RESUMEN
Ehlers-Danlos syndrome (EDS) is characterized by joint hypermobility, alterations in the skin and additional signs of connective tissue involvement. EDS type VI was the first connective tissue disorder for which a specific defect in collagen metabolism was identified, namely a deficiency of lysyl hydroxylase activity. We now report a homozygous single basepair substitution converting the CGA codon (Arg319) to a TGA termination codon in two siblings with EDS type VI. The healthy parents, who are first cousins, and two of the three healthy siblings of the patients are heterozygous. The mutation leads to an almost complete absence of lysyl hydroxylase activity in extracts derived from fibroblasts of the patients.
Asunto(s)
Síndrome de Ehlers-Danlos/genética , Mutación Puntual/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Adolescente , Secuencia de Bases , Niño , Consanguinidad , ADN Complementario/análisis , Síndrome de Ehlers-Danlos/enzimología , Femenino , Fibroblastos/química , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/análisis , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Análisis de Secuencia de ADNRESUMEN
Biochemical abnormalities were studied in two brothers with bladder divericulas, inguinal hernias, slight skin laxity, and hyperelasticity and skeletal abnormalities including occipital exostoses. Lysyl oxidase activity was low in the medium of cultured skin fibroblasts, this abnormality being accompanied by reduced conversion of the newly synthesized collagen into the soluble form. Copper concentrations were markedly elevated in the cultured skin fibroblasts, but decreased in the serum and hair. Serum cerulophasmin levels were also low. The reduced lysyl oxidase activity is suggested to be responsible for ther clinical manifestations, but the deficiency in this copper-dependent enzyme may be secondary to the abnormalities in the metabolism of the cation. Nevertheless, a mutation directly affecting both lysyl oxidase and an intracellular copper transport protein cannot be excluded. The disease is tentatively classified as one subtype of the Ehlers-Danlos syndrome.
Asunto(s)
Aminoácido Oxidorreductasas/deficiencia , Enfermedades del Tejido Conjuntivo/genética , Cobre/metabolismo , Proteína-Lisina 6-Oxidasa/deficiencia , Adulto , Niño , Enfermedades del Tejido Conjuntivo/clasificación , Divertículo/genética , Síndrome de Ehlers-Danlos/clasificación , Humanos , Masculino , Síndrome del Pelo Ensortijado/diagnóstico , Mutación , Enfermedades de la Vejiga Urinaria/genéticaRESUMEN
Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.
Asunto(s)
Arteriosclerosis/metabolismo , Colágeno/biosíntesis , Vasos Coronarios/metabolismo , Mimosina/farmacología , Músculo Liso Vascular/metabolismo , Factores de Iniciación de Péptidos/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno/biosíntesis , Pironas/farmacología , Proteínas de Unión al ARN , Angioplastia de Balón , Animales , Arteriosclerosis/patología , Arteriosclerosis/cirugía , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Colágeno/antagonistas & inhibidores , Vasos Coronarios/citología , Vasos Coronarios/patología , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Hidroxilación , Inmunohistoquímica , Músculo Liso Vascular/citología , Músculo Liso Vascular/patología , Micotoxinas/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Procolágeno/análisis , Procolágeno-Prolina Dioxigenasa/biosíntesis , Procolágeno-Prolina Dioxigenasa/aislamiento & purificación , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Spodoptera , Transfección , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
UDP-glucuronic acid from the carboxyl group was coupled to agarose via a six-carbon atom spacer, and columns prepared from this material were used in an affinity chromatography of collagen glucosyltransferase. The enzyme was found to have a high affinity for such columns in the presence of Mn2+ in the buffer, whereas a considerably lower affinity was noted in the absence of such ions. The enzyme could be eluted from the column with either EDTA, UDP-glucose, or small peptides prepared from collagen, the peptides being the most effective eluting agent. After elution the enzyme was separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase putification of about 3000-fold was obtained from extract of chick embryos by relatively simple steps. Collagen galactosyltransferase was found to have no affinity for the column, suggesting that the binding was not only due to the UDP moiety, but that the uronic acid derivate of glucose also contributed to its specificity.
Asunto(s)
Glucosiltransferasas/aislamiento & purificación , Uridina Difosfato Ácido Glucurónico , Azúcares de Uridina Difosfato , Animales , Tampones (Química) , Embrión de Pollo , Cromatografía de Afinidad , Colágeno , Ácido Edético , Manganeso , Fragmentos de Péptidos , Unión Proteica , Sefarosa , Uridina Difosfato GlucosaRESUMEN
Lysyl oxidase is an extracellular enzyme that initiates crosslink formation in the major connective tissue proteins, the collagens and elastin. This enzyme activity accumulated in a fresh medium of cultured human skin fibroblasts for at least 24 h, but the accumulation was distinctly non-linear after the first 12 h. Most of the total enzyme activity was present in the medium, the activity found in the cell layer representing about 30% of the total activity at 4 h, and about 10-15% at 24 h. The bulk of the cell-layer-associated activity appeared to be extracellular, as more than half was lost upon trypsinization. Culturing of the cells for 8 h in the presence of either monensin or nigericin, ionophores known to inhibit the secretion of many proteins at the level of the Golgi complex, markedly reduced the accumulation of lysyl oxidase activity in the medium. Monensin was particularly effective, as it produced a distinct inhibition even at a 10 nM concentration, reaching 50% at 30 nM. Both ionophores also reduced enzyme activity in the cell layer, whereas no definite decrease was seen in the activity of the trypsinized cells. The effect of monensin was evidently not due to any general toxicity on the part of the drug, since even a 500 nM concentration gave no inhibition of the incorporation of [3H]leucine into total protein. Tunicamycin also reduced lysyl oxidase activity in the medium and to a lesser extent in the cell layer, but the effective dose, 1-10 micrograms/ml, also inhibited the incorporation of [3H]leucine into total protein. The reduced enzyme activity may therefore not be due to a direct effect of tunicamycin on the glycosylation of the lysyl oxidase protein itself but may be mediated through other actions of the drug. Colchicine caused no inhibition in lysyl oxidase activity secretion even at a 10 microM concentration, although it has been reported to inhibit collagen secretion at doses more than one order of magnitude lower.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Antibacterianos/farmacología , Colchicina/farmacología , Furanos/farmacología , Glucosamina/análogos & derivados , Monensina/farmacología , Nigericina/farmacología , Proteína-Lisina 6-Oxidasa/metabolismo , Piel/enzimología , Tunicamicina/farmacología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Leucina/metabolismo , Piel/efectos de los fármacos , Factores de TiempoRESUMEN
The kinetics of the lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) reaction were studied using enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs involving Fe2+, alpha-ketoglutarate, O2 and the peptide substrate, whereas parallel lines were obtained for pairs comprising ascorbate and each of the other substrates. The pair composed of Fe2+ and alpha-ketoglutarate gave an asymmetrical initial veolcity pattern, indicating binding of these two reactants in this order, that of Fe2+ being at thermodynamic equilibrium. The initial velocity patterns are identical with those reported for prolyl 4-hydroxylase, and the apparent Km and Kd values calculated from these data are also very similar. The largest difference was fo-nd in Km and Kd for alpha-ketoglutarate, which were about 4 times the corresponding values for prolyl 4-hydroxylase. Ascorbate was found to be a quite specific requirement for lysyl hydroxylase, but the enzyme catalyzed its reaction for a short time at a high rate in the complete absence of this vitamin, suggesting that the reaction with ascorbate does not occur during each catalytic cycle. Lysyl hydroxylase catalyzed an uncoupled decarboxylation of alpha-ketoglutarate in the absence of the peptide substrate, the rate being about 4% of that observed in the presence of a saturating concentration of the peptide substrate. This uncoupled decarboxylation required the same cosubstrates as the complete reaction.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Animales , Ácido Ascórbico/farmacología , Embrión de Pollo , Hierro/farmacología , CinéticaRESUMEN
Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO2, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe2+, alpha-ketoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO2, succinate and Fe2+, in which Fe2+ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO2 is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO2 and succinate or after the release of all products, including Fe2+, and dehydroascorbate is released before the binding of Fe2+. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical. Zn2+, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe2+, alpha-ketoglutarate, O2 and ascorbate respectively, and epinephrine non-competitively with regard to all cosubstrates. Apparent Ki values are given for the product and other inhibitors.
Asunto(s)
Oxigenasas de Función Mixta/antagonistas & inhibidores , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/antagonistas & inhibidores , Animales , Ácido Ascórbico/farmacología , Dióxido de Carbono/farmacología , Embrión de Pollo , Ácido Deshidroascórbico/farmacología , Epinefrina/farmacología , Ácido Homogentísico/farmacología , Cinética , Nitroazul de Tetrazolio/farmacología , Péptidos/farmacología , Succinatos/farmacologíaRESUMEN
Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.
Asunto(s)
Glucosiltransferasas/aislamiento & purificación , Animales , Embrión de Pollo , Cromatografía de Afinidad , Colágeno , Glucosiltransferasas/metabolismo , Peso Molecular , PolietilenglicolesRESUMEN
Lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) has a high affinity for columns of concanavalin A-agarose, which was markedly reduced in the presence of alpha-methyl-D-mannoside, suggesting that the enzyme is a glycoprotein. Once bound, the enzyme could not be eluted with the glycoside alone, whereas an effective elution was achieved by a combination of alpha-methyl-D-mannoside and ethylene glycol. The data thus suggest that hydrophobic interaction stabilized the complex of the enzyme with the column. This information was applied to obtain a lysyl hydroxylase purification of about 3000-fold with a recovery of more than 10% from extract of chick embryos by relatively simple steps.
Asunto(s)
Oxigenasas de Función Mixta/aislamiento & purificación , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/aislamiento & purificación , Animales , Embrión de Pollo , Cromatografía de Afinidad , Cromatografía en Gel , Concanavalina A , Glicoles de Etileno , Metilmanósidos , Peso Molecular , SefarosaRESUMEN
The effect of cortisol on the cellular concentration of translatable procollagen mRNAs was studied in cultured human skin fibroblasts. Cortisol selectively decreased the amount of procollagen mRNAs, in comparison to the total mRNA activity, when the cells were grown in enriched medium conditions, i.e., with 10% newborn calf serum. The selective decrease was first observed after 6 h exposure to 1 microM cortisol. In depleted medium conditions, i.e., with 2% newborn calf serum, the initial response was a stimulatory one, followed after about 12 h by a decrease in the procollagen mRNA activity. The results suggest that the selective inhibitory effect of cortisol on the cellular concentration of translatable procollagen mRNA species needs an optimal serum concentration. Furthermore, the results give support to the hypothesis that the decrease in the procollagen mRNA concentration after cortisol administration is a secondary response, preceded by the induction of some intracellular regulation system.
Asunto(s)
Hidrocortisona/farmacología , Procolágeno/genética , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Piel/metabolismo , Transcripción Genética/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , CinéticaRESUMEN
The activities of five intracellular enzymes of collagen biosynthesis were determined during cartilage and bone formation induced in rats by demineralized bone matrix. The five enzymes, prolyl 4-hydroxylase, prolyl 3-hydroxylase, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosyl-hydroxylysyl glucosyltransferase, exhibited broadly parallel profiles; the activities rising steeply from day one to reach their highest values on day nine and decreasing gradually thereafter. The maximal enzyme activity correlated with the period of chondrogenesis and hypertrophic cartilage characterized by the synthesis of cartilage-specific type II collagen. Prolyl 4-hydroxylase was also studied in respect of its tissue distribution and cellular location using indirect immunofluorescence. The enzyme was mainly located in the mesenchymal cells on day three, in the chondrocytes and hypertrophic chondrocytes on days seven to nine, and in the osteoblasts on day eleven and thereafter.
Asunto(s)
Desarrollo Óseo , Matriz Ósea/metabolismo , Huesos/enzimología , Cartílago/enzimología , Colágeno/biosíntesis , Animales , Colágeno/metabolismo , Galactosiltransferasas/metabolismo , Glucosiltransferasas/metabolismo , Hidroxilisina/metabolismo , Masculino , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , RatasRESUMEN
The addition of Zn2+ inhibited lysine hydroxylation markedly less effectively than it did proline hydroxylation in chick embryo tendon cells, 3T6 fibroblasts and lysyl hydroxylase-deficient Ehlers-Danlos Syndrome Type VI fibroblasts. With low Zn2+ concentrations, a similar difference was also seen in chick embryo cartilage cells, whereas with high concentrations both hydroxylations were affected to the same extent in this cell type. Ascorbate deficiency likewise had a much less effect on lysine than proline hydroxylation when studied with 3T6 fibroblasts. As these two effectors involve quite different mechanisms, it is suggested that relative insensitivity to inhibition may be a property of lysine hydroxylation seen in many cell types with a number of agents. Studies on the mechanism of the difference in the inhibition indicates that the phenomenon is probably not due to differences in the kinetic constants of Zn2+ and ascorbate for the two enzymes. Neither is it probably to any major extent due to delayed procollagen triple helix formation nor a difference in the location of the two hydroxylases within the cisternae of the rough endoplasmic reticulum. The difference similarly cannot be explained solely by an excess of lysyl hydroxylase in the cell. It may thus be due either to some other intracellular property or to the combined effect of several factors.
Asunto(s)
Ácido Ascórbico/farmacología , Colágeno/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Zinc/farmacología , Animales , Deficiencia de Ácido Ascórbico/enzimología , Cartílago/enzimología , Células Cultivadas , Embrión de Pollo , Pollos , Síndrome de Ehlers-Danlos/enzimología , Fibroblastos/enzimología , Cinética , Ratones , Piel/enzimología , Tendones/enzimologíaRESUMEN
The activities of the four enzymes catalyzing intracellular post-translational modifications of the collagen polypeptide chains were assayed in the kidneys of rats with streptozotocin diabetes. When the changes in the four enzyme activities were expressed per milligram of protein in the 15,000 X g supernatant of the kidney homogenates, there were no changes in any of the enzyme activities at four weeks and only slight increases in the prolyl and lysyl hydroxylase activities at 12 weeks after the induction of diabetes. When the changes were expressed as total enzyme activities per two kidneys, again no changes were found in any enzyme activity at four weeks, but at 12 weeks significant increases were found in all four enzyme activities, namely prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase, and collagen glucosyltransferase. The data would be consistent with an increased collagen synthesis in diabetic kidneys, but they do not support the hypothesis that there might be specific changes in some of these enzyme activities or in the level of certain posttranslational modifications of the collagen polypeptide chains in this disease.
Asunto(s)
Colágeno/biosíntesis , Diabetes Mellitus/metabolismo , Hexosiltransferasas/metabolismo , Riñón/enzimología , Oxigenasas de Función Mixta/metabolismo , Animales , Peso Corporal , Diabetes Mellitus/inducido químicamente , Galactosiltransferasas/metabolismo , Glucosiltransferasas/metabolismo , Riñón/anatomía & histología , Masculino , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Biosíntesis de Proteínas , Ratas , Estreptozocina , Factores de TiempoRESUMEN
Prolyl 4-hydroxylases (EC 1.14,11.2) catalyze the formation of 4-hydroxyproline in collagens and other proteins with collagen-like sequences. The vertebrate type I and type II enzymes are [alpha (I)]2 beta 2 and [alpha (II)]2 beta 2 tetramers, respectively, whereas the enzyme from the nematode Caenorhabditis elegans is an alpha beta dimer. The type I enzyme is the major form in most but not all vertebrate tissues. The catalytic properties of the various enzyme forms are highly similar, but there are distinct, although small, differences in K(m) values for various peptide substrates between the enzyme forms and major differences in Ki values for the competitive inhibitor, poly(L-proline). Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. Kinetic studies and theoretical considerations have led to elucidation of the reaction mechanism, and recent extensive site-directed mutagenesis studies have identified five critical residues at the cosubstrate binding sites. A number of compounds have been characterized that inhibit it competitively with respect to some of the cosubstrates, and three groups of suicide inactivators have also been identified. The beta subunit in all forms of prolyl 4-hydroxylase is identical to protein disulfide isomerase (PDI), a multifunctional polypeptide that also serves as a subunit in the microsomal triglyceride transfer protein, as a chaperone-like polypeptide that probably assists folding of a number of newly synthesized proteins, and in several other functions. The main role of the PDI polypeptide as a protein subunit is probably related to its chaperone function. Recent expression studies of recombinant human prolyl 4-hydroxylase subunits in a yeast have indicated that the formation of a stable enzyme tetramer in vivo requires coexpression of collagen polypeptide chains.
Asunto(s)
Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Animales , Humanos , Modelos Químicos , Conformación Proteica , Relación Estructura-ActividadRESUMEN
We report the complete cDNA sequence of the human lysyl oxidase-like 4 (LOXL4) gene, a new member of the lysyl oxidase (LO) gene family. The predicted polypeptide is 756 amino acids long, including a 24-residue signal peptide. The C-terminal region contains a LO domain similar to those of LOX, LOXL, LOXL2 and LOXL3. The N-terminal region has four subregions similar to scavenger receptor cysteine-rich domains that are highly conserved with LOXL2 and LOXL3. The LOXL4 mRNA is approximately 4 kb in size and is expressed in many tissues, the highest levels among the tissues studied being in the skeletal muscle, testis and pancreas. Recombinant LOXL4 expressed in HT-1080 cells was secreted into the culture medium with no evident proteolytic processing.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Cromosomas Humanos Par 10 , Cisteína/genética , Proteínas de la Membrana , Receptores de Lipoproteína , Aminoácido Oxidorreductasas/clasificación , Aminoácido Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cisteína/metabolismo , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Masculino , Ratones , Datos de Secuencia Molecular , Péptidos/clasificación , Péptidos/genética , Estructura Terciaria de Proteína , Proteína-Lisina 6-Oxidasa , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens and related proteins by the hydroxylation of lysine residues in peptide linkages. Three isoenzymes of LH have so far been characterized. We report here that the human LH3 gene is 11.6 kb in size and consists of 19 exons. Transcription is initiated at one major site and several minor sites, the first exon containing 249-335 bp of untranslated sequences and 109 bp of a translated sequence. Exons 2-18 are similar in size to those of the human LH1 gene, whereas the introns are markedly shorter. The LH3 gene contains a total of 15 full length Alu retroposons or partial Alu fragments of more than 100 bp, in introns 5, 6, 12, 15 and 17. These generate a potential for genomic rearrangements, as has been shown for the LH1 gene in Ehlers-Danlos syndrome type VI. The 5'-flanking region of the LH3 gene was found to be entirely different from that of the LH1 gene, suggesting different regulation of these two genes.
Asunto(s)
Exones , Intrones , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Complementario , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Recent coexpression studies of the subunits of human prolyl 4-hydroxylase (4-PH) in the yeast Pichia pastoris have indicated that only a minor fraction of them were present in the alpha2beta2 tetramer, while coexpression with type III procollagen markedly increased their assembly level. We report here that the half-life of the recombinant 4-PH tetramer in Pichia when studied by pulse-chase experiments was only 50 min. Coexpression with the pro alpha1(III) chains increased this half-life to 12.5 h. Coexpression with the pro alpha1(I) chains, which were produced at half the level of the pro alpha1(III) chains, gave a half-life of 6.5 h. Coexpression with collagen thus markedly increases the half-life of the 4-PH tetramer, and the half-life may be related to the level of collagen expression.
Asunto(s)
Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno/genética , Procolágeno/metabolismo , Dominio Catalítico , Estabilidad de Enzimas , Expresión Génica , Semivida , Humanos , Técnicas In Vitro , Pichia/genética , Procolágeno-Prolina Dioxigenasa/química , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule.
Asunto(s)
Pichia/genética , Procolágeno/metabolismo , Pliegue de Proteína , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vectores Genéticos , Humanos , Membranas Intracelulares/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Microscopía Inmunoelectrónica , Feromonas , Pichia/metabolismo , Procolágeno/genética , Procolágeno-Prolina Dioxigenasa/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
Insect cells coinfected with two baculoviruses, one coding for the pro alpha chains of human type II procollagen and the other for both the alpha and beta subunits of human prolyl 4-hydroxylase, produced the cartilage-specific type II collagen with a stable triple helix. The highest expression levels, up to 50 mg/l of type II collagen, were obtained in suspension culture using a modified construct in which sequences coding for the signal peptide and N propeptide of type II procollagen had been replaced by those for type III procollagen. The type III N propeptide artificially generated into type II procollagen was found to be cleaved at a much higher rate than the wild-type type II N propeptide, probably because the former interacted poorly with the triple-helical domain of type II procollagen. The amino acid composition of the recombinant type II collagen was very similar to that of the non-recombinant protein, but the hydroxylysine content was only 17% and that of glycosylated hydroxylysines was equally low. The hydroxylysine content was increased to the level found in the non-recombinant collagen by using an additional baculovirus coding for lysyl hydroxylase, and a substantial increase was also found in the glycosylated hydroxylysine content. No difference in thermal stability was found between the low- and high-hydroxylysine collagens.
Asunto(s)
Colágeno/biosíntesis , Hidroxilisina/análisis , Proteínas Recombinantes/biosíntesis , Animales , Catálisis , Línea Celular , Colágeno/química , Vectores Genéticos , Glucosiltransferasas/metabolismo , Glicosilación , Humanos , Nucleopoliedrovirus , Procolágeno/biosíntesis , Procolágeno/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Spodoptera , TemperaturaRESUMEN
Lysyl oxidase activity in the culture medium of eight malignantly transformed human cell lines was very low compared with that in four control fibroblast lines, being 9-16% in five sarcoma cell lines and 7-11% in three other tumour cell lines. The low enzyme activity was probably due to deficient enzyme synthesis rather than impaired secretion into the cell medium, as low activity was also found in urea extracts of the cell pellets. Lysyl oxidase production thus appears to be closely regulated with deficient collagen gene expression in malignant transformation.