RESUMEN
α2 -Macroglobulin (α2 M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2 M (hα2 M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2 M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.
Asunto(s)
alfa-Macroglobulinas/química , Animales , Pollos , Quimotripsina/metabolismo , Cristalización , Cristalografía por Rayos X , Endopeptidasas/metabolismo , Glicósido Hidrolasas/metabolismo , Humanos , Hidrolasas/metabolismo , Metilaminas/química , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas , Termolisina , alfa-Macroglobulinas/aislamiento & purificaciónRESUMEN
The efficiency of six maternal serum markers for Down's syndrome (DS), alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), free beta-hCG, pregnancy-associated plasma protein-A (PAPP-A), the proform of eosinophil major basic protein (ProMBP), pregnancy-specific-beta-1-glycoprotein (SP(1)), and combinations thereof, was examined. Discriminant analysis in 156 DS pregnancies and 546 controls defined three effective combinations of serum marker logMoMs (multiples of the median in control samples) in three gestational age windows, i.e. Index I (weeks 7-9) = 0.52 logMoM ProMBP + 0.28 logMoM PAPP-A - logMoM SP(1); Index II (weeks 10-12) = 1.94 logMoM free beta-hCG - logMoM SP(1), and Index III (weeks 15-19) = 0.78 logMoM free beta-hCG + 1.12 logMoM ProMBP - logMoM AFP. The estimated detection rates of indices and age for a false-positive rate (FPR) of 5% were 73% for Index I, 69% for Index II, and 60% for Index III. Including the ultrasound marker nuchal translucency, using a DS at term risk of 1 : 400 as cut-off, the detection rates of the indices increased to 86, 83, and 82% for FPRs of 4.3, 4.1, and 5.8%, respectively. The indices are promising markers for screening for DS.
Asunto(s)
Biomarcadores/sangre , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Pruebas Genéticas , Diagnóstico Prenatal , Adulto , Factores de Edad , Reacciones Falso Positivas , Femenino , Humanos , Cariotipificación , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Sensibilidad y EspecificidadRESUMEN
The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.
Asunto(s)
Proteína Plasmática A Asociada al Embarazo/química , Proteína Plasmática A Asociada al Embarazo/clasificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Análisis Mutacional de ADN , Humanos , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Proteína Plasmática A Asociada al Embarazo/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Zinc/químicaRESUMEN
Pregnancy-associated plasma protein-A (PAPP-A) has recently been identified as the proteinase responsible for cleavage of insulin-like growth factor binding protein (IGFBP)-4, an inhibitor of IGF action, in several biological fluids. Cleavage of IGFBP-4 by PAPP-A is believed to occur only in the presence of IGF. We here report that in addition to IGFBP-4, PAPP-A also cleaves IGFBP-5. Cleavage occurs at one site, between Ser-143 and Lys-144 of IGFBP-5. In the presence of IGF, IGFBP-4 and -5 are cleaved with similar rates by PAPP-A. Interestingly, cleavage of IGFBP-5 by PAPP-A does not require the presence of IGF, but is slightly inhibited by IGF. These findings have implications for the mechanism of proteolysis of IGFBP-4 by PAPP-A, suggesting that IGFBP-4 binds IGF, which then becomes a PAPP-A substrate. Using highly purified, recombinant proteins, we establish that (1) PAPP-A cleavage of IGFBP-4 can occur in the absence of IGF, although the rate of hydrolysis is very slow, and (2) IGF is unable to bind to PAPP-A. We thus conclude that IGF enhances proteolysis by binding to IGFBP-4, not by interaction with PAPP-A, which could not previously be ruled out.
Asunto(s)
Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína Plasmática A Asociada al Embarazo/metabolismo , Somatomedinas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Hidrólisis , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/química , Unión ProteicaRESUMEN
A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.
Asunto(s)
Endopeptidasas/química , Endopeptidasas/genética , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Proteínas Gestacionales/química , Proteínas Gestacionales/genética , Secuencia de Aminoácidos , Cartilla de ADN , Bases de Datos Factuales , Endopeptidasas/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Etiquetas de Secuencia Expresada , Humanos , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Proteínas Gestacionales/metabolismo , Proteína Plasmática A Asociada al Embarazo/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Proteínas Sanguíneas/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasas , Proteínas Sanguíneas/química , Western Blotting , Línea Celular , Cromatografía por Intercambio Iónico , ADN Complementario/metabolismo , Disulfuros , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos , Ensayo de Inmunoadsorción Enzimática , Proteínas en los Gránulos del Eosinófilo , Eosinófilos/química , Femenino , Humanos , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/química , Plásmidos/metabolismo , Embarazo , Proteína Plasmática A Asociada al Embarazo/antagonistas & inhibidores , Proteína Plasmática A Asociada al Embarazo/metabolismo , Proteína Plasmática A Asociada al Embarazo/fisiología , Somatomedinas/metabolismo , TransfecciónRESUMEN
The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.
Asunto(s)
Asparagina/química , Cistina/química , Eosinófilos/enzimología , Peroxidasas/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Disulfuros/química , Peroxidasa del Eosinófilo , Glicoproteínas/química , Glicosilación , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ProteínaRESUMEN
The proform of eosinophil major basic protein (proMBP), the most abundant protein in the eosinophil specific granule, is synthesized by the placenta and secreted into the maternal circulation, where it is found complex-bound to pregnancy-associated plasma protein-A (PAPP-A) and other proteins. We examined the potential of proMBP as a maternal serum marker for fetal Down syndrome (DS) by determining its maternal serum concentration (MSpMBP) in 25 Down syndrome (DS) pregnancies and 152 control pregnancies in the first trimester, and in 105 DS pregnancies and 156 control pregnancies in the second trimester. The median (95 per cent confidence interval) MSpMBP MoM in DS pregnancies (n=15) was 0.66 (0.49-0.79) in gestational weeks 5-9; 1.06 (0.71-1.97) in weeks 10-12 (n=10) and 1.62 (1.18-1.98) in weeks 14-20 (n=105). Using parameterized receiver operator characteristics analysis for proMBP as a single marker for DS, detection rates (DRs) of 22 per cent and 38 per cent, for false-positive rates (FPRs) of 5 per cent, were found in weeks 5-9 (using MSpMBP=cut-off) and weeks 14-20 (using MSpMBP>/=cut-off), respectively. When age and MSpMBP were used as markers in combination, a DR of 36.8 per cent for an FPR of 5.5 per cent was obtained in weeks 5-9 using a risk cut-off of 1:250. In weeks 14-20 the DR was 48.4 per cent for an FPR of 5.3 per cent using the same risk cut-off. This makes proMBP a marker comparable in diagnostic efficiency to human chorionic gonadotrophin (hCG), and exceeding that of alpha-fetoprotein (AFP) and unconjugated oestriol (uE3), in the second trimester.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Síndrome de Down/sangre , Eosinófilos/metabolismo , Intercambio Materno-Fetal/fisiología , Proteínas Gestacionales/sangre , Diagnóstico Prenatal/métodos , Ribonucleasas , Envejecimiento/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Síndrome de Down/diagnóstico , Proteínas en los Gránulos del Eosinófilo , Femenino , Edad Gestacional , Humanos , Modelos Lineales , Tamizaje Masivo , Embarazo , Primer Trimestre del Embarazo , Factores de RiesgoRESUMEN
PAPP-A/proMBP, the complex of pregnancy-associated plasma protein-A (PAPP-A) and the proform of eosinophil major basic protein (proMBP), circulates at increasing levels during pregnancy. The major site of synthesis is the placenta, in which PAPP-A mRNA has been localized to the syncytiotrophoblast and the placental X cells, whereas proMBP mRNA has been localized to the placental X cells only. The function of PAPP-A/proMBP and its components has remained speculative for years. Recently, however, it has been shown that PAPP-A specifically cleaves insulin-like growth factor (IGF) binding protein-4 in an IGF-dependent manner. Female reproductive and nonreproductive tissues have previously been reported to contain PAPP-A immunoreactivity, based on studies using preparations of anti(PAPP-A/proMBP), now known to recognize both PAPP-A and proMBP, and other irrelevant antigens. To analyze for the presence of PAPP-A and proMBP mRNA, a sensitive semiquantitative reverse transcription (RT) polymerase chain reaction (PCR) method was developed. Reverse-transcribed poly(A)(+) RNA was used as a template in a competitive PCR. PAPP-A and proMBP mRNA levels were normalized against the level of beta-actin mRNA. Both mRNA species were significantly more abundant in term placenta than in other tissues analyzed. All analyzed tissues, including endometrium, myometrium, colon, and kidney, contained both PAPP-A and proMBP mRNA.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Mediadores de Inflamación/metabolismo , Proteína Plasmática A Asociada al Embarazo/biosíntesis , ARN Mensajero/metabolismo , Actinas/biosíntesis , Actinas/genética , Proteínas en los Gránulos del Eosinófilo , Eosinófilos/metabolismo , Femenino , Humanos , Reacción en Cadena de la Polimerasa , Embarazo , Ribonucleasas/metabolismoRESUMEN
The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. Mass analysis of isolated heme bispeptides supports the hypothesis of a heme b linked through two esters to the polypeptide. Mass analysis of heme monopeptides reveals that >90% have a nonderivatized methyl group at the position of the light chain linkage. Apparently, an ester had not been formed during biosynthesis. The light chain linkage could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.
Asunto(s)
Ácido Aspártico/química , Eosinófilos/enzimología , Ácido Glutámico/química , Hemo/química , Peroxidasas/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Peroxidasa del Eosinófilo , Ésteres , Humanos , Lactoperoxidasa/química , Datos de Secuencia Molecular , Oligopéptidos/química , Análisis de SecuenciaRESUMEN
Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.
Asunto(s)
Astacoidea/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Sistema Digestivo/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Vitelogeninas/genética , Secuencia de Aminoácidos , Animales , Astacoidea/metabolismo , Secuencia de Bases , Proteínas Sanguíneas/química , Clonación Molecular , Reactivos de Enlaces Cruzados , ADN Complementario , Femenino , Biblioteca de Genes , Lipoproteínas/química , Masculino , Mamíferos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oviposición , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transglutaminasas/metabolismo , Vitelogeninas/química , Factor de von Willebrand/química , Factor de von Willebrand/genéticaRESUMEN
Proteolytic cleavage of the six known insulin-like growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblast-conditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.
Asunto(s)
Fibroblastos/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/aislamiento & purificación , Proteína Plasmática A Asociada al Embarazo/genética , Secuencia de Aminoácidos , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Espectrometría de Masas , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Embarazo , Proteína Plasmática A Asociada al Embarazo/química , Análisis de SecuenciaRESUMEN
Human complement component C5 has been crystallized using a low-salt batch technique. The crystals are large hexagonal bi-pyramids often larger than 1.5 mm. Although these crystals were grown in low salt (0.1 M NaCl), they are remarkably stable for at least 2 months at 281 K and they are not dissolved in aqueous buffers containing up to 2 M sodium chloride. The space group is P3121 or P3221, and the cell parameters were determined to be a = 144.9, b = 144.9, c = 243.1 A; alpha = 90 degrees, beta = 90, gamma = 120 degrees. At room temperature and cryo-temperatures the crystals diffract at best to 6 A using rotating-anode X-ray sources. Using synchrotron radiation with cryoprotection using 40%(v/v) PEG 400 the resolution limit can be extended to 3.3 A. In both cases the crystals show significant anisotropy, with relatively weaker reflections at higher resolution in the a*b* plane.
Asunto(s)
Complemento C5/química , Anisotropía , Complemento C5/aislamiento & purificación , Complemento C5/efectos de la radiación , Cristalización , Cristalografía por Rayos X , Humanos , Conformación Proteica , SincrotronesRESUMEN
BACKGROUND: The large plasma proteinase inhibitors of the alpha 2-macroglobulin superfamily inhibit proteinases by capturing them within a central cavity of the inhibitor molecule. After reaction with the proteinase, the alpha-macroglobulin-proteinase complex binds to the alpha-macroglobulin receptor, present in the liver and other tissues, and becomes endocytosed and rapidly removed from the circulation. The complex binds to the receptor via recognition sites located on a separate domain of approximately 138 residues positioned at the C terminus of the alpha-macroglobulin subunit. RESULTS: The crystal structure of the receptor-binding domain of bovine alpha 2-macroglobulin (bRBD) has been determined at a resolution of 1.9 A. The domain primarily comprises a nine-strand beta structure with a jelly-roll topology, but also contains two small alpha helices. CONCLUSIONS: The surface patch responsible for receptor recognition is thought to involve residues located on one of the two alpha helices of the bRBD as well as residues in two of the beta strands. Located on this alpha helix are two lysine residues that are important for receptor binding. The structure of bRBD is very similar to the approximately 100-residue C-terminal domain of factor XIII, a transglutaminase from the blood coagulation system.
Asunto(s)
Fragmentos de Péptidos/química , alfa-Macroglobulinas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Factor XIII/química , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Receptores Inmunológicos/metabolismo , Receptores de LDL , Homología de Secuencia de Aminoácido , alfa-Macroglobulinas/metabolismoRESUMEN
We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-1 plasminogen-activator inhibitor (PAI-1) to the non-target proteinases trypsin, endoproteinase Asp-N, proteinase K and subtilisin. This analysis has allowed us to detect conformational differences between the different forms of PAI-1 outside the reactive-centre loop and beta-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around alpha-helix E, beta-strand 1A, and the flanking loops, which are believed to form flexible joints during movements of beta-sheet A. Secondly, hypersensitive sites were observed in the loop between alpha-helix I and beta-strand 5A. Thirdly, the gate region, encompassing beta-strands 3C and 4C, was highly susceptible to trypsin in latent PAI-1, but not in the other conformations. The digestion patterns differed among all four forms of PAI-1, indicating that each represents a unique conformation. The differential proteolytic susceptibility of the flexible-joint region may be coupled to the differential affinity to vitronectin, binding in the same region. The analysis also allowed detection of conformational differences between reactive-centre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogen-activator-complexed PAI-1 was different from that of active PAI-1, but indistinguishable from that of one of the reactive-centre-cleaved forms, as the complexed and this particular cleaved PAI-1 were completely resistant to all the non-target proteinases tested. This observation is in agreement with the notion that complex formation involves reactive-centre cleavage and a large degree of insertion of the reactive-centre loop into beta-sheet A. Our analysis has allowed the identification of some flexible regions that appear to be implicated in the conformational changes during the movements of beta-sheet A and during the inhibitory reaction of serpins with their target proteinases.
Asunto(s)
Endopeptidasas/metabolismo , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/metabolismo , Conformación Proteica , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Humanos , Cinética , Metaloendopeptidasas , Modelos Moleculares , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Análisis de Secuencia , Serpinas/metabolismo , Subtilisinas/metabolismo , Tripsina/metabolismoRESUMEN
Yeast is widely used in molecular biology. Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants. We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature.
Asunto(s)
Cartilla de ADN , ADN de Hongos/análisis , Pichia/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Detergentes , Calor , Moldes GenéticosRESUMEN
A 155-kDa proteinase inhibitor, pacifastin, from plasma of the freshwater crayfish, Pacifastacus leniusculus, was found to be composed of two covalently linked subunits. The two subunits are encoded by two different mRNAs, which were cloned and sequenced. The heavy chain of pacifastin (105 kDa) is related to transferrins, containing three transferrin lobes, two of which seem to be active for iron binding. The light chain of pacifastin (44 kDa) is the inhibitory subunit, and has nine cysteine-rich inhibitory domains that are homologous to each other and to low molecular weight proteinase inhibitors isolated from the grasshopper, Locusta migratoria. The nine light chain domains and the Locusta inhibitors share a characteristic cysteine array (Cys-Xaa9-12-Cys-Xaa2-Cys-Xaa-Cys-Xaa6-8-Cys-Xaa4++ +-Cys) distinct from any described proteinase inhibitor family, suggesting that they constitute a new family of proteinase inhibitors. Pacifastin is the first known protein that has combined properties of a transferrin-like molecule and a proteinase inhibitor.
Asunto(s)
Inhibidores de Proteasas/química , Proteínas/química , Transferrina/química , Secuencia de Aminoácidos , Animales , Astacoidea , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Inhibidores de Proteasas/aislamiento & purificación , Proteínas/genética , Proteínas/aislamiento & purificación , Análisis de SecuenciaRESUMEN
The complete amino acid sequence of human placental oxytocinase (placental leucine aminopeptidase) has been determined by cDNA cloning and sequencing. Oxytocinase is a type II integral membrane protein of 1025 amino acid residues, consisting of an acidic intracellular region of 110 amino acids followed by a hydrophobic transmembrane segment of 22 residues and 893 extracellular residues containing the characteristic Zn2+ coordination sequence element His-Glu-Xaa-Xaa-His-(18 residues)-Glu found in gluzincins. Two sets of cDNA clones with different 5'-ends were isolated and suggested to represent different spliced products of 3.6 kb (mature mRNA) and 12 kb, respectively. Oxytocinase mRNA is present in large amounts in placenta, heart and skeletal muscle and in small amounts in brain, kidney, liver and pancreas. A conserved sequence element, the GAMEN motif, which distinguishes the aminopeptidase family among gluzincins from other gluzincins, has been identified.
Asunto(s)
Cistinil Aminopeptidasa/química , Placenta/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cistinil Aminopeptidasa/genética , ADN Complementario/química , Femenino , Humanos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Monoclonal and polyclonal antibodies were produced against osteocalcin purified from bovine bone. The antibodies were used for the development of a two-site ELISA. A monoclonal antibody recognising residues 15-49 of bovine osteocatcin generated by cleavage with 70% formic acid was used as capture antibody and a biotinylated polyclonal antibody recognising the same peptide was used for detection. The two-site assay did not detect any tryptic peptides of bovine osteocalcin (residues 1-19, 20-43, and 45-49) nor did it detect the peptide 1-14 or the synthetic peptide 37-49. Cross-reaction was observed for osteocalcin in plasma from sheep, goat, chicken and human but not for osteocalcin from rat and pig.
RESUMEN
A partial bovine SSRP1 (structure-specific recognition protein 1) cDNA has been isolated from a osteoblast cDNA library and sequenced. The bovine SSRP1 cDNA of 1870 bp encoding 460 amino acid residues showed 86% and 98% sequence identity with human SSRP1 at the DNA and protein level, respectively. Expression of SSRP1 mRNA was detected in many human tissues, with a particularly high level in kidney.