RESUMO
The prion protein (PrP) misfolding to its infectious form is critical to the development of prion diseases, whereby various ligands are suggested to participate, such as copper and nucleic acids (NA). The PrP globular domain was shown to undergo NA-driven liquid-liquid phase separation (LLPS); this latter may precede pathological aggregation. Since Cu(II) is a physiological ligand of PrP, we argue whether it modulates phase separation altogether with nucleic acids. Using recombinant PrP, we investigate the effects of Cu(II) (at 6 M equivalents) and a previously described PrP-binding GC-rich DNA (equimolarly to protein) on PrP conformation, oligomerization, and phase transitions using a range of biophysical techniques. Raman spectroscopy data reveals the formation of the ternary complex. Microscopy suggests that phase separation is mainly driven by DNA, whereas Cu(II) has no influence. Our results show that DNA can be an adjuvant, leading to the structural conversion of PrP, even in the presence of an endogenous ligand, copper. These results provide new insights into the role of Cu(II) and NA on the phase separation, structural conversion, and aggregation of PrP, which are critical events leading to neurodegeneration.
Assuntos
Cobre/química , Oligonucleotídeos/química , Proteínas da Gravidez/química , Agregados Proteicos , Animais , Cátions Bivalentes , Clonagem Molecular , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Placenta growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in pathological conditions. PlGF exerts its biological function through the binding and activation of the seven immunoglobulin-like domain receptor Flt-1, also known as VEGFR-1. Here, we report the first detailed mutagenesis studies that provide a basis for understanding molecular recognition between PlGF-1 and Flt-1, highlighting some of the residues that are critical for receptor recognition. Mutagenesis analysis, performed on the basis of a structural model of interaction between PlGF and the minimal binding domain of Flt-1, has led to the identification of several PlGF-1 residues involved in Flt-1 recognition. The two negatively charged residues, Asp-72 and Glu-73, located in the beta3-beta4 loop, are critical for Flt-1 binding. Other mutations, which bring about a significant decrease in PlGF binding activity, are Gln-27, located in the N-terminal alpha-helix, and Pro-98 and Tyr-100 on the beta6 strand. The mutation of one of the two glycosylated residues of PlGF, Asn-84, generates a PlGF variant with reduced binding activity. This indicates that, unlike in VEGF, glycosylation plays an important role in Flt-1 binding. The double mutation of residues Asp-72 and Glu-73 generates a PlGF variant unable to bind and activate the receptor molecules on the cell surface. This variant failed to induce in vitro capillary-like tube formation of primary endothelial cells or neo-angiogenesis in an in vivo chorioallantoic membrane assay.
Assuntos
Proteínas da Gravidez/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Alantoide/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Embrião de Galinha , Córion/fisiologia , DNA Complementar/genética , Feminino , Ácido Glutâmico , Glutamina , Humanos , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Placenta , Fator de Crescimento Placentário , Gravidez , Proteínas da Gravidez/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.
Assuntos
Alelos , DNA/genética , Isoenzimas/química , Isoenzimas/genética , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Substituição de Aminoácidos/genética , Arginina/genética , DNA Complementar/genética , Dimerização , Feminino , Proteínas Ligadas por GPI , Genótipo , Ácido Glutâmico/genética , Glicina/genética , Humanos , Cinética , Placenta/enzimologia , Gravidez , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Prolina/genética , Estrutura Quaternária de Proteína/genéticaRESUMO
Ferritin is a ubiquitous iron storage protein existing in multiple isoforms composed of 24 heavy and light chain subunits. We describe here a third ferritin-related subunit cloned from human placenta cDNA library and named PLIF (placental immunomodulatory ferritin). The PLIF coding region is composed of ferritin heavy chain (FTH) sequence lacking the 65 C-terminal amino acids, which are substituted with a novel 48 amino acid domain (C48). In contrast to FTH, PLIF mRNA does not include the iron response element in the 5'-untranslated region, suggesting that PLIF synthesis is not regulated by iron. The linkage between the FTH and C48 domains created a restriction site for EcoRI. PLIF protein was found to localize in syncytiotrophoblasts of placentas (8 weeks of gestation) at the fetal-maternal interface. Increased levels of PLIF transcript and protein were also detected in the breast carcinoma cell lines T47D and MCF-7 but not in the benign corresponding cell line HBL-100. In vitro, PLIF was shown to down-modulate mixed lymphocyte reactions and to inhibit the proliferation of peripheral blood mononuclear cells stimulated with OKT3. The accumulated data indicate that PLIF is an embryonic immune factor involved in down-modulating the maternal immune recognition of the embryo toward anergy. This mechanism may have been adapted by breast cancer cells over expressing PLIF.
Assuntos
Ferritinas/isolamento & purificação , Tolerância Imunológica , Placenta/metabolismo , Proteínas da Gravidez/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Mama/citologia , Mama/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Feminino , Ferritinas/química , Ferritinas/genética , Ferritinas/fisiologia , Humanos , Dados de Sequência Molecular , Oxirredutases , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Proteínas da Gravidez/fisiologiaRESUMO
Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.
Assuntos
Substâncias de Crescimento , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas da Gravidez/química , Proteínas da Gravidez/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cisteína/química , DNA Complementar/metabolismo , Dissulfetos , Isomerismo , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Conformação Proteica , Relaxina/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Raios UltravioletaRESUMO
A full-length cDNA encoding a novel human protein was cloned from placenta cDNA. The corresponding 1542 amino acid protein sequence was termed 'pregnancy-associated plasma protein-E' (PAPP-E) as it shows a 62% homology to the human pregnancy-associated plasma protein-A (PAPP-A) that is a diagnostic marker for trisomies, especially Down syndrome. The conserved domain structure contains five motifs related to the short consensus repeats of complement proteins and selectins, three motifs related to the lin-notch motifs of proteins regulating early tissue differentiation, and a putative zinc-binding motif and active site of the metzincin-superfamily of metalloproteases. The PAPP-E gene was localized to chromosome 1q23-25. Northern blot analysis showed that PAPP-E is predominantly expressed in placenta.
Assuntos
Endopeptidases , Proteínas da Gravidez/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 1 , Clonagem Molecular , DNA Complementar/química , Feminino , Humanos , Metaloproteinase 9 da Matriz/genética , Dados de Sequência Molecular , Placenta/química , Placenta/metabolismo , Proteínas da Gravidez/química , Proteína Plasmática A Associada à Gravidez/genética , Alinhamento de SequênciaRESUMO
We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.
Assuntos
Anticoncepcionais/metabolismo , Glicoproteínas/biossíntese , Proteínas da Gravidez/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Sequência de Carboidratos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Anticoncepcionais/química , Cricetinae , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Glucosamina/farmacologia , Glucose/farmacologia , Glicodelina , Glicoproteínas/química , Glicosilação , Humanos , Imunoensaio , Immunoblotting , Lectinas/química , Dados de Sequência Molecular , Monossacarídeos/química , Proteínas da Gravidez/química , Proteínas Recombinantes/química , Espectrometria de Massas de Bombardeamento Rápido de Átomos , TransfecçãoRESUMO
The preparation of a series of sialylated and fucosylated N,N'-diacetyllactosediamine-type diantennary glycopeptides is reported. By sequential enzymatic action of jack bean beta-galactosidase, snail beta 4-N-acetyl-galactosaminyltransferase, bovine colostrum alpha 6-sialyltransferase and human milk alpha 3-fucosyltransferase, a diantennary glycopeptide obtained from asialo fibrinogen was converted at a 5-mumol scale to a series of structures occurring on the glycoprotein glycodelin A, which potentially inhibit human sperm-egg binding.