RESUMEN
Human meprin ß (h-meprin ß), a single-zinc metalloendoprotease of the astacin family, is potentially involved in disorders such as fibrosis and Alzheimer's disease. Here, we describe the expression of the enzyme in the yeast Pichia pastoris. The N-terminal signal sequence was replaced by the α-leader of Saccharomyces, enabling efficient secretion of the mature enzyme, harboring either an N-terminal or C-terminal His-tag. The purification by affinity and hydrophobic interaction chromatography resulted in isolation of 58.4 mg/l of homogenous human pro-meprin ß from fermentation broth. The activated enzyme isolated from yeast (yh-meprin ß) displayed virtually identical enzymatic activity as h-meprin from a mammalian cell line. Furthermore, the yh-meprin ß was N-glycosylated and secreted as a dimer with a molecular mass of 148 kDa. Endoglycosidase H treatment generated a protein with a molecular mass of 133 kDa, but essentially unchanged kinetic parameters. Thus, our data suggest that human meprin ß expressed in P. pastoris displays virtually identical parameters as meprin from other sources. The high yield of protein expression, the ease of purification and the deglycosylation in its native state appear to favor further studies aiming at inhibitor screening and structure-based inhibitor refinement.
Asunto(s)
Clonación Molecular/métodos , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Pichia/genética , Electroforesis en Gel de Poliacrilamida , Glicosilación , Humanos , Metaloendopeptidasas/química , Metaloendopeptidasas/aislamiento & purificación , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Glucagón/metabolismo , Animales , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacología , Dipeptidil Peptidasa 4/sangre , Dipeptidil Peptidasa 4/aislamiento & purificación , Dipeptidil Peptidasa 4/farmacología , Glucagón/química , Glucagón/farmacología , Humanos , Isoleucina/análogos & derivados , Isoleucina/farmacología , Cinética , Masculino , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ratas , Ratas Wistar , Inhibidores de Serina Proteinasa/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos , Tiazoles/farmacologíaRESUMEN
The hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP)-1 act on the pancreas to potentiate glucose-induced insulin secretion (enteroinsular axis). These hormones (incretins) are rapidly hydrolyzed by the circulating enzyme dipeptidyl peptidase IV (DP IV) into biologically inactive NH2-terminally truncated fragments. This study describes the effect of inhibiting endogenous DP IV with a specific DP IV inhibitor, isoleucine thiazolidide (Ile-thiazolidide), on glucose tolerance and insulin secretion in the obese Zucker rat. In initial studies, the specificity of Ile-thiazolidide as an inhibitor of incretin degradation was determined using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These results showed that inhibiting DP IV activity with Ile-thiazolidide blocked the formation of NH2-terminally truncated GIP and GLP-1. Oral administration of Ile-thiazolidide resulted in rapid inhibition of circulating DP IV levels by 65% in obese and lean Zucker rats. Suppression of DP IV levels enhanced insulin secretion in both phenotypes with the most dramatic effect occurring in obese animals (150% increase in integrated insulin response vs. 27% increase in lean animals). Ile-thiazolidide treatment improved glucose tolerance in both phenotypes and restored glucose tolerance to near-normal levels in obese animals. This was attributed to the glucose-lowering actions of increasing the circulating half-lives of the endogenously released incretins GIP and, particularly, GLP-1. This study suggests that drug manipulation of plasma incretin activity by inhibiting the enzyme DP IV is a valid therapeutic approach for lowering glucose levels in NIDDM and other disorders involving glucose intolerance.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Intolerancia a la Glucosa/fisiopatología , Isoleucina/análogos & derivados , Ratas Zucker/fisiología , Tiazoles/farmacología , Administración Oral , Animales , Glucemia/análisis , Femenino , Glucosa/fisiología , Insulina/sangre , Isoleucina/farmacología , Masculino , Obesidad/sangre , Obesidad/fisiopatología , Ratas , Valores de ReferenciaRESUMEN
A novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting Ki values in the microM range. Boc-protected (omega-N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching Ki values of 30 nM to 60 microM. Additionally, alpha-N-BOC-(omega-N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction.