RESUMO
Human FSH consists of a mixture of isoforms that can be separated on the basis of differences in negative charge conferred by variations in the numbers of sialic acid residues that terminate oligosaccharide branches. Western analysis of human FSH isoforms separated by chromatofocusing revealed the presence of two human FSHbeta isoforms that differed in size. A low mol wt human FSHbeta isoform was associated with all FSH isoform fractions. A high mol wt human FSHbeta isoform was associated with the more acidic fractions and increased in relative abundance as the pI decreased. Characterization of representative human FSHbeta isoforms by mass spectrometry and automated Edman degradation revealed a low mol wt isoform that was not glycosylated. A high mol wt isoform was N-glycosylated at Asn residues 7 and 24. These results indicate that pituitary human FSH consists of two classes of molecules: those that possess a nonglycosylated beta-subunit and those that possess a glycosylated beta-subunit. Glycoprotein hormones are known to be elliptical molecules, and the beta-subunit oligosaccharides project outward from the short diameter, thereby increasing it. It is interesting to speculate that this change in shape might affect ultrafiltration rates, leading to differences in delivery rates to target tissues and elimination by filtration in the kidney.
Assuntos
Hormônio Foliculoestimulante/química , Animais , Western Blotting , Gonadotropina Coriônica/metabolismo , Cromatografia de Afinidade , Dietilestilbestrol/farmacologia , Eletroforese em Gel de Poliacrilamida , Feminino , Hormônio Foliculoestimulante/isolamento & purificação , Hormônio Foliculoestimulante/farmacologia , Subunidade beta do Hormônio Folículoestimulante , Glicosilação , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Humanos , Ensaio Imunorradiométrico , Masculino , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Ensaio Radioligante , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Testículo/metabolismoRESUMO
The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.
Assuntos
Complemento C3b/metabolismo , Complemento C3d/metabolismo , Herpesvirus Humano 4/metabolismo , Receptores de Complemento 3d/metabolismo , Proteínas da Matriz Viral/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Baculoviridae/genética , Biotinilação , Soluções Tampão , Complemento C3c/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Cinética , Ligantes , Lisina/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Receptores de Complemento 3d/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Cloreto de Sódio/metabolismo , Ressonância de Plasmônio de Superfície , Proteínas da Matriz Viral/genéticaRESUMO
Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains. Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg. Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a beta-sheet secondary structure with a melting temperature of 59 degrees C. Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2. Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form. These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand. Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2. Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric. Its sedimentation coefficient was determined to be 1.36 S. The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed. Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.
Assuntos
Complemento C3b/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores de Complemento 3d/química , Receptores de Complemento 3d/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Clonagem Molecular/métodos , Simulação por Computador , Sequência Consenso , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Pichia/genética , Ligação Proteica , Receptores de Complemento 3d/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Soluções , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , UltracentrifugaçãoRESUMO
To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifth component of complement from a rainbow trout (Oncorhynchus mykiss) liver cDNA library; this is the first report of C5 cloning in a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25kb), representing approximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validate the molecular information derived from the cloning we developed an improved purification protocol. Mass spectrometric analysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partial cDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liver and Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence of C5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish.
Assuntos
Complemento C5/genética , Oncorhynchus mykiss/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Complemento C3/genética , Complemento C4/genética , Complemento C5/classificação , Complemento C5/imunologia , DNA Complementar , Humanos , Dados de Sequência Molecular , Oncorhynchus mykiss/imunologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , alfa-Macroglobulinas/genéticaRESUMO
We have previously identified a 13-residue cyclic peptide, Compstatin, that binds to complement component C3 and inhibits complement activation. Herein, we describe the binding kinetics, structure-activity relationship, and biotransformation of Compstatin. Biomolecular interaction analysis using surface-plasmon resonance showed that Compstatin bound to native C3 and its fragments C3b and C3c, but not C3d. While binding of Compstatin to native C3 was biphasic, binding to C3b and C3c followed the 1:1 Langmuir binding model; the affinities of Compstatin for C3b and C3c were 22- and 74-fold lower, respectively, than that of native C3. Analysis of Compstatin analogs synthesized for structure-function studies indicated that 1) the 11-membered ring between disulfide-linked Cys2-Cys12 constitutes a minimal structure required for optimal activity; 2) retro-inverso isomerization results in loss of inhibitory activity; and 3) some residues of the type I beta-turn segment also interact with C3. In vitro studies of Compstatin in human blood indicated that a major pathway of biotransformation was the removal of Ile1, which could be blocked by N-acetylation of the peptide. These findings indicate that acetylated Compstatin is stable against enzymatic degradation and that the type I beta-turn segment is not only critical for preservation of the conformational stability, but also involved in intermolecular recognition.
Assuntos
Proteínas Inativadoras do Complemento/química , Proteínas Inativadoras do Complemento/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Biotransformação , Complemento C3/metabolismo , Cisteína/metabolismo , Hemólise , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/sangue , Ligação Proteica/imunologia , Relação Estrutura-AtividadeRESUMO
Compstatin, a newly described C3-binding peptide, inhibits complement activation by blocking C3 convertase-mediated cleavage of C3. As the complement activation is an essential part of the rejection reaction, we evaluated the ability of Compstatin to delay or prevent hyperacute rejection in an ex vivo xenograft model. Pig kidneys were perfused with fresh human blood containing either Compstatin (n=6) or a control agent (n=6). Graft survival and activation of complement, leukocytes and platelets both in the fluid-phase and in the tissue were examined. The survival of the Compstatin-perfused kidneys (median, 380 min) was significantly (P=0.0036) longer than that of the controls (median, 90 min). The classical complement pathway (C1rs-C1inhibitor and C4bc) was significantly and equally activated in both groups during the first 60 min. C3 activation products increased fivefold and terminal complement complex eightfold in the control group, but no increase occurred in the Compstatin group during this period. Immunohistochemistry showed less C3 and fibrin deposition and immune electron microscopy showed less terminal SC5b-9 complement complex deposition in the Compstatin group. A significant change in total white cells, neutrophils, myeloperoxidase, and expression of the surface activation markers CD11b (CR3) and CD35 (CR1) and CD62L (L-selectin) was observed in both groups. Leukocyte activation was lower in the Compstatin group but the difference was not statistically significant. There were no differences in platelet counts, thrombospondin, soluble P-selectin or beta-thromboglobulin between the groups. We conclude that Compstatin prolongs graft survival and suggest that it may be a useful agent for attenuating hyperacute rejection by inhibiting C3 and thus terminal complement pathway activation.
Assuntos
Proteínas Inativadoras do Complemento/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Complemento C3b/metabolismo , Complemento C3b/urina , Complemento C3c/metabolismo , Complemento C3c/urina , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/urina , Feminino , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Transplante de Rim/imunologia , Transplante de Rim/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Masculino , Microscopia Imunoeletrônica , Modelos Biológicos , Perfusão , Transplante HeterólogoRESUMO
Hybrid hormones were created using combinations of equine (e) LH, eFSH, and eCG alpha- and beta-subunit preparations. The efficiency of eFSH beta association was highest with eLH alpha (64-72%) and was lowest with eCG alpha (37-50%). Selective removal of alphaAsn56 oligosaccharide increased heterodimerization efficiency by 9-20% for eLH alpha, by 21-28% for eFSH alpha, and by 28-41% for eCG alpha. Both alpha and beta subunits contributed significantly to FSH receptor-binding activities of the hybrids. Purified hybrid hormone preparations consisting of either eFSH beta or eLH beta combined with eLH alpha, eFSH alpha, or eCG alpha were prepared. Equine FSH beta hybrids were more active in the FSH radioreceptor assay than eLH beta hybrids; within each beta-subunit group the eLH alpha hybrids were the most active, followed by eFSH alpha hybrids, while the least active were eCG alpha hybrids. A truncated, des(121-149) eLH beta derivative (eLH beta t) combined with native alpha-subunit preparations exhibited the same effect of alpha-subunit type on FSH receptor binding. Hybrids combining the eLH beta t derivative with Asn56-deglycosylated (N56dg-)eLH alpha, N56dg-eFSH alpha, and N56dg-eCG alpha preparations possessed 2.2- to 4.3-fold increased FSH receptor-binding activities as compared with the same hybrid preparations possessing the Asn56 carbohydrate. Granulosa cell bioassay of purified native eFSH beta and eLH beta hybrid hormones indicated no significant effect of the alpha-subunit carbohydrate differences on progesterone production. The alpha-subunit Asn56 oligosaccharide exerts a hormone-specific inhibitory influence on in vitro subunit reassociation and FSH receptor binding related to the size of its Man(alpha1-6)Man antenna.
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/genética , Gonadotropinas Equinas/metabolismo , Oligossacarídeos/genética , Receptores do FSH/genética , Receptores do FSH/metabolismo , Animais , Galinhas , Cromatografia em Gel , Cricetinae , Eletroforese em Gel de Poliacrilamida , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Hibridização Genética , Hormônio Luteinizante/metabolismo , Mutação , Oligossacarídeos/síntese química , Ratos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The third component of the complement system, C3, is a common denominator in the activation of the classical, alternative, and lectin pathways. The ability of C3 molecule to interact with at least 20 different proteins makes it the most versatile component of this system. Since these interactions are important for phagocytic, immunoregulatory, and immune evasion mechanisms, the analysis of its structure and functions has been a subject of intense research. Here we review our current work on the C3-ligand interactions, C3-related viral molecular mimicry, evolution of the complement system, and identification of C3-based complement inhibitors.
Assuntos
Complemento C3 , Animais , Antígenos Virais/imunologia , Complemento C3/genética , Complemento C3/imunologia , Complemento C3/metabolismo , Evolução Molecular , Humanos , Mimetismo MolecularRESUMO
Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Células CHO , Chlorocebus aethiops , Cromatografia em Gel , Cricetinae , Conformação Proteica , Coelhos , Membro 14 de Receptores do Fator de Necrose Tumoral , Células Vero , Proteínas do Envelope Viral/químicaRESUMO
The examples presented indicate that MALDI-MS is a useful tool for evaluating the progress of peptide synthesis at all the necessary levels: automated assembly, cleavage and deprotection chemistries, RP-HPLC analyses and purifications, and structural validation of the final product. The technique, if judiciously applied, permits the evaluation of complex peptide mixtures and often provides a semiquantitative overview. We have found that the availability of this method has enabled the provision of high-quality peptide reagents for use in the local research environment. The integration of this methodology into our peptide synthesis facility has also enabled and encouraged us to undertake more challenging synthetic problems such as phosphopeptide synthesis, peptide cyclizations, and peptide modification chemistries that would not ordinarily be offered if the laboratory lacked this technology. MALDI-MS is one of the more versatile and readily integrable mass spectrometric methods that can be incorporated into the average peptide synthesis laboratory.
Assuntos
Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Peptídeos/análise , Peptídeos/síntese química , Peptídeos/químicaRESUMO
There are two species for which both pituitary and placental gonadotropins are readily available, humans and horses. The human gonadotropins are better characterized than equine gonadotropins. Nevertheless, the latter are very interesting because they provide exceptions to some of the general structure-function principles derived from studies on human and other mammalian gonadotropins. For example, separate genes encode the hLH beta and hCG beta subunits while a single gene encodes eLH beta and eCG beta. Thus, eCG and eLH differ only in their oligosaccharide moieties and eLH is the only LH that possesses the O-glycosylated C-terminal extension previously believed to be restricted to chorionic gonadotropins. Truncation experiments involving eLH beta and hCG beta have suggested the C-terminal extension has no effect on receptor binding. However, the largest of three eCG forms which differ only in the extent of O-glycosylation possessed reduced affinity for LH and FSH receptors. This result suggested that effects of O-glycosylation need to be considered when examining the glycosylation differences between eLH and eCG responsible for the 10-fold lower eCG receptor binding affinity compared with that of eLH. Contribution of alpha Asn56 N-linked oligosaccharides to the different biological activities of eLH and eCG has been evaluated following selective removal using peptide-N-glycanase digestion of native equine alpha-subunit preparations. Hormones-specific patterns of glycosylation were observed on alpha Asn56 of eLH, eFSH, and eCG. Removal of alpha Asn56 oligosaccharides increased the rate of subunit association, the extent of association, and receptor binding activity. Some unassociated alpha-subunit oligosaccharides were identified which may interfere with subunit association because they were more abundant in unassociated subunit oligosaccharide maps than in a total oligosaccharide map. This was most striking in the case of eCG alpha in which two minor peaks became the major oligosaccharide peaks detectable in the unassociated eCG alpha fraction following association with eLH beta and eFSH beta. The biological activities exhibited by hybrid hormones, eLH alpha reassociated with oLH beta and pLH beta, found to be greater than those of oLH and pLH provided an interesting exception to the general rule that the beta-subunit determines the potency of the heterodimer. LH receptor binding activities of eLH beta-chimeric ovine/equine alpha-subunits suggested that the equine alpha-subunit N-terminal domain may be responsible for this effect. Equine FSH has higher FSH receptor binding activity than human, ovine, and porcine FSH preparations. This probably results from two factors. First, the presence of the equine alpha-subunit promotes receptor binding as noted above. Second, the overall -2 charge of the eFSH beta determinant loop, which is less negative that the -3 observed in other species, results from the presence of an Asn residue at position 88 instead of Asp. This apparently facilitates binding to the FSH receptor.
Assuntos
Gonadotropinas/química , Sequência de Aminoácidos , Animais , Gonadotropina Coriônica/química , Gonadotropina Coriônica/metabolismo , Hormônio Foliculoestimulante/química , Hormônio Foliculoestimulante/metabolismo , Glicosilação , Cavalos , Humanos , Hormônio Luteinizante/química , Hormônio Luteinizante/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de FusãoRESUMO
Both human neutrophil elastase (HNE) and free chymotrypsin (Chtr) proteolyze Chtr within the complex that Chtr forms with antichymotrypsin (ACT). As free Chtr is stable both to self-digestion and to digestion by HNE, these results are indicative of a stability and/or conformational change in Chtr that accompanies complex formation. As determined by both N-terminal sequence analysis and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS), the major initial sites of HNE cleavage of complexed Chtr are between gamma-chain residues A158/S159 and V188/S189. Significantly, this latter site is at the base of the S1 site that recognizes the P1 position of the serpin. A slower cleavage in the beta-chain between T139/G140 is also found. In addition, rACT is cleaved between residues V22/D23. The gamma-chain of complexed Chtr is also cleaved by free Chtr, but at different sites: L162/L163 and W172/G173. beta-Chain cleavages were also found between residues Q81/K82 and F114/S115. Cleavages similar to those described above were also found when Chtr was complexed with the L358F-rACT variant, but not for Chtr complexed with either of the smaller inhibitors bovine pancreatic trypsin inhibitor or turkey ovomucoid third domain, nor for the covalent adduct of Chtr with N-p-tosylphenylalanyl chloromethyl ketone. We conclude that the structural change in Chtr making it a proteinase substrate is coupled with the large conformational change in ACT following complex formation. Complexed Chtr is much less reactive toward proteolytic digestion in the presence of high salt than in its absence, in accord with the high-salt induced release of active enzyme from the Chtr.rACT complex and the suggestion that electrostatic interactions mediate the coupling of structural change between rACT and Chtr within the Chtr.rACT complex. Potential physiological consequences of this work are explored.
Assuntos
Quimotripsina/química , alfa 1-Antiquimotripsina/química , Sequência de Aminoácidos , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Humanos , Hidrólise , Elastase de Leucócito , Modelos Moleculares , Dados de Sequência Molecular , Elastase Pancreática/metabolismo , Fosfatos/química , Conformação ProteicaRESUMO
A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through cysteine 8, combined with mass spectrometry and Edman degradation, showed that disulfide bonds link cysteine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cysteine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.
Assuntos
Simplexvirus/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Dissulfetos , Humanos , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities, G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about 1/30th the mitogenic activity of bovine PRL; G-ePRL was approximately 1/10th as active as ePRL. Glycosylation of G-ePRL at Asn31 was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29-37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man3, GlcNAc2, GalNAc1, Fuc0.6, Gal0.2, NeuAc0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn31 carbohydrate unit is a fucosylated complex mono- and /or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.
Assuntos
Monossacarídeos/química , Prolactina/química , Aminoácidos/análise , Animais , Divisão Celular/efeitos dos fármacos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Glicosilação , Cavalos , Peso Molecular , Hipófise/química , Prolactina/isolamento & purificação , Prolactina/farmacologia , Células Tumorais CultivadasRESUMO
Three equine CG (eCG) forms with identical amino acid sequences but different mol wt and monosaccharide compositions were isolated from a crude eCG preparation and designated eCG-L (low mol wt), eCG-M (medium mol wt), and eCG-H (high mol wt). No differences in primary structure between each form and the known sequence of eCG were observed. SDS-PAGE of these preparations under reducing conditions revealed that the mol wt differences between them were due only to the different sizes of their beta-subunits. Carbohydrate compositions suggested an increase in O-glycosylation in the higher mol wt forms. N-Linked glycopeptide fragments obtained from eCG beta-subunits by endoproteinase Lys-C digestion had identical electrophoretic mobilities. Thus, the different molecular sizes of the beta-subunits were associated only with disparities in O-glycosylation of their C-terminal extension. When tested in a LH and several FSH radioligand assay systems, eCG-H proved to have significantly lower receptor-binding activities than eCG-L and eCG-M. Endo-beta-galactosidase digestion increased the FSH receptor-binding activity of all eCG forms; however, partially deglycosylated eCG-H remained the least active form. Thus, the O-linked oligosaccharides of eCG-H exert a negative influence on its receptor-binding activity.
Assuntos
Gonadotropina Coriônica/química , Gonadotropina Coriônica/metabolismo , Glicosídeo Hidrolases , Oligossacarídeos/química , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Cavalos , Masculino , Metaloendopeptidases/metabolismo , Peso Molecular , Monossacarídeos/análise , Ratos , Ovinos , Relação Estrutura-Atividade , beta-Galactosidase/metabolismoRESUMO
Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP). Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protection against tryptic cleavage. Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates. Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes. The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling.
Assuntos
Trifosfato de Adenosina/metabolismo , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Conformação Proteica , Salmonella typhimurium/enzimologia , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Clonagem Molecular , Expressão Gênica , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Pentosiltransferases/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Fosforilação , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , TripsinaRESUMO
Phospho- and unphospho- peptides were used to define the essential sequence for a tau epitope, which is recognized by Tau-1 antibody and phosphorylated in Alzheimer's disease (AD). The epitope was mapped within the amino acid residues 192-199 of tau and was phosphorylated by the p34cdc2/p58cyclin A proline directed kinase (PDPK), but not by purified mitogen activated protein kinase (p42mapk). Addition of phosphate to the last serine of the epitope was the most effective in abolishing the reactivity of the epitope to Tau-1 antibody. Our results suggest that one and possibly more members of the PDPK family may play a role in the pathogenesis of AD.
Assuntos
Doença de Alzheimer/metabolismo , Neuropeptídeos/metabolismo , Fosfopeptídeos/metabolismo , Proteínas tau/metabolismo , Sequência de Aminoácidos , DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases Direcionadas a Prolina , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
We previously described a monoclonal antibody (MAb) library generated by infecting BALB/c mice with rubella virus (RV) and selected by an enzyme-linked immunosorbent assay (ELISA) using purified virion targets. Plasmid pARV02-01, which expresses the fusion protein RecA1-35-GIGDLGSP-E1(202)-E1(283)-GDP-LacZ9-1015 in Escherichia coli, was shown to be a ligand for MAbs E1-18 and E1-20 (J. S. Wolinsky, M. McCarthy, O. Allen-Cannady, W. T. Moore, R. Jin, S. N. Cao, A. Lovett, and D. Simmons, J. Virol. 65:3986-3994, 1991). Both of these MAbs neutralize RV infectivity. A series of five overlapping synthetic peptides was made to further explore the requirements of this MAb binding domain. One of these peptides (SP15; E1(208) to E1(239)) proved an effective ligand for both MAbs in the ELISA. Stepwise synthesis of SP15 defined the minimal amino-terminal requirement for binding MAb E1-18 as E1(221) and that of MAb E1-20 as E1(223); the minimal carboxyl-terminal requirement is uncertain but does not exceed E1(239). Immunization of mice and rabbits with SP15 induced polyvalent antibody reactive with SP15, with other overlapped and related but not unrelated synthetic peptides, and with RV. The rabbit anti-SP15 antibody showed neutralization activity to RV similar to that of MAbs E1-18 and E1-20 but lacked hemagglutination inhibition activity. These data define a neutralization domain on E1 and suggest that the RV epitopes conserved by SP15 may be critical for protective host humoral immune responses.