico , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Nefrocalcinosis/complicaciones , Nefrocalcinosis/diagnóstico , Nefrocalcinosis/genética , Índice de Severidad de la EnfermedadRESUMEN
Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously, we reported that tgm2 -/- mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2's transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca(2+)] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2's role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Transglutaminasas/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Unión al GTP/química , Glucosa/metabolismo , Humanos , Secreción de Insulina , Células Secretoras de Insulina/química , Espectrometría de Masas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ratas , Especificidad por Sustrato , Transglutaminasas/químicaRESUMEN
OBJECTIVE: Heterozygous, gain-of-function mutations of the insulin gene can cause permanent diabetes with onset ranging from the neonatal period through adulthood. The aim of our study was to screen for the insulin gene in patients who had been clinically classified as type 1 diabetic but who tested negative for type 1 diabetes autoantibodies. RESEARCH DESIGN AND METHODS: We reviewed the clinical records of 326 patients with the diagnosis of type 1 diabetes and identified seven probands who had diabetes in isolation and were negative for five type 1 diabetes autoantibodies. We sequenced the INS gene in these seven patients. RESULTS: In two patients whose diabetes onset had been at 2 years 10 months of age and at 6 years 8 months of age, respectively, we identified the mutation G(B8)S and a novel mutation in the preproinsulin signal peptide (A(Signal23)S). CONCLUSIONS: Insulin gene mutations are rare in absolute terms in patients classified as type 1 diabetic (0.6%) but can be identified after a thorough screening of type 1 diabetes autoantibodies.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Insulina/genética , Mutación , Adolescente , Edad de Inicio , Niño , Preescolar , Diabetes Mellitus Tipo 1/sangre , Tamización de Portadores Genéticos , Humanos , Lactante , Anticuerpos Insulínicos/sangre , Precursores de Proteínas/genética , Estudios RetrospectivosAsunto(s)
Diabetes Mellitus/epidemiología , Retinopatía Diabética/epidemiología , Vitreorretinopatía Proliferativa/epidemiología , Adolescente , Adulto , Diabetes Mellitus/tratamiento farmacológico , Femenino , Estudios de Seguimiento , Gliburida/uso terapéutico , Humanos , Lactante , Recién Nacido , Insulina/uso terapéutico , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: To investigate the prevalence of maturity-onset diabetes of the young (MODY) in Italian children with incidental hyperglycemia. RESEARCH DESIGN AND METHODS: Among 748 subjects age 1-18 years with incidental hyperglycemia, minimal diagnostic criteria for MODY were met by 172 families. Mutational analyses of the glucokinase (GCK) and hepatocyte nuclear factor 1alpha (HNF1A) genes were performed. RESULTS: We identified 85 GCK gene mutations in 109 probands and 10 HNF1A mutations in 12 probands. In GCK patients, the median neonatal weight and age at the first evaluation were lower than those found in patients with HNF1A mutations. Median fasting plasma glucose and impaired fasting glucose/impaired glucose tolerance frequency after oral glucose tolerance testing were higher in GCK patients, who also showed a lower frequency of diabetes than HNF1A patients. CONCLUSIONS: GCK mutations are the prevailing cause of MODY (63.4%) when the index case is recruited in Italian children with incidental hyperglycemia.
Asunto(s)
Diabetes Mellitus Tipo 2/epidemiología , Hiperglucemia/complicaciones , Adolescente , Niño , Preescolar , Diabetes Mellitus Tipo 2/genética , Femenino , Glucoquinasa/genética , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Hiperglucemia/genética , Lactante , Italia , Masculino , MutaciónRESUMEN
Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.