RESUMEN
The Aristaless-related homeobox gene (ARX) is indispensable for interneuron development. Patients with ARX polyalanine expansion mutations of the first two tracts (namely PA1 and PA2) suffer from intellectual disability of varying severity, with seizures a frequent comorbidity. The impact of PA1 and PA2 mutations on the brain development is unknown, hindering the search for therapeutic interventions. Here, we characterized the disturbances to cortical interneuron development in mice modeling the two most common ARX polyalanine expansion mutations in human. We found a consistent â¼40-50% reduction of calbindin-positive interneurons, but not Stt+ or Cr+ interneurons, within the cortex of newborn hemizygous mice (p=0.024) for both mutant strains compared to wildtype (p=0.011). We demonstrate that this was a consequence of calbindin precursor cells being arrested or delayed at the ventral subpallium en route of tangential migration. Ex-vivo assay validated this migration deficit in PA1 cells (p=0.0002) suggesting that the defect is contributed by intrinsic loss of Arx function within migrating cells. Both humans and mice with PA1 mutations present with severe clinical features, including intellectual disability and infantile spasms. Our data further demonstrated the pathogenic mechanism was robustly shared between PA1 and PA2 mutations, as previously reported including Arx protein reduction and overlapping transcriptome profiles within the developing mouse brains. Data from our study demonstrated that cortical calbindin interneuron development and migration is negatively affected by ARX polyalanine expansion mutations. Understanding the cellular pathogenesis contributing to disease manifestation is necessary to screen efficacy of potential therapeutic interventions.
Asunto(s)
Movimiento Celular/fisiología , Corteza Cerebral/crecimiento & desarrollo , Neuronas GABAérgicas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Interneuronas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Calbindinas/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Neuronas GABAérgicas/patología , Humanos , Interneuronas/patología , Mutación con Pérdida de Función , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Péptidos/genéticaRESUMEN
There is considerable genetic and phenotypic heterogeneity associated with intellectual disability (ID), specific learning disabilities, attention-deficit hyperactivity disorder, autism and epilepsy. The intelligence quotient (IQ) motif and SEC7 domain containing protein 2 gene (IQSEC2) is located on the X-chromosome and harbors mutations that contribute to non-syndromic ID with and without early-onset seizure phenotypes in both sexes. Although IQ and Sec7 domain mutations lead to partial loss of IQSEC2 enzymatic activity, the in vivo pathogenesis resulting from these mutations is not known. Here we reveal that IQSEC2 has a key role in dendritic spine morphology. Partial loss-of-function mutations were modeled using a lentiviral short hairpin RNA (shRNA) approach, which achieved a 57% knockdown of Iqsec2 expression in primary hippocampal cell cultures from mice. Investigating gross morphological parameters after 8 days of in vitro culture (8DIV) identified a 32% reduction in primary axon length, in contrast to a 27% and 31% increase in the number and complexity of dendrites protruding from the cell body, respectively. This increase in dendritic complexity and spread was carried through dendritic spine development, with a 34% increase in the number of protrusions per dendritic segment compared with controls at 15DIV. Although the number of dendritic spines had normalized by 21DIV, a reduction was noted in the number of immature spines. In contrast, when modeling increased dosage, overexpression of wild-type IQSEC2 led to neurons with shorter axons that were more compact and displayed simpler dendritic branching. Disturbances to dendritic morphology due to knockdown of Iqsec2 were recapitulated in neurons from Iqsec2 knockout mice generated in our laboratory using CRISPR/Cas9 technology. These observations provide evidence of dosage sensitivity for IQSEC2, which normally escapes X-inactivation in females, and links these disturbances in expression to alterations in the morphology of developing neurons.
Asunto(s)
Espinas Dendríticas/patología , Epilepsia/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Discapacidad Intelectual/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Dendritas/patología , Espinas Dendríticas/metabolismo , Femenino , Hipocampo/metabolismo , Discapacidad Intelectual/patología , Masculino , Ratones , Ratones Noqueados , Mutación , Neurogénesis/fisiología , Neuronas/patología , Fenotipo , ARN Interferente Pequeño/genéticaRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway was originally discovered by virtue of its ability to rapidly degrade aberrant mRNAs with premature termination codons. More recently, it was shown that NMD also directly regulates subsets of normal transcripts, suggesting that NMD has roles in normal biological processes. Indeed, several NMD factors have been shown to regulate neurological events (for example, neurogenesis and synaptic plasticity) in numerous vertebrate species. In man, mutations in the NMD factor gene UPF3B, which disrupts a branch of the NMD pathway, cause various forms of intellectual disability (ID). Using Epstein Barr virus-immortalized B cells, also known as lymphoblastoid cell lines (LCLs), from ID patients that have loss-of-function mutations in UPF3B, we investigated the genome-wide consequences of compromised NMD and the role of NMD in neuronal development and function. We found that ~5% of the human transcriptome is impacted in UPF3B patients. The UPF3B paralog, UPF3A, is stabilized in all UPF3B patients, and partially compensates for the loss of UPF3B function. Interestingly, UPF3A protein, but not mRNA, was stabilised in a quantitative manner that inversely correlated with the severity of patients' phenotype. This suggested that the ability to stabilize the UPF3A protein is a crucial modifier of the neurological symptoms due to loss of UPF3B. We also identified ARHGAP24, which encodes a GTPase-activating protein, as a canonical target of NMD, and we provide evidence that deregulation of this gene inhibits axon and dendrite outgrowth and branching. Our results demonstrate that the UPF3B-dependent NMD pathway is a major regulator of the transcriptome and that its targets have important roles in neuronal cells.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Discapacidad Intelectual/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Proteínas de Unión al ARN/genética , Encéfalo/crecimiento & desarrollo , Línea Celular , Línea Celular Transformada , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Expresión Génica/genética , Hipocampo/anatomía & histología , Hipocampo/crecimiento & desarrollo , Humanos , Mutación , Neuronas/citología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genéticaRESUMEN
ARX mutations cause a diverse spectrum of human disorders, ranging from severe brain and genital malformations to non-syndromic intellectual disability (ID). ARX is a transcription factor with multiple domains that include four polyalanine (pA) tracts, the first two of which are frequently expanded by mutations. We progressively screened DNA samples from 613 individuals with ID initially for the most frequent ARX mutations (c.304ins(GCG)(7)'expansion' of pA1 and c.429_452dup 'dup24bp' of pA2). Five hundred samples without pA1 or pA2 mutations had the entire ARX ORF screened by single stranded polymorphism conformation (SSCP) and/or denaturing high pressure liquid chromatography (dHPLC) analysis. Overall, eight families with six mutations in ARX were identified (1.31%): five duplication mutations in pA2 (0.82%) with three new clinical reports of families with the dup24bp and two duplications larger than the dup24bp mutation discovered (dup27bp, dup33bp); and three point mutations (0.6%), including one novel mutation in the homeodomain (c.1074G>T). Four ultraconserved regions distal to ARX (uc466-469) were also screened in a subset of 94 patients, with three unique nucleotide changes identified in two (uc466, uc467). The subcellular localization of full length ARX proteins was assessed for 11 variants. Protein mislocalization increased as a function of pA2 tract length and phenotypic severity, as has been previously suggested for pA1. Similarly, protein mislocalization of the homeodomain mutations also correlated with clinical severity, suggesting an emerging genotype vs cellular phenotype correlation.
Asunto(s)
Discapacidades del Desarrollo/genética , Pruebas Genéticas/métodos , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Trastorno Autístico/diagnóstico , Trastorno Autístico/genética , Secuencia de Bases , Niño , Preescolar , Duplicación Cromosómica , Estudios de Cohortes , Secuencia Conservada , Discapacidades del Desarrollo/diagnóstico , Femenino , Estudios de Asociación Genética , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Lactante , Masculino , Mutación , Tasa de Mutación , Linaje , Polimorfismo Conformacional Retorcido-Simple , Factores de Transcripción/metabolismoRESUMEN
Mutations in the UPF3B gene, which encodes a protein involved in nonsense-mediated mRNA decay, have recently been described in four families with specific (Lujan-Fryns and FG syndromes), nonspecific X-linked mental retardation (XLMR) and autism. To further elucidate the contribution of UPF3B to mental retardation (MR), we screened its coding sequence in 397 families collected by the EuroMRX consortium. We identified one nonsense mutation, c.1081C>T/p.Arg361(*), in a family with nonspecific MR (MRX62) and two amino-acid substitutions in two other, unrelated families with MR and/or autism (c.1136G>A/p.Arg379His and c.1103G>A/p.Arg368Gln). Functional studies using lymphoblastoid cell lines from affected patients revealed that c.1081C>T mutation resulted in UPF3B mRNA degradation and consequent absence of the UPF3B protein. We also studied the subcellular localization of the wild-type and mutated UPF3B proteins in mouse primary hippocampal neurons. We did not detect any obvious difference in the localization between the wild-type UPF3B and the proteins carrying the two missense changes identified. However, we show that UPF3B is widely expressed in neurons and also presents in dendritic spines, which are essential structures for proper neurotransmission and thus learning and memory processes. Our results demonstrate that in addition to Lujan-Fryns and FG syndromes, UPF3B protein truncation mutations can cause also nonspecific XLMR. We also identify comorbidity of MR and autism in another family with UPF3B mutation. The neuronal localization pattern of the UPF3B protein and its function in mRNA surveillance suggests a potential function in the regulation of the expression and degradation of various mRNAs present at the synapse.
Asunto(s)
Trastorno Autístico/genética , Codón sin Sentido/genética , Discapacidad Intelectual/genética , Neuronas/metabolismo , Proteínas de Unión al ARN/genética , Adulto , Sustitución de Aminoácidos/genética , Animales , Trastorno Autístico/complicaciones , Línea Celular , Espinas Dendríticas/metabolismo , Regulación hacia Abajo , Femenino , Hipocampo/metabolismo , Humanos , Discapacidad Intelectual/complicaciones , Masculino , Ratones , Persona de Mediana Edad , Linaje , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
Aristaless-related homeobox gene (ARX) is an important paired-type homeobox gene involved in the development of human brain. The ARX gene mutations are a significant contributor to various forms of X-chromosome-linked mental retardation with and without additional features including epilepsy, lissencephaly with abnormal genitalia, hand dystonia or autism. Here we demonstrate that the human ARX protein is a potent transcriptional repressor, which binds to Groucho/transducin-like enhancer of split (TLE) co-factor proteins and the TLE1 in particular through its octapeptide (Engrailed homology repressor domain (eh-1) homology) domain. We show that the transcription repression activity of ARX is modulated by two strong repression domains, one located within the octapeptide domain and the second in the region of the polyalanine tract 4, and one activator domain, the aristaless domain. Importantly, we show that the transcription repression activity of ARX is affected by various naturally occurring mutations. The introduction of the c.98T>C (p.L33P) mutation results in the lack of binding to TLE1 protein and relaxed transcription repression. The introduction of the two most frequent ARX polyalanine tract expansion mutations increases the repression activity in a manner dependent on the number of extra alanines. Interestingly, deletions of alanine residues within polyalanine tracts 1 and 2 show low or no effect. In summary we demonstrate that the ARX protein is a strong transcription repressor, we identify novel ARX interacting proteins (TLE) and offer an explanation of a molecular pathogenesis of some ARX mutations, including the most frequent ARX mutations, the polyalanine tract expansion mutations, c.304ins(GCG)7 and c.428_451dup.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Proteínas de Homeodominio/genética , Mutación , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Transcripción Genética/fisiología , Factores de Edad , Alanina/genética , Animales , Encéfalo/citología , Células Cultivadas , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Inmunoprecipitación/métodos , Hibridación in Situ/métodos , Ratones , Neuronas/metabolismo , Transducina/metabolismo , Transfección/métodosRESUMEN
During early postnatal development, the intestine is highly responsive to LR(3)IGF-I administration but refractory to IGF-I, in contrast to the mature intestine. Given that LR(3)IGF-I is an IGF-I analog that binds poorly to IGF binding proteins, the response of the intestine is likely to reflect regulation of IGF-I bioactivity by IGF binding proteins. This study measures the delivery of exogenous IGF-I peptides to the intestine in preweaning (d-19) and adult rats to determine whether a correlation exists with the potency advantage of LR(3)IGF-I in the intestine during postnatal development. IGF-I or LR(3)IGF-I (2.6 microg/kg) was spiked with corresponding (125)I-labeled peptide (10 x 10(6) cpm) and administered iv as a bolus (n = 5-6/group) with blood and tissue samples collected 5 and 10 min post injection. In both age groups, the levels of (125)I-IGF-I retained in the blood at both 5 and 10 min were higher than the levels of (125)I-LR(3)IGF-I, consistent with the slower clearance rate for the native peptide. In the gastrointestinal tract, the levels of (125)I-LR(3)IGF-I per gram of tissue were 37-50% higher than (125)I-IGF-I. Surprisingly, there was little difference in the relative delivery of LR(3)IGF-I to IGF-I to the intestine, across developmental age. Although bolus iv-injected LR(3)IGF-I was cleared more rapidly from the circulation than IGF-I and was subsequently delivered to the intestine in higher amounts than the native peptide, the ratio of LR(3)IGF-I to IGF-I in gut tissues was approximately 2:1 in both age groups. Hence, selective delivery to the gut is unlikely to explain the markedly higher potency of (125)I-LR(3)IGF-I in stimulating growth of the preweaning vs. adult intestine.
Asunto(s)
Envejecimiento/metabolismo , Animales Lactantes/metabolismo , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Mucosa Intestinal/metabolismo , Animales , Disponibilidad Biológica , Femenino , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fragmentos de Péptidos/sangre , Ratas , Ratas WistarRESUMEN
In contrast to the adult gut, the immature intestine is refractory to subcutaneously infused insulin-like growth factor I (IGF-I). IGF binding protein (IGFBP) mRNA expression was characterized in intestinal tissues from 6-, 19-, and 90-day-old rats to determine if changes in local expression could account for this age-related change in IGF-I potency. For all age groups, IGFBP-3 to -6, but not IGFBP-1 or -2, were detected by Northern blot analysis. IGFBP-3, -4, and -5 were more intensely expressed in the 6-day-old rat intestine compared with weanling or adult tissue. In contrast, IGFBP-6 expression peaked at the time of weaning. In situ hybridization showed IGFBP-3 to -6 expression was confined to cells of the lamina propria and submucosa and also in the muscularis layer for IGFBP-5. Furthermore, the pattern of IGFBP-5 localization in the intestine changed with development. The findings indicate that the expression of IGFBP-3 to -6 is higher in the immature intestine compared with the adult intestine, suggesting locally produced IGFBPs may inhibit systemically derived IGF-I action in the intestine. Therefore, changes to local IGFBP expression may contribute to the varying response of the rat intestine to IGF-I peptides during postnatal development.
Asunto(s)
Expresión Génica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Intestino Delgado/crecimiento & desarrollo , Envejecimiento , Animales , Northern Blotting , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Hibridación in Situ , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Intestino Delgado/química , Masculino , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
Recently, glucagon-like peptide 2 has emerged as a potent stimulator of epithelial growth, joining insulin-like growth factor I, hepatocyte growth factor and keratinocyte growth factor as potential treatment modalities for intestinal disorders associated with loss of mucosal mass, such as short bowel syndrome. Investigations into other members of the expanded epidermal growth factor peptide family, the development of more potent peptide analogues, and advances in the development of enterally administered bioactive growth factor formulations further expands the repertoire of epithelial growth factors applicable to conditions associated with epithelial insufficiency.
Asunto(s)
Sustancias de Crecimiento/fisiología , Enfermedades Intestinales/patología , Mucosa Intestinal/patología , Mucinas , Proteínas Musculares , Neuropéptidos , Adaptación Fisiológica , Factor de Crecimiento Epidérmico/fisiología , Factor 7 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/fisiología , Factores de Crecimiento de Fibroblastos/uso terapéutico , Péptido 2 Similar al Glucagón , Péptidos Similares al Glucagón , Sustancias de Crecimiento/uso terapéutico , Factor de Crecimiento de Hepatocito/fisiología , Factor de Crecimiento de Hepatocito/uso terapéutico , Humanos , Enfermedades Intestinales/tratamiento farmacológico , Péptidos/fisiología , Péptidos/uso terapéutico , Regeneración , Síndrome del Intestino Corto/tratamiento farmacológico , Síndrome del Intestino Corto/patología , Somatomedinas/fisiología , Somatomedinas/uso terapéutico , Factor de Crecimiento Transformador alfa/fisiología , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
The growth mitogenic properties of IGF-I on tissues of the gastrointestinal tract are well established; however, IGF effects on enzyme maturation are less clear. To test whether IGF-I peptide administration stimulates disaccharidase activity, we administered IGF-I or the more potent analog, long [Arg3]IGF-I, at doses ranging between 2 and 12.5 micrograms g-1 d-1 to suckling Wistar rat pups by either continuous s.c. infusion or by three times daily orogastric gavage. Peptides were administered for approximately 6 d starting on d 6 or 12 postpartum with six to nine rats per group. The results of the study demonstrated that systemically but not orally administered IGF-I stimulated duodenal wet tissue weight (up to 85%) and length (up to 36%). Enzyme maturation was assessed by measuring disaccharidase biochemically in tissue homogenates. Enzyme activity was also localized histocytochemically in cryostat-sectioned duodenum. After systemic infusion of IGF-I, intestinal lactase activity increased proportional to mucosal mass in both age groups. Systemic infusion of the more potent analog, long [Arg3]IGF-I, precociously induced the decline in lactase activity and accelerated the appearance of sucrase activity in the rat pups infused during the later suckling period. These findings indicate that enzyme maturation can be accelerated by systemically derived IGF-I peptides. Orogastrically IGF-I peptides, delivered at pharmacologic doses, did not affect intestinal growth or digestive enzyme maturation in suckling rat pups treated between 6 and 18 d postpartum, indicating the efficacy of IGF-I peptides may depend on the route of delivery and postnatal age of the recipient.
Asunto(s)
Disacaridasas/metabolismo , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/farmacología , Intestinos/enzimología , Administración Oral , Animales , Animales Lactantes , Duodeno/efectos de los fármacos , Duodeno/enzimología , Duodeno/crecimiento & desarrollo , Mucosa Intestinal/crecimiento & desarrollo , Intestinos/efectos de los fármacos , Yeyuno/citología , Yeyuno/efectos de los fármacos , Yeyuno/enzimología , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacologíaRESUMEN
This study describes developmental changes in gastrointestinal response to insulin-like growth factor I (IGF-I) peptide administration. Neonatal rats were infused with IGF-I or long [Arg3]IGF-I (LR(3)IGF-I) for 6.5 days starting on day 6 or 12 postpartum. Peptides were delivered by mini osmotic pumps at 0, 2, 5, or 12.5 microg x g(-1) x day(-1). IGF-I infusion increased plasma IGF-I levels in both age groups but stimulated body weight gain only in the older rats. Infusion of LR(3)IGF-I did not change plasma IGF-I levels. Both peptides enhanced expression of IGF-binding proteins (IGFBP) 1 and 2 and induced IGFBP-3 in the older rats. For both age groups, weights of the kidney and spleen increased by up to 85 and 76%, respectively. IGF-I treatment also stimulated gut weight and length by up to 60 and 32%, respectively, but dose dependency was observed only in the older rats. LR(3)IGF-I was more potent for all growth parameters in both age groups. Histological observations included thickening of the mucosa and muscularis externa after infusion of IGF-I peptides. Thymidine labeling in the younger rats indicated that proliferative activity increased proportionately with crypt cell growth. These results show that IGF-I peptides selectively stimulate growth of gastrointestinal tissues in suckling rats and that the proximal gut was the most peptide-responsive region.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Factor I del Crecimiento Similar a la Insulina/farmacología , Intestinos/crecimiento & desarrollo , Factores de Edad , Animales , Animales Lactantes , División Celular/efectos de los fármacos , Duodeno/anatomía & histología , Duodeno/crecimiento & desarrollo , Íleon/anatomía & histología , Íleon/crecimiento & desarrollo , Insulina/sangre , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Mucosa Intestinal/citología , Peso Molecular , Tamaño de los Órganos/efectos de los fármacos , RatasRESUMEN
This study describes the biosynthesis of a human epidermal growth factor fusion protein, Long EGF, that has a 53 amino acid extension peptide derived from the 46 N-terminal amino acids of porcine GH. The approach allowed the production of Long EGF at high efficiency due to the expression of the fusion protein in high yield as inclusion bodies in Escherichia coli. Long EGF had a slightly lower potency compared with native EGF in a range of assays, including binding to anti-EGF antibodies or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat intestinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta 1. Degradation of Long EGF and native EGF was compared in gastrointestinal flushings as an indication of whether the EGF domain of the fusion protein would be protected from proteolytic cleavage and be useful as a trophic agent in the gut. Incubation with flushings from the stomach or jejunum of rats caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg53 in the stomach and a removal of the C-terminal pentapeptide (49 Trp-Trp-Glu-Leu-Arg53) in the small bowel was demonstrated by N-terminal sequencing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent binding activities to the EGF receptor and anti-EGF antibodies. The data show that a human EGF fusion protein can be produced efficiently in a bacterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EGF in both stomach and small bowel producing similar biological activity to native EGF, it could not prevent subsequent degradation of the EGF domain. Other strategies are being investigated to develop an effective oral form of EGF that resists digestion by proteases in the gastrointestinal tract.
Asunto(s)
Sistema Digestivo/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Arginina , Secuencia de Bases , Clonación Molecular , Factor de Crecimiento Epidérmico/biosíntesis , Factor de Crecimiento Epidérmico/farmacología , Escherichia coli , Humanos , Yeyuno , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacología , Eliminación de Secuencia , PorcinosRESUMEN
To investigate the potential of IGF-I peptides as therapeutics in the gut, the survival profiles of a bolus of 125I-labelled IGF-I (8.6 ng) in vivo in various ligated gut segments of fasted adult rats have been examined. The intactness of IGF-I tracer in the flushed luminal contents was estimated by trichloroacetic acid precipitation, antibody and receptor binding assays. It was found that IGF-I was degraded very rapidly in duodenum and ileum segments with a half-life (t1/2) of 2 min by all three methods. IGF-I was slightly more stable in the stomach (t1/2 = 8, 5 and 2.5 min by the above three methods), and considerably more stable in the colon (t1/2 = 38, 33 and 16 min as judged by the three methods). Rates of degradation in gut flushings in vitro were similar to the in vivo rates except for the colon, where IGF-I was proteolysed more rapidly in vivo. As a means of developing gut-stable and active forms of IGF-I, several approaches were examined for their effectiveness in prolonging IGF-I survival in the upper gut. It was found that the extension peptide on the analogue, LR3IGF-I did not protect IGF-I, nor did association with IGF-binding protein-3. However, an IGF-I antiserum was effective in prolonging IGF-I half-life in duodenum fluid by 28-fold. Charge interaction between IGF-I and heparin could also protect IGF-I in the stomach but not in duodenum flushings. Furthermore, casein (a non-specific dietary protein) and to a lesser extent, BSA and lactoferrin, were effective in preserving IGF-I structural integrity and receptor binding activity in both stomach and duodenum fluids. It can be concluded that IGF-I cannot be expected to retain bioactivity if delivered orally because of rapid proteolysis in the upper gut, but the use of IGF antibodies and casein could represent useful approaches for IGF-I protection in oral formulae.