RESUMO
Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.
Assuntos
Dissulfetos/química , Glicoproteínas/química , Glicoproteínas/genética , Mapeamento de Peptídeos/métodos , Receptores Citoplasmáticos e Nucleares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Dimerização , Ácido Ditionitrobenzoico/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Peso Molecular , Osteoprotegerina , Fragmentos de Peptídeos/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral , Sensibilidade e Especificidade , Análise de Sequência , Serina Endopeptidases/metabolismo , Termolisina/metabolismoRESUMO
Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.
Assuntos
Citocinas/farmacologia , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/enzimologia , Linfocinas/farmacologia , Metaloendopeptidases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Interleucina-1/farmacologia , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Receptores Proteína Tirosina Quinases/química , Receptor de TIE-1 , Receptores de TIE , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador alfa/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Procedures for HPLC peptide map analysis of recombinant human granulocyte colony stimulating factor include reduction and S-carboxymethylation of the denatured protein, as well as protease digestion with Staphylococcus aureus endoproteinase Glu-C followed by reverse-phase liquid chromatographic separations. Under nonoptimized experimental conditions analytical problems including methionine modification during carboxymethylation, as well as generation of large, insoluble fragments and nonspecific cleavages during proteolytic digestion, occurred. These problems have complicated the analysis of peptide digests and affected the performance of HPLC columns. This report describes the elimination of these problems by optimizing peptide mapping procedures. We found that mild reduction and alkylation conditions can prevent methionine modification, while protease digestion in the presence of urea at room temperature alleviates generation of peptides derived from incomplete digestion and nonspecific cleavage by endoproteinase Glu-C. Peptide maps generated using the optimized procedures contain fewer peptide peaks with higher recovery. Elimination of incomplete digestion, which generates fewer larger, insoluble peptides, substantially extends the life of reverse-phase columns. The optimized method reproducibly produced peptide maps suitable for routine analysis.
Assuntos
Fator Estimulador de Colônias de Granulócitos/análise , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Humanos , Metionina , Dados de Sequência Molecular , Proteínas Recombinantes/análiseRESUMO
The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.