RESUMO
BACKGROUND: Phosphatidylethanolamine-binding protein (PEBP) is a basic protein found in numerous tissues from a wide range of species. The screening of gene and protein data banks defines a family of PEBP-related proteins that are present in a variety of organisms, including Drosophila and inferior eukaryotes. PEBP binds to phosphatidylethanolamine and nucleotides in vitro, but its biological function in vivo is not yet known. The expression of PEBP and related proteins seems to be correlated with development and cell morphogenesis, however. To obtain new insights into the PEBP family and its potential functions, we initiated a crystallographic study of bovine brain PEPB. RESULTS: The X-ray crystal structure of bovine brain PEBP has been solved using multiple isomorphous replacement methods, and refined to 1.84 A resolution. The structure displays a beta fold and exhibits one nonprolyl cis peptide bond. Analysis of cavities within the structure and sequence alignments were used to identify a putative ligand-binding site. This binding site is defined by residues of the C-terminal helix and the residues His85, Asp69, Gly109 and Tyr119. This site also corresponds to the binding site of phosphorylethanolamine, the polar head group of phosphatidylethanolamine. CONCLUSIONS: This study shows that PEBP is not related to the G-protein family nor to known lipid-binding proteins, and therefore defines a novel structural family of phospholipid-binding proteins. The PEBP structure contains no internal hydrophobic pocket, as described for lipocalins or small phospholipid-transfer proteins. Nevertheless, in PEBP, a small cavity close to the protein surface has a high affinity for anions, such as phosphate and acetate, and also phosphorylethanolamine. We suggest that this cavity corresponds to the binding site of the polar head group of phosphatidylethanolamine.
Assuntos
Proteína de Ligação a Androgênios , Encéfalo/metabolismo , Proteínas de Transporte/química , Conformação Proteica , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Bovinos , Cristalografia por Raios X/métodos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidiletanolaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
K99 lectin from Escherichia coli was purified and biotinylated via the amino groups of lysine residues using N-biotinyl-6-amino-caproic acid N-hydroxysuccinimide ester (BcapNHS). Biotin was detected on Lys-47 and Lys-87. It was previously demonstrated (Jacobs, A.A.C., Van den Berg, P.A., Bak, H.J. and De Graaf, F.K. (1986) Biochim. Biophys. Acta 872, 92-97) that modification of lysine residues 132 and 133 with 4-chloro-3,5-dinitrobenzoate (CDNB) resulted in the loss of the binding capacity of K99 fimbriae. Due to the higher size of the biotin derivative compared to CDNB, Lys-132 or Lys-133, essential for the biological activity, were not modified. The biotinylation did not cause the loss of the haemagglutinating activity but was sufficient to permit detection of the lectin by streptavidin. A flow cytometric analysis was used for the detection of the receptors on the surface of erythrocytes.
Assuntos
Antígenos de Bactérias/química , Antígenos de Superfície/química , Proteínas da Membrana Bacteriana Externa/química , Toxinas Bacterianas , Escherichia coli/química , Lectinas/química , Adesinas de Escherichia coli , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/biossíntese , Antígenos de Superfície/biossíntese , Proteínas da Membrana Bacteriana Externa/biossíntese , Biotina , Sequência de Carboidratos , Eritrócitos/química , Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/análise , Cavalos , Lisina/análise , Dados de Sequência Molecular , Peptídeos/análiseRESUMO
The complete amino acid sequence (458 amino acid residues) of human group-specific component 2 (Gc2) protein was determined. Computer analyses established a three-fold internal homology of Gc2 protein as well as an extensive homology between the overall structures of Gc2 protein, human serum albumin and human alpha-fetoprotein.
Assuntos
Serina Endopeptidases , Albumina Sérica , Proteína de Ligação a Vitamina D , alfa-Fetoproteínas , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Brometo de Cianogênio , Endopeptidases , Humanos , Fragmentos de Peptídeos , TripsinaRESUMO
From rape (Brassica napus) seedlings proteins able to bind fatty acids and their CoA-esters were purified by gel filtration and cation-exchange chromatography. Among the four proteins detected, one of them (peak IV) appeared purified to homogeneity. This protein is a monomer with a molecular mass of about 9 kDa, as estimated by gel filtration and by polyacrylamide gel electrophoresis. The isoelectric point of the rape protein was higher than 10.5 as determined by chromatofocusing. The pure rape protein appeared furthermore to be able to transfer several phospholipids (phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine) between membranes. The rape protein, having a multifunctional property, was thus called acyl-binding/lipid-transfer protein (AB-LTP). In order to compare this protein to plant lipid-transfer proteins (LTPs), its structure was determined. The amino acid analysis of the rape AB-LTP revealed a high amount of alanine, an absence of histidine and tryptophan and the presence of eight cysteine residues. The N-terminal amino acid sequence of the rape protein revealed a high homology to plant LTPs. These observations led us to propose that the rape AB-LTPs belong to a category of plant proteins interacting with lipids and playing a role in the fatty acid dynamics.
Assuntos
Brassica/química , Proteínas de Transporte/isolamento & purificação , Proteínas de Neoplasias , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Antígenos de Plantas , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cromatografia em Gel , Cromatografia por Troca Iônica , Coenzima A/metabolismo , Proteínas de Ligação a Ácido Graxo , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Fosfolipídeos/metabolismo , Proteínas de Plantas/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The C-terminal part (residues 106-171) of sheep kappa-casein, called caseinoglycopeptide (CGP), inhibits thrombin- and collagen-induced platelet aggregation in a dose-dependent manner (mean inhibitory concentration (IC50) 215 microM and 100 microM, respectively). An enzymatic hydrolysate of CGP was fractionated by reverse phase high performance liquid chromatography: three peptides KDQDK (residues 112-116), TAQVTSTEV (residues 163-171) and QVTSTEV (residues 165-171) completely inhibited thrombin-induced platelet aggregation. CGP at a concentration near its IC50 had a very long life when incubated in human or guinea-pig plasma. An ex vivo experiment showed that 17% of CGP was found 60 min after its i.v. bolus injection in guinea-pig. By hydrophobic cluster analysis, human fibrinogen and sheep kappa-casein peptides, inhibitors of platelet aggregation, were compared and we observed similarities for their C-terminal parts and for their short peptides (RGDF and KDQDK).
Assuntos
Caseínas/farmacologia , Fragmentos de Peptídeos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária , Sequência de Aminoácidos , Animais , Sangue , Caseínas/administração & dosagem , Caseínas/química , Cromatografia Líquida de Alta Pressão , Colágeno/farmacologia , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/química , Cobaias , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , Estrutura Secundária de Proteína , Ovinos , Trombina/farmacologia , Tripsina/metabolismoRESUMO
In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures. These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins. The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities. However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds. This structural variety should correspond to the recognition of multiple cellular partners.
Assuntos
Proteína de Ligação a Androgênios , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Escherichia coli , Escherichia coli/química , Sequência de Aminoácidos , Ânions/metabolismo , Proteínas Arqueais/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Transporte/genética , Clonagem Molecular , Biologia Computacional , Sequência Conservada/genética , Cristalografia por Raios X , Citoplasma/química , Dimerização , Escherichia coli/citologia , Escherichia coli/genética , Genes Bacterianos/genética , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Periplasma/química , Proteína de Ligação a Fosfatidiletanolamina , Proteínas de Transferência de Fosfolipídeos , Prostateína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Secretoglobinas , Alinhamento de Sequência , Homologia de Sequência , Especificidade por Substrato , UteroglobinaRESUMO
A cytosolic 21-23 kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. The protein was encountered in numerous tissues of several species. High expression of the mRNA encoding the 21-23 kDa protein was found in rat testes. Immunohistochemical studies showed the presence of the 21-23 kDa protein in the elongated spermatids and epididymal fluid of rat testis and in brain oligodendrocytes of developing rats. As the bovine, human and rat brain 21-23 kDa proteins had only few sequence homologies with already know proteins, ti was concluded that they belong to a new protein family. In order to get additional information on the structural features of the 21-23 kDa protein, we built a molecular model which displayed a nucleotide binding site. The affinity of the bovine brain 21-23 kDa protein towards nucleotides as well as its association with cytosolic proteins and small GTP-binding proteins were demonstrated. Recently, significant sequence homologies were found with an antigen from Onchocerca volvulus, a fruit fly odorant-binding protein and the yeast protein TFS1 which is a dosage-dependent suppressor of CDC25 mutations. A positive regulation of RAS is carried out by CDC25 product which facilitates the GDP/GTP exchange on RAS proteins. These results imply that 21-23 kDa proteins function in oxidoreduction reactions and signal mechanisms during cell growth and maturation.
Assuntos
Proteína de Ligação a Androgênios , Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Citosol/química , Ligantes , Masculino , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Proteína de Ligação a Fosfatidiletanolamina , Proteínas de Transferência de Fosfolipídeos , Prostateína , Secretoglobinas , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Testículo/química , Distribuição Tecidual , UteroglobinaRESUMO
41 Amino acid long N-terminal sequences of the three major human vitamin D-binding proteins (group-specific components Gc1F, Gc1S and Gc2) were characterized: they were identical. By computer analyses, the alignment of this N-terminal sequence with several sequences of human serum pre-proalbumin and human pre-alpha-fetoprotein was established.
Assuntos
Albumina Sérica , Proteína de Ligação a Vitamina D/sangue , alfa-Fetoproteínas , Sequência de Aminoácidos , Computadores , Humanos , Pré-Albumina , Precursores de ProteínasAssuntos
Caseínas , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Cromatografia em Gel , Cromatografia por Troca Iônica , Quimotripsina , Feminino , Fibrinogênio , Glicopeptídeos , Humanos , Fragmentos de Peptídeos/análise , Peptídeo Hidrolases , Ovinos , Especificidade da Espécie , EspectrofotometriaRESUMO
A cytosolic 21-kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. It was encountered in several tissues and species; however, its accurate function remained partially unknown. In order to obtain information from its structural features, we built a molecular model which revealed it to possess a nucleotide-binding site. In the present research, we describe the affinity of the bovine brain 21-kDa protein for nucleotides, and its association with cytosolic proteins, small GTP-binding proteins and lipid droplets. Our results suggest that, through its association with small GTP-binding proteins, the 21-kDa protein is implicated in signal mechanisms during cell growth and maturation.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Citosol/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Nucleotídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Conformação Proteica , Ratos , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The complete amino acid sequence (185 amino acid residues) of the good-type lysozyme from the egg white of ostrich (Struthio camelus) has been determined from the study of tryptic and Staphylococcus aureus V8 protease peptides as well as of CNBr and BNPS-skatole fragments. A slight sequence homology between the goose-type and chicken-type lysozymes has been observed.
Assuntos
Aves/metabolismo , Clara de Ovo , Muramidase/classificação , Sequência de Aminoácidos , Animais , GalinhasRESUMO
1. According to the important biological role of fatty acids and phospholipids in cell membranes, two cytosolic proteins implicated in their binding and transport in brain were considered, namely: Fatty Acid-Binding Protein and basic 21 kDa protein. 2. They were reviewed as well as their related protein families. 3. Although the two protein groups do not present significant sequence homologies, they share several similar properties and might thus be implicated in common physiological functions.
Assuntos
Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Proteínas de Neoplasias , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Ligação a Ácido Graxo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
A fatty acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified protein migrated as a single protein band in 15% polyacrylamide gel electrophoresis with an apparent molecular mass of 14.7 kDa. To ascertain that the purified protein was a FABP, it was submitted to fatty acid-binding tests. Oleic and palmitic acids bound to brain FABP but this was not the case for palmitoyl CoA. By Scatchard analysis the ligand binding values were: Kd = 0.28 microM, Bmax (mol/mol) = 0.6 for oleic acid and Kd = 0.8 microM, Bmax (mol/mol) = 2.1 for palmitic acid. The complete amino acid sequence of the brain FABP was determined and a microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP.
Assuntos
Química Encefálica , Proteínas de Transporte/metabolismo , Ligantes , Miocárdio/metabolismo , Proteínas de Neoplasias , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Bovinos , Citosol/química , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Dados de Sequência MolecularRESUMO
A fatty-acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified FABP behaved as an anionic protein with an apparent molecular mass of 14.7 kDa; its complete amino acid sequence was determined and microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP and bovine mammary-derived growth inhibitor (MDGI).
Assuntos
Química Encefálica , Proteínas de Transporte/isolamento & purificação , Ácidos Graxos/metabolismo , Proteínas de Neoplasias , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Bovinos , Proteínas de Ligação a Ácido Graxo , Dados de Sequência Molecular , Miocárdio/análiseRESUMO
The amino acid sequence of lysozyme c from chachalaca egg white was determined. Like other bird lysozymes c, that of the chachalaca has 129 amino acid residues. It differs from other avian lysozymes c by 2 to 31 amino acid substitutions as well as by being devoid of phenylalanine. It contains substitutions at 9 positions which are invariant in the other 7 bird lysozymes of known sequence. Although the chachalaca is classified zoologically in the order Galliformes, which includes chickens and other pheasant-like birds, its lysozyme differs more from those of pheasant-like birds than do the lysozymes c of ducks. Phylogenetic analysis of the sequence comparisons confirms that the lineage leading to chachalaca lysozyme c separated from that leading to other galliform lysozymes c before the duck lysozyme c lineage did. This indicates a contrast between protein evolution and evolution at the organismal level. Immunological comparison of chachalaca lysozyme c with other lysozymes of known sequence provides further support for the proposal that immunological cross-reactivity is strongly dependent on degree of sequence resemblance among bird lysozymes.
Assuntos
Aves/metabolismo , Clara de Ovo/análise , Muramidase/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Reações Cruzadas , Muramidase/imunologia , Fragmentos de Peptídeos/análise , Filogenia , Especificidade da Espécie , TripsinaRESUMO
Sequence studies of the N-terminal halves of the lysozymes isolated from Bombyx mori, Gallera mellonella and Spodoptera littoralis (Lepidoptera) allow us to classify these enzymes among the c (chicken) type lysozymes.
Assuntos
Lepidópteros/enzimologia , Muramidase/análise , Sequência de Aminoácidos , Animais , Evolução Biológica , Bombyx/enzimologia , Galinhas , Mariposas/enzimologia , Especificidade da EspécieRESUMO
Hydrosoluble transplantation antigens were prepared from membranes and microsomes of C(3)H mice (BP8 tumoral cells) and purified by isoelectric focusing. A biologically active fraction which seems homogenous by acrylamide gel electrophoresis was characterized: it specifically inhibits anti C(3)H hemagglutinin antibodies and provokes a highly significant prolongation of skin graft.
RESUMO
The complete amino acid sequence (186 amino acid residues) of a basic cytosolic protein from bovine brain has been determined. It was previously described as a phosphatidylethanolamine binding protein. Computer analyses have been used to calculate its hydropathy profile and to predict its secondary structure. Comparison with other proteins did not detect any significant sequence similarity, except for a short region which presents 53% sequence homology with bovine phosphatidylcholine transfer protein.
Assuntos
Proteína de Ligação a Androgênios , Química Encefálica , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Proteínas de Transporte , Bovinos , Citosol/análise , Peso Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Fosfatidilcolinas , Proteínas de Transferência de Fosfolipídeos , Homologia de Sequência do Ácido Nucleico , TripsinaRESUMO
A gamma-globulin fraction was isolated from the antiserum raised against a 21-kilodalton (kDa) basic protein which was purified from bovine brain cytosol. This fraction was employed to study the immunocytochemical localization of the 21-kDa protein during the development of rat brain. Immunostaining was observed on oligodendrocytes and their processes at all stages of development investigated. This immunostaining was less prominent in very young and adult brains. Myelin fibers were always moderately stained; neurons and astrocytes were not immunolabelled. The electron microscopic study revealed that the labelling covers the entire cytoplasm of the oligodendrocytes, being more dense along the membranes of the rough endoplasmic reticulum and the plasma membrane. Other cytoplasmic organelles were unstained. The present report emphasizes that 21-kDa protein may serve as a specific marker for oligodendroglial cells in the central nervous system despite its presence in peripheral organs.