RESUMO
Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.
Assuntos
Acrosina/genética , Acrosina/isolamento & purificação , DNA Complementar/genética , Espermatozoides/enzimologia , Perus/genética , Acrosina/química , Acrosina/metabolismo , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fenômenos Químicos , Cromatografia em Gel , Clonagem Molecular , Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Inibidor da Tripsina Pancreática de Kazal/metabolismoRESUMO
The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.
Assuntos
Proteínas Aviárias/química , Sêmen/química , Inibidor da Tripsina Pancreática de Kazal/química , Perus , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/isolamento & purificação , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Masculino , Dados de Sequência Molecular , Alinhamento de Sequência , Testículo/química , Inibidor da Tripsina Pancreática de Kazal/genética , Inibidor da Tripsina Pancreática de Kazal/isolamento & purificação , Perus/genéticaRESUMO
Azurocidin is an inactive serine protease homolog with primary sequence similarity to neutrophil elastase, cathepsin G, and proteinase 3. The aim of this study was to investigate possible consequences of differential glycosylation of azurocidin in regard to its secretion, protein stability as measured by susceptibility to proteolysis, and antibacterial activity. Site-directed mutagenesis was employed to generate mutant azurocidin variants lacking individual N-glycosylation sites. Our results show that N-linked glycans may play a role in proper azurocidin folding and subsequent secretion by insect cells. We also demonstrate that N-linked glycosylation contributes to azurocidin stability by protecting it from proteolysis. The lack of N-glycosylation at individual sites does not significantly influence the azurocidin antibacterial activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Proteínas de Transporte/genética , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Glicosilação , Hidrólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Tripsina/metabolismoRESUMO
Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.
Assuntos
Sequência Conservada , Dissulfetos/química , Evolução Molecular , Ferro/metabolismo , Répteis/genética , Transferrina/química , Transferrina/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cisteína , Glicosilação , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Filogenia , SolubilidadeRESUMO
Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile (Crocodilus niloticus, C. rhombifer) and one snake (Python molurus bivittatus) ovotransferrins. The proteins were purified by anion exchange and hydrophobic chromatography, and their N-terminal amino-acid sequences, molecular mass and isoelectric points were determined. All three proteins are glycosylated and their N-glycan chromatographic profiles show the largest contribution of neutral oligosaccharides in crocodile and disialylated glycans in python ovotransferrin. The absorption spectra of iron-saturated transferrins were analysed. Iron release from these proteins is pH-dependent, showing a biphasic character in crocodile ovotransferrins and a monophasic type in the python protein. The reason for the different types of iron release is discussed.
Assuntos
Jacarés e Crocodilos , Boidae , Conalbumina/isolamento & purificação , Animais , Conalbumina/metabolismo , Ovos/análise , Feminino , HomeostaseRESUMO
Transferrins form an important class of iron-binding proteins widely distributed in the physiological fluids of vertebrates and invertebrates. In vertebrates they are present mostly in serum as serotransferrins. In birds and reptiles transferrins are also found in eggs as ovotransferrins. However, until now only chicken and duck ovotransferrin sequences have been published. This paper presents data on the purification, biochemical analysis, cloning and sequencing of ovotransferrins from red-eared turtle, African ostrich and turkey, revealing their significant homology with other known ovotransferrin sequences. The proteins were purified by size-exclusion and anion-exchange chromatography. Isoelectric points, iron-saturated and iron-free spectra, as well as the mRNA nucleotide sequences of 2,409 nt (ORF: 2,106 nt encoding a 701-amino-acid polypeptide; ), 2,418 nt (ORF 2,118 nt encoding a 705-amino-acid polypeptide; ), and 2,397 nt (ORF: 2,118 nt encoding a 705-amino-acid polypeptide; ) were determined for ostrich (OtrF), red-eared turtle (TtrF), and turkey (MtrF) ovotransferrin, respectively.
Assuntos
Conalbumina/genética , Conalbumina/isolamento & purificação , Struthioniformes/genética , Perus/genética , Tartarugas/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/isolamento & purificação , Proteínas do Ovo/isolamento & purificação , Glicosilação , Ferro/metabolismo , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Análise EspectralRESUMO
Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.
Assuntos
Oncorhynchus mykiss/metabolismo , Sêmen/enzimologia , Inibidores da Tripsina , alfa 1-Antitripsina , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Complementar/análise , Glicosilação , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência , Inibidores da Tripsina/química , Inibidores da Tripsina/genética , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/metabolismoRESUMO
Azurocidin, also known as cationic antimicrobial protein 37 kDa (CAP37) or heparin-binding protein (HBP) is an inactive homolog of serine proteinases residing in granulocytes. The ability to cleave peptide bond was lost due to replacement of two of the three residues from the conserved catalytic triad characteristic for serine proteinases. Azurocidin has a broad spectrum of antimicrobial activity, mainly against Gram-negative bacteria. It is also recognized as a multifunctional inflammatory mediator for its contracting effects on endothelial cells causing an increase of vascular permeability, capacity to bind endotoxin and ability to attract monocytes to inflammation sites.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Granulócitos/metabolismo , Mediadores da Inflamação/metabolismo , Serina Endopeptidases/análise , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Permeabilidade Capilar/imunologia , Permeabilidade Capilar/fisiologia , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Humanos , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The properties of plant purple acid phosphatases (PAPs), metallophosphoesterases present in some bacteria, plants and animals are reviewed. All members of this group contain a characteristic set of seven amino-acid residues involved in metal ligation. Animal PAPs contain a binuclear metallic center composed of two irons, whereas in plant PAPs one iron ion is joined by zinc or manganese ion. Among plant PAPs two groups can be distinguished: small PAPs, monomeric proteins with molecular mass around 35 kDa, structurally close to mammalian PAPs, and large PAPs, homodimeric proteins with a single polypeptide of about 55 kDa. Large plant PAPs exhibit two types of structural organization. One type comprises enzymes with subunits bound by a disulfide bridge formed by cysteines located in the C-terminal region around position 350. In the second type no cysteines are located in this position and no disulfide bridges are formed between subunits. Differences in structural organisation are reflected in substrate preferences. Recent data reveal in plants the occurrence of metallophosphoesterases structurally different from small or large PAPs but with metal-ligating sequences characteristic for PAPs and expressing pronounced specificity towards phytate or diphosphate nucleosides and inorganic pyrophosphate.
Assuntos
Fosfatase Ácida/genética , Glicoproteínas/genética , Plantas/enzimologia , Fosfatase Ácida/química , Fosfatase Ácida/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Glicoproteínas/química , Glicoproteínas/fisiologia , Dados de Sequência Molecular , Filogenia , Plantas/química , Plantas/genética , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Acid phosphatase (AP) and diphosphonucleoside phosphatase/phosphodiesterase (PPD1) were purified from yellow lupin (Lupinus luteus L.) immature green seeds (40 days after blooming), dry seeds (40 days later) and dry seeds stored for 160 days. Both enzymes are known to differ in the type of N-glycosylation: the first has an N-glycosylation pattern typical for a vacuolar protein, while the second enzyme has a pattern typical for an extracellular or membrane-bound protein. N-Glycans were released from each of the enzyme preparations, fluorescence labeled, separated and identified by HPLC (GlycoSep N and GlycoSep H columns). Changes in the level of each N-glycan during seed maturation and dormancy were compared. The results show that N-glycan processing in the case of AP and PPD1-two proteins residing in the same plant organ, but possibly in different compartments-is not synchronized and performed not only in metabolically active maturing seeds, but also in metabolically inactive dormant seeds.
Assuntos
Fabaceae/embriologia , Monoéster Fosfórico Hidrolases/metabolismo , Polissacarídeos/metabolismo , Sementes/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Hidrólise , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/isolamento & purificação , Polissacarídeos/química , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Espectrometria de FluorescênciaRESUMO
N-glycans of human neutrophil azurocidin, enzymatic inactive homolog of serine proteinase playing important and multifunctional roles in antimicrobial defense, endotoxin binding, monocyte, and T-cell activation, were isolated by hydrazinolysis and fluorescence labeled. An ion-exchange chromatography on GlycoSep C column separated neutral, mono-, and disialylated glycans. The glycans from each group were separated subsequently on GlycoSep N and GlycoSep H columns. Sequential exoglycosidase treatment and HPLC mapping allowed determining 21 different glycan structures, majority of them being neutral (79.8%), the rest-mono- (13.1%) and disialylated (1.2%).