RESUMO
Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques to highlight differences in neonatal nutrition. We developed a milk proteomics technique that overcomes previous technical barriers including pervasive post-translational modifications and limited sample volume. We identified 1606 and 518 proteins in human and macaque milk, respectively. During analysis of detected protein orthologs, we identified 88 differentially abundant proteins. Of these, 93% exhibited increased abundance in human milk relative to macaque and include lactoferrin, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, vitamin D-binding protein, and haptocorrin. Furthermore, proteins more abundant in human milk compared with macaque are associated with development of the gastrointestinal tract, the immune system, and the brain. Overall, our novel proteomics method reveals the first comprehensive macaque milk proteome and 524 newly identified human milk proteins. The differentially abundant proteins observed are consistent with the perspective that human infants, compared with nonhuman primates, are born at a slightly earlier stage of somatic development and require additional support through higher quantities of specific proteins to nurture human infant maturation.
Assuntos
Lactação/fisiologia , Leite Humano/química , Anotação de Sequência Molecular , Proteoma/isolamento & purificação , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Desenvolvimento Infantil/fisiologia , Cromatografia Líquida , Feminino , Trato Gastrointestinal/crescimento & desenvolvimento , Trato Gastrointestinal/metabolismo , Humanos , Sistema Imunitário/crescimento & desenvolvimento , Sistema Imunitário/metabolismo , Lactente , Lactoferrina/isolamento & purificação , Lactoferrina/metabolismo , Macaca mulatta/crescimento & desenvolvimento , Macaca mulatta/metabolismo , Leite Humano/metabolismo , Proteoma/metabolismo , Receptores de Imunoglobulina Polimérica/isolamento & purificação , Receptores de Imunoglobulina Polimérica/metabolismo , Especificidade da Espécie , Espectrometria de Massas em Tandem , Transcobalaminas/isolamento & purificação , Transcobalaminas/metabolismo , Proteína de Ligação a Vitamina D/isolamento & purificação , Proteína de Ligação a Vitamina D/metabolismo , alfa 1-Antiquimotripsina/isolamento & purificação , alfa 1-Antiquimotripsina/metabolismoRESUMO
Alpha 1-antichymotrypsin (ACT) inhibits chymotrypsin-like enzymes, particularly neutrophil cathepsin G. Moreover, ACT in its native form suppresses chemotaxis of neutrophils and decreases neutrophil production of superoxide radicals. We recently showed that Moraxella catarrhalis ubiquitous surface protein (Usp) A1 is able to specifically bind ACT in the context of a novel virulence mechanism. In this study, we report that recombinant UspA1(557-704) coupled to CNBr-Sepharose can be used in a simple one-step purification of ACT from human plasma. UspA1(557-704)-purified ACT remains intact and active as shown by binding to M. catarrhalis and a chymotrypsin inhibition assay. The novel method for ACT isolation from plasma has important advantages in simplicity and time as compared to conventional methods.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cromatografia de Afinidade/métodos , Moraxella catarrhalis/química , alfa 1-Antiquimotripsina/isolamento & purificação , Western Blotting , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Humanos , alfa 1-Antiquimotripsina/sangueAssuntos
Albuminas/química , Albuminas/isolamento & purificação , Artefatos , Eletroforese em Gel Bidimensional/métodos , Glicoproteínas/sangue , Glicoproteínas/isolamento & purificação , Lectinas/química , Glicoproteínas/química , Humanos , Espectrometria de Massas/métodos , Peso Molecular , Proteômica/métodos , Sensibilidade e Especificidade , alfa 1-Antiquimotripsina/sangue , alfa 1-Antiquimotripsina/química , alfa 1-Antiquimotripsina/isolamento & purificaçãoRESUMO
Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicoproteínas/análise , Glicoproteínas/isolamento & purificação , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Glicoproteínas/química , Dados de Sequência Molecular , Oxirredução , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Polissacarídeos/análise , Polissacarídeos/química , Polissacarídeos/metabolismo , Resinas Sintéticas , alfa 1-Antiquimotripsina/análise , alfa 1-Antiquimotripsina/química , alfa 1-Antiquimotripsina/isolamento & purificação , alfa 1-Antitripsina/análise , alfa 1-Antitripsina/química , alfa 1-Antitripsina/isolamento & purificação , alfa-2-Glicoproteína-HSRESUMO
Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.
Assuntos
Endopeptidases/metabolismo , Substâncias de Crescimento/metabolismo , Macrófagos Peritoneais/enzimologia , Precursores de Proteínas/metabolismo , alfa 1-Antiquimotripsina/sangue , alfa 1-Antiquimotripsina/farmacologia , Animais , Precursores Enzimáticos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Processamento de Proteína Pós-Traducional , Inibidores de Serina Proteinase/sangue , Inibidores de Serina Proteinase/farmacologia , alfa 1-Antiquimotripsina/isolamento & purificaçãoRESUMO
Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.
Assuntos
Medula Suprarrenal/química , Grânulos Cromafim/química , Cisteína Endopeptidases/metabolismo , Inibidores de Serina Proteinase/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , Medula Suprarrenal/ultraestrutura , Animais , Bovinos , Quimotripsina/metabolismo , Encefalina Metionina/análise , Encefalinas/metabolismo , Imunofluorescência , Ponto Isoelétrico , Precursores de Proteínas/metabolismo , Inibidores de Serina Proteinase/farmacologia , alfa 1-Antiquimotripsina/farmacologiaRESUMO
An alpha1-antichymotrypsin-like serpin has been implicated in Alzheimer's disease (AD) based on immunochemical detection of alpha1-antichymotrypsin (ACT) in amyloid plaques from the hippocampus of AD brains. The presence of neuroendocrine isoforms of ACTs and reported variations in human liver ACT cDNA sequences raise the question of the molecular identity of ACT in brain. In this study, direct reverse transcription-polymerase chain reaction and cDNA sequencing indicate that the hippocampus ACT possesses the reactive site loop that is characteristic of serpins, with Leu as the predicted P1 residue interacting with putative chymotrypsin-like target proteases. The deduced primary sequence of the human hippocampus ACT possesses more than 90% homology with reported primary sequences for the human liver ACT. Moreover, identical ACT primary sequences deduced from the cDNAs were demonstrated in the hippocampus of control and AD brains. Northern blots showed that ACT mRNA expression in hippocampus was 900 times lower than that in liver. Also, hippocampus and liver ACT proteins demonstrated differential sensitivities to deglycosylation. Overall, reverse transcription-polymerase chain reaction combined with cDNA and primary sequence analyses have defined the molecular identity of human hippocampus ACT in control and AD brains. The determined reactive site loop domain of hippocampus ACT will allow prediction of potential target proteases inhibited by ACT in AD.
Assuntos
Doença de Alzheimer/metabolismo , Hipocampo/química , Fígado/química , Inibidores de Serina Proteinase/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , DNA Complementar/química , Glicosilação , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , alfa 1-Antiquimotripsina/química , alfa 1-Antiquimotripsina/genéticaRESUMO
alpha 1-Antichymotrypsin, a member of the serpins, is the predominant plasma inhibitor of neutrophil cathepsin G. The aim of this study was to purify ostrich alpha 1-antichymotrypsin and to compare its biochemical properties with those of other species. Ostrich alpha 1-antichymotrypsin was purified from serum by ammonium sulphate fractionation, QAE-Sephadex C-50 and phenyl-Toyopearl chromatography. N-terminal sequence, amino acid composition, molecular mass, isoelectric point and reaction with cathepsin G, elastase and chymotrypsin were determined. SDS-PAGE revealed a M, of 55,000 for ostrich alpha 1-antichymotrypsin and pI values of 6.8 and 4.1-4.3 were obtained. The amino acid composition revealed 444 residues and the N-terminal sequence of the first 20 residues revealed a homology of 30% when compared with several other alpha 1-antichymotrypsin sequences. Total inhibition of cathepsin G by ostrich alpha 1-antichymotrypsin was found at a 4:1 molar ratio of inhibitor to enzyme which was similar to that found for commercial alpha 1-antichymotrypsin. Immunological studies highlighted the lack of cross-reactivity between ostrich and human alpha 1-antichymotrypsin. The study indicated that ostrich alpha 1-antichymotrypsin-like molecule exhibited similar properties to human alpha 1-antichymotrypsin although there were notable differences.
Assuntos
Inibidores de Serina Proteinase/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Aves , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Inibidores de Serina Proteinase/sangue , Especificidade da Espécie , alfa 1-Antiquimotripsina/sangueRESUMO
The current assays for serum prostate specific antigen (PSA) have failed to produce the same PSA values on the same specimens because of problems with antibody specificity and calibrator preparation. To eliminate these problems, we proposed to replace the current serum PSA assay with an assay specific for the PSA-ACT (PSA-alpha 1-antichymotrypsin) complex in the serum. An assay specific for the PSA-ACT complex was established using the anti-PSA antibody to coat the microplate for capturing the PSA complex and anti-ACT polyclonal antibodies for quantification. There was an excellent correlation between serum concentrations of PSA-ACT and total PSA, using either the Hybritech calibrator (gamma = 0.996) or a serum calibrator prepared in house (gamma = 0.993), in random as well as in serial specimens from 14 individual patients. Even though we did not find a gradual increase in the percentage of PSA-ACT with the increase of total PSA in cancer patients, a slightly higher percentage of free PSA was measured in pooled normal sera (18%) and in pooled sera containing only 12 ng/ml of total PSA (12%), compared to serum pools containing elevated PSA (> 100 ng/ml) level, in which most PSA was in the complex form (95%). Therefore, using an assay that specifically measures the PSA-ACT complex in the serum not only simplifies the preparation of calibrator but eliminates the difficulty of antibody selection, it also allows various assay kits to produce identical PSA values and also improve the test specificity for prostate cancer.
Assuntos
Antígeno Prostático Específico/isolamento & purificação , Hiperplasia Prostática/sangue , Neoplasias da Próstata/sangue , Humanos , Masculino , Antígeno Prostático Específico/sangue , Kit de Reagentes para Diagnóstico , alfa 1-Antiquimotripsina/sangue , alfa 1-Antiquimotripsina/isolamento & purificaçãoRESUMO
We have explored various chromatographic procedures with the intention of establishing an isolation procedure that would allow us to isolate a large quantity of PSA-ACT (prostate specific antigen-alpha 1-antichymotrypsin) complex either from patients' sera or from incubation mixtures of free PSA and protease inhibitors. We found that at pH 7.2, both free PSA and PSA-ACT molecules are negatively charged and bind to the DEAE-Sepharose column. However, they could be separated from each other using a linear gradient of NaCl at pH 7.2. Both free PSA and PSA-ACT molecules were also found to be retained by the Con A Sepharose column because of the carbohydrate moiety of the PSA molecule. These two molecules were not separable by Con A chromatography. These two molecules apparently differ in their isoelectric points and were well separated by chromatofocusing using a pH gradient from pH 9 to 6. It appears that chromatofocusing can also be used to identify the isoforms of free PSA because of its high resolving power. The large difference in molecular size between free PSA and PSA-ACT complex allowed their separation by gel filtration chromatography on a column containing either S-100, S-200, or S-300 gel. S-200 gel appeared to be the best for the separation of free PSA from PSA-ACT and for the removal of other contaminating serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antígeno Prostático Específico/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , Cromatografia , Cromatografia em Gel , Concanavalina A , Dextranos , Etanolaminas , Humanos , Antígeno Prostático Específico/sangue , Sefarose , alfa 1-Antiquimotripsina/sangueRESUMO
Evidence is presented showing that alpha 1-antichymotrypsin (ACT) inhibits a novel prohormone thiol protease (PTP) involved in processing the enkephalin precursor. Colocalization of ACT immunoreactivity with PTP within isolated secretory vesicles of bovine adrenal medulla and pituitary indicated that endogenous ACT could regulate PTP in vivo. The endogenous 60 kDa bovine ACT (bACT)-like protein was purified from pituitary by chromatography on DEAE-Sepharose, chromatofocusing, butyl-Sepharose, and Sephacryl S-200. Characterization showed that the bACT-like protein was a potent inhibitor of PTP (Ki,app value of 2.2 nM) as well as an effective inhibitor of chymotrypsin (Ki,app value of 2.3 nM). Furthermore, the bACT-like protein formed sodium dodecyl sulfate-stable complexes with chymotrypsin, which is typical of serpin protease inhibitors. Importantly, PTP formed sodium dodecyl sulfate-stable complexes with human ACT, suggesting that PTP's cleavage specificity may resemble the reactive center of ACT. PTP cleavage of enkephalin-containing peptides at the NH2-terminal side of paired basic residues (Lys-Arg, Arg-Arg, Lys-Lys), flanking the COOH terminus of (Met)enkephalin (Tyr-Gly-GLy-Phe-Met), indicates methionine at the P1 position. PTP cleavage of peptide-methylcoumarin amide and peptide-p-nitroanilide substrates demonstrated specificity for paired basic and monobasic residues, as well as a role for methionine in PTP's cleavage site. These results showing PTP's ability for processing at a methionine residue which resembles the P1 specificity of ACT are compatible with inhibition of PTP by ACT. These findings are the first demonstration of the involvement of a protease inhibitor in neuropeptide precursor processing. The known developmental regulation of ACT in brain and significant amounts of ACT in amyloid plaques of Alzheimer's disease suggest a possible role for PTP in the maturation of peptidergic neurons.
Assuntos
Medula Suprarrenal/metabolismo , Cisteína Endopeptidases , Endopeptidases/metabolismo , Encefalinas/biossíntese , Oligopeptídeos/metabolismo , Hipófise/metabolismo , alfa 1-Antiquimotripsina/isolamento & purificação , alfa 1-Antiquimotripsina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia em Gel , Cromatografia por Troca Iônica , Quimotripsina/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
alpha 1-Antichymotrypsin was detected as a minor but normal component of pancreatic juice and found, with the aid of monoclonal antibodies and lectins, to differ from serum alpha 1-antichymotrypsin with respect to its carbohydrate moiety. alpha 1-Antichymotrypsin carries blood group determinants, as is typical for glycoproteins of pancreatic origin.
Assuntos
Antígenos de Grupos Sanguíneos/imunologia , Pâncreas/imunologia , alfa 1-Antiquimotripsina/imunologia , Anticorpos Monoclonais/imunologia , Carboidratos/análise , Digoxigenina , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas Imunoenzimáticas , Lectinas , Pâncreas/química , Suco Pancreático/química , Polissacarídeos/análise , alfa 1-Antiquimotripsina/sangue , alfa 1-Antiquimotripsina/isolamento & purificaçãoRESUMO
Aberrantly phosphorylated tau proteins (i.e., A68 or PHF-tau) and beta-amyloid or A4 (beta A4) peptides are major components of pathologic lesions in Alzheimer disease (AD). Although A68 and beta A4 colocalize in AD neurofibrillary tangles (NFTs) and amyloid-rich senile plaques (SPs), the mechanisms leading to the convergence of A68, beta A4, and other proteins in the same AD lesions are unknown. To probe the biological properties of A68 in vivo, and to assess interactions of A68 with endogenous proteins in the rodent brain, we injected A68, dephosphorylated A68 (DEP-A68), and normal adult human tau protein into the hippocampus and neocortex of rats. In marked contrast to DEP-A68 and tau, A68 resisted rapid proteolysis and induced codeposits of three rodent proteins--i.e., beta A4, ubiquitin, and alpha 1-antichymotrypsin (ACT)--that accumulate in AD NFTs and SPs together with A68. These findings suggest that A68 may interact with beta A4, ubiquitin, and ACT in neuronal perikarya as well as in the extracellular space after release of A68 from degenerating neurons. The model system described here will facilitate efforts to elucidate mechanisms leading to the convergence of A68, beta A4, ubiquitin, and ACT in hallmark lesions of AD.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proteínas do Tecido Nervoso/farmacologia , Peptídeos beta-Amiloides/isolamento & purificação , Animais , Química Encefálica , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Imuno-Histoquímica , Modelos Biológicos , Emaranhados Neurofibrilares , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Ubiquitinas/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , Proteínas tau/farmacologiaRESUMO
Genetic variation of human alpha 1-antichymotrypsin (ACT) was investigated in sera using thin-layer polyacrylamide gel isoelectric focusing (pH range 4.0-6.5) followed by immunoprinting with a monospecific anti-human ACT antibody. Sialidase-treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant "ACT Bochum" was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.
Assuntos
alfa 1-Antiquimotripsina/genética , alfa 1-Antiquimotripsina/isolamento & purificação , Feminino , Variação Genética , Humanos , Imunoeletroforese , Focalização Isoelétrica , Pneumopatias Obstrutivas/sangue , Pneumopatias Obstrutivas/genética , Masculino , Linhagem , Fenótipo , alfa 1-Antiquimotripsina/deficiênciaRESUMO
The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.
Assuntos
Substâncias de Crescimento/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , Anticorpos , Neoplasias da Mama , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Meios de Cultura Livres de Soro , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Estradiol/farmacologia , Feminino , Humanos , Imunoeletroforese Bidimensional , Focalização Isoelétrica , Peso Molecular , alfa 1-Antiquimotripsina/biossíntese , alfa 1-Antiquimotripsina/farmacologiaRESUMO
alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.
Assuntos
Oligossacarídeos/química , Polissacarídeos/química , alfa 1-Antiquimotripsina/química , Amidoidrolases , Asparagina , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia de Afinidade , Humanos , Hidrogênio , Imunoeletroforese Bidimensional , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Polissacarídeos/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , alfa 1-Antitripsina/química , alfa 1-Antitripsina/isolamento & purificaçãoRESUMO
While an inhibitory effect on natural killer (NK) cell activity was demonstrated with partially purified alpha 1 Achy, neither highly purified alpha 1 Achy from two healthy donors nor from one patient with giant-cell arteritis, which carries more highly branched glycans, inhibited the NK cytotoxicity. Our purification procedure, based on immunoaffinity chromatography and gel filtration, was not in question since the pure alpha 1-proteinase inhibitor (alpha 1PI) prepared in our laboratory by using a similar procedure continued to inhibit the NK cytotoxicity. If an inhibitory effect not related to antiprotease activity occurs with alpha 1PI, it is surprising that it is not shared by alpha 1 Achy which, like alpha 1PI, belongs to the serpin family and which possesses a strong structural homology with alpha 1PI. Our finding that alpha 1PI is able to affect human NK cytotoxicity while alpha 1 Achy (even with more highly branched glycans) is unable to suggests that events controlling NK activity may involve other enzymes than chymotrypsin-like enzymes.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , alfa 1-Antiquimotripsina/farmacologia , alfa 1-Antitripsina/farmacologia , Arterite/sangue , Sobrevivência Celular , Células Gigantes , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Albumina Sérica/farmacologia , alfa 1-Antiquimotripsina/isolamento & purificação , alfa 1-Antitripsina/isolamento & purificaçãoRESUMO
Our recent studies demonstrated that alpha 1-antichymotrypsin (ACT), a serine protease inhibitor, was associated with the beta-protein in the brain amyloid deposits of Alzheimer's disease, aged human controls and aged monkeys, suggesting a role for the inhibitor in the amyloid deposition. In the present study we used immunohistochemistry to test for the presence of ACT in the amyloid deposits which contain, as their major component, a protein different from the beta-protein. ACT was not found in the amyloid deposits in primary or secondary amyloidosis, familial and amyloidotic polyneuropathy or Creutzfeldt-Jakob disease (non-beta-protein amyloidoses), but was found (together with beta-protein) in Alzheimer's disease, Down's syndrome, normal aging, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin. These results suggest a specific association of ACT with beta-protein amyloid. We next examined the distribution of the inhibitor in normal human brain and in various human neuropathological states in order to identify cells that express this protein during brain degeneration. In addition to its association with amyloid, ACT immunoreactivity was also located in astrocytes near areas of neuronal or tissue loss, in a few neurons and pericytes and in the epithelium of the choroid plexus.
Assuntos
Doença de Alzheimer/patologia , Amiloide/análise , Amiloidose/patologia , alfa 1-Antiquimotripsina/análise , Doença de Alzheimer/imunologia , Amiloide/imunologia , Amiloide/isolamento & purificação , Peptídeos beta-Amiloides , Amiloidose/imunologia , Astrócitos/análise , Astrócitos/imunologia , Humanos , Imuno-Histoquímica , Inibidores de Serina Proteinase/imunologia , alfa 1-Antiquimotripsina/imunologia , alfa 1-Antiquimotripsina/isolamento & purificaçãoRESUMO
Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.
Assuntos
Inibidores de Proteases/isolamento & purificação , alfa-Globulinas/isolamento & purificação , Antitrombina III/isolamento & purificação , Cromatografia de Afinidade , Proteínas Inativadoras do Complemento 1/isolamento & purificação , Humanos , Cininogênios/isolamento & purificação , Macroglobulinas/isolamento & purificação , Plasminogênio/isolamento & purificação , Inibidores de Proteases/sangue , Inibidores da Tripsina/isolamento & purificação , alfa 1-Antiquimotripsina/isolamento & purificação , alfa 1-Antitripsina/isolamento & purificação , alfa 2-Antiplasmina/isolamento & purificaçãoRESUMO
Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.