RESUMO
The many milk-clotting proteases from plant are glycosylated; attachment of monosaccharides to enzyme is an advantage for its activity and stability. In this study, gas chromatography coupled to mass spectrometry-electrospray ionization was used to identify glycans bond to proteases purified from Balanites aegyptiaca fruits pulp through cation exchange chromatography. Carbohydrates were identified according to the retention time and the ion at m/z after derivation by heptafluorobutyric acid. The chromatograms obtained from monosaccharides analysis revealed the presence of galactose, mannose, arabinose, xylose, rhamnose and glucuronic acid. The mass spectrometry-electrospray ionization spectra corroborated these findings.
Assuntos
Balanites/enzimologia , Frutas/enzimologia , Peptídeo Hidrolases/metabolismo , Glicosilação , Monossacarídeos/análise , Monossacarídeos/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Ácidos Urônicos/análise , Ácidos Urônicos/metabolismoRESUMO
We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.
Assuntos
Hemeritrina/análogos & derivados , Hemeritrina/genética , Sanguessugas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Biblioteca Gênica , Hemeritrina/química , Sanguessugas/fisiologia , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/análise , Reprodução , Alinhamento de SequênciaRESUMO
Although lactoferrins (Lfs) isolated from milk of various mammals exhibit a close structural relationship, they show species-specific binding to cells. To define the specificity of recognition of human (hLf), bovine (bLf) and murine (mLf) lactoferrin by human intestinal cells, we analysed the binding of the three proteins to a subclone derived from human carcinoma cell line HT29. We observed that hLf and bLf interact with two types of binding sites (K(d): 63+/-22 nM; 0.7+/-0.2 microM) while mLf was recognized only by the lowest affinity binding sites with a lower number of binding sites. Using N-terminal deleted human Lf variants, we found that the sequence G(1)RRRR(5) is mainly responsible for the interactions with HT29 cells. Lactoferrin-binding sites on the surface of HT29 cells were further identified as heparan sulphate and chondroitin sulphate glycosaminoglycans. We conclude that the presence of the sequence A(1)PRK(4) in bLf and K(1)ATT(4) in mLf provides an insight into why the interaction of bLf with cell membrane-associated glycosaminoglycans is similar to that of hLf and why binding of these lactoferrin species differs from that of murine Lf.
Assuntos
Enterócitos/metabolismo , Glicosaminoglicanos/metabolismo , Lactoferrina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Sulfatos de Condroitina/química , Glicosaminoglicanos/química , Células HT29 , Heparitina Sulfato/química , Humanos , Lactoferrina/química , Lactoferrina/genética , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade da EspécieRESUMO
In order to determine the N-glycosylation potential of maize, a monocotyledon expression system for the production of recombinant glycoproteins, human lactoferrin was used as a model. The human lactoferrin coding sequence was inserted into the pUC18 plasmid under control of the wheat glutenin promoter. Maize was stably transformed and recombinant lactoferrin was purified from the fourth generation seeds. Glycosylation was analysed by gas chromatography, lectin detection, glycosidase digestions and mass spectrometry. The results indicated that both N-glycosylation sites of recombinant lactoferrin are mainly substituted by typical plant paucimannose-type glycans, with beta1,2-xylose and alpha1,3-linked fucose at the proximal N-acetylglucosamine, and that complex-type glycans with Lewis(a) determinants are not present in maize recombinant lactoferrin.
Assuntos
Lactoferrina/metabolismo , Zea mays/metabolismo , Western Blotting , Glicosilação , Humanos , Modelos Químicos , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The serum of the freshwater prawn contains a sialic acid specific lectin (MrL) that agglutinates erythrocytes from rat and rabbit, as well as some Gram negative and positive bacterial strains. In this work, we performed the chemical characterization of the MrL purified by affinity chromatography on stroma from rat erythrocytes and by ion exchange chromatography. In its active form, MRL is a dimeric glycoprotein with 9.5 kDa per subunit. The amino acid sequence of the lectin was deduced from peptides obtained after trypsin treatment by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF). The predicted amino acid sequence of the lectin showed 54% homology with the hyperglycemic hormone from Macrobrachium rosenbergii. It also showed homology with the variable region of the human immunoglobulin kappa (22%) and lambda (27%) light chains. The lectin is a glycoprotein with 11% (w/w) carbohydrate content and is constituted by Gal, Man, GlcNAc, GalNAc and NeuAc in a molar ratio of 4:3:2:1:0.6. The primary structure of the carbohydrate chains of the lectin from the freshwater prawn was determined by affinity chromatography of MrL-glycopeptides on Con A and LCA lectin columns, which indicated that the main carbohydrate chains conforming the lectin are N-glycosidically linked. Man3 GlcNAc2.1 oligosaccharides were the most abundant structures with 57%) followed by Gal1.3 Man3 GlcNAc2.8 with 24%. Our results suggest that the freshwater prawn possess a lectin in the hemolymph plasma, related to those from the immunoglobulin superfamily.
Assuntos
Lectinas/química , Lectinas/isolamento & purificação , Palaemonidae/química , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Glicoproteínas/química , Humanos , Cadeias gama de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/química , Coelhos , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.
Assuntos
Calcineurina/metabolismo , Ciclofilinas/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Ciclofilinas/genética , Ciclofilinas/metabolismo , Ciclosporina/metabolismo , Ciclosporina/farmacologia , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Peptidilprolil Isomerase , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Mutação Puntual , Ligação Proteica/genéticaRESUMO
We previously characterized a receptor of Mr 105,000 for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of R2R3R4R5 of hLf in the interaction with cells, we studied the binding of hLf variants obtained either by tryptic proteolysis (hLf-2N, hLF-3N and hLf-4N) or by mutagenesis (rhLf-5N). Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. The binding parameters of bovine Lf and native hLf did not differ, whereas the binding parameters of murine Lf resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulfation, reduced the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N indicating that the hLf binding sites include sulfated molecules. The results suggest that the interaction of hLf with about 80,000 binding sites per Jurkat cell, mainly sulfated molecules, is dependent on R2R3R4, but not on R5. Interaction with about 20,000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. We conclude that the deletion of R2-R5 from hLf may serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.
Assuntos
Lactoferrina/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Humanos , Células Jurkat , Lactoferrina/química , Dados de Sequência Molecular , Ligação Proteica , Receptores de Superfície Celular/química , Linfócitos T/patologiaRESUMO
Production and characterization of human lactoferrin (hLf) in transgenic tobacco is reported. We have engineered two constructs containing either the native signal peptide from human lactoferrin or the signal peptide from sweet potato sporamin fused to human lactoferrin encoding cDNA. N-terminal sequences of rhLf purified from tobacco were identical to Lf from human milk for both constructs. The tobacco rhLf presents a molecular mass closely identical to native protein. Overall sugar composition shows the presence of plant specific xylose while sialic acid is absent. Binding parameters of the recombinant molecule to both Jurkat lymphoblastic T-cells or HT29-18-C1 enterocytes are similar to those of human lactoferrin isolated from milk.
Assuntos
Lactoferrina/genética , Nicotiana/genética , Plantas Tóxicas , Sequência de Aminoácidos , Sequência de Bases , Carboidratos/análise , Primers do DNA , DNA Complementar , Glicosilação , Humanos , Lactoferrina/química , Lactoferrina/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Lactoferrin, an iron-binding 80-kDa glycoprotein, is a major component of human milk whose structure is now well defined. The binding site of lactoferrin to the membrane receptor of lymphocyte has been located in the region 4-52, but the amino acids directly involved in the interaction have not been identified yet. To gain further insights into the structure-function relationships of the lactoferrin binding site, we first expressed the cDNA encoding human lactoferrin in the lepidoptera Spodoptera frugiperda cells (Sf9) using a recombinant baculovirus. The selected transformant secreted and N-glycosylated protein of 78 kDa which was immunoprecipitated by specific anti-lactoferrin antibodies. To confirm the structure and the function of the recombinant lactoferrin, the protein was purified by ion-exchange chromatography and its physical, biochemical, and biological properties were compared with those of the native protein. In particular, the N-terminal amino acid sequence and the iron-binding stability as a function of pH, of both proteins, were identical. The main difference concerns the glycosylation which leads to glycans of lower molecular masses as detected by the electrophoretic mobility of lactoferrin after N-glycosidase F treatment and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Despite the different glycosylation features, the recombinant lactoferrin retained the binding property to the Jurkat human lymphoblastic T-cell line of the native lactoferrin. On the basis of these analyses, production of protein mutants generated by site-directed mutagenesis is now in process.
Assuntos
Baculoviridae/genética , Lactoferrina/biossíntese , Lactoferrina/química , Animais , Linhagem Celular , Clonagem Molecular , DNA Complementar/isolamento & purificação , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Glicosilação , Humanos , Ferro/metabolismo , Células Jurkat/metabolismo , Lactoferrina/genética , Lactoferrina/isolamento & purificação , Espectrometria de Massas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência , Espectrofotometria , Espectrofotometria Ultravioleta , Spodoptera/genéticaRESUMO
We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg2-Arg3-Arg4-Arg5 of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf-2N, hLf-3N and hLf-4N) from native hLf that had been treated with trypsin. Native hLf bound to 102000 sites on Jurkat cells with a dissociation constant (Kd) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf-5N) bound to 17000 sites with a Kd of 12 nM. The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; Kd 30 nM) resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N, indicating that the hLf-binding sites include sulphated molecules. We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg2-Arg3-Arg4, but not on Arg5. Interaction with approx. 20000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. Moreover, the affinity of hLf for the latter binding site is enhanced approx. 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.
Assuntos
Arginina/metabolismo , Lactoferrina/metabolismo , Receptores de Superfície Celular/metabolismo , Sulfatos/metabolismo , Sequência de Aminoácidos , Animais , Arginina/genética , Sítios de Ligação , Bovinos , Cloratos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Células Jurkat , Lactoferrina/química , Lactoferrina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Tripsina/metabolismoRESUMO
1. Using direct, inhibiting and competitive enzyme-linked immunosorbent assay (ELISA), two steps were involved in the mapping of the recognition site of a polyclonal antibody against oxytocin (OT). 2. The percentage of cross-reactivity between OT and the N-terminal or the C-terminal fragment of OT demonstrated that the C-terminal fragment is the antigenic part of OT. 3. The percentage of cross-reactivity between OT and other molecules of the OT family indicated that the amino acid in the 8-position and the C-terminal amide of the OT molecule contribute to the recognition. 4. In the two sex segmental ganglia of the leech Erpobdella octoculata, where cells immunoreactive to the anti-OT are detected, the antibody has allowed to characterize an epitope close to the mammalian OT by its C-terminal part.
Assuntos
Anticorpos/química , Epitopos/química , Sanguessugas/química , Neuropeptídeos/química , Ocitocina/química , Ocitocina/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Gânglios/química , Gânglios/imunologia , Gônadas/inervação , Indicadores e Reagentes , Sanguessugas/anatomia & histologia , Dados de Sequência Molecular , Neuropeptídeos/imunologia , Mapeamento de Peptídeos , TitulometriaRESUMO
Theromyzon tessulatum vitellin was identified as a lipoglycoprotein of 490 kDa. The insolubility of this molecule in low-ionic-strength media was used to extract it from the ovaries. Antiserum prepared against vitellin was shown to react with a coelomic fluid component of 520 kDa. This vitellin precursor, or vitellogenin, was purified by gel permeation and ion-exchange column chromatography. These two lipoglycoproteins were characterized by amino acid, carbohydrate and lipid analysis and subunit composition. In spite of differences in terms of native molecular mass, solubility and isoelectric point, the lipoglycoproteins isolated from the coelomic fluid and the ovary were similar in their subunit components (a single polypeptide of 165 kDa) and in their amino acid and carbohydrate compositions. However, vitellogenin was found to be more highly lipidated (31.8% by mass) than vitellin (24% by mass) and lipid analysis indicated a higher amount of sterols and phospholipids in vitellogenin. From these data, we conclude that vitellogenin and vitellin are probably dimers of two identical subunit polypeptides plus lipid and that, after vitellogenin is sequestered in the oocyte, part of its lipid component is stripped from the molecule to give vitellin. Furthermore, electrophoretic analysis seems to indicate that vitellogenin synthesis and secretion is initiated following the third and last blood meal of the animal but that vitellogenin significantly accumulates in the coelomic fluid before being incorporated in the oocytes suggesting a complex mode of vitellogenesis regulation.