RESUMO
Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 A resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric 'head-to-head' association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor.
Assuntos
Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/química , Fator de Células-Tronco/metabolismo , Sequência de Aminoácidos , Citocinas/química , Citocinas/metabolismo , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N-linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.
Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator Estimulador de Colônias/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Células CHO , Cromatografia de Afinidade , Cricetinae , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Escherichia coli , Feminino , Glicosilação , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-kit , Receptores Proteína Tirosina Quinases/química , Receptores de Fator Estimulador de Colônias/química , Proteínas Recombinantes/química , Valores de Referência , Sensibilidade e Especificidade , SolubilidadeRESUMO
The gene for human stem cell factor (SCF) encodes a leader sequence followed by 248 amino acids (Martin et al., 1990, Cell 63, 203). Of these 248 amino acids, the first 189 correspond to an extracellular domain and the remainder correspond to a hydrophobic transmembrane domain plus a cytoplasmic domain. A naturally occurring soluble form, released by proteolytic cleavage after amino acid 165, has been described. An alternatively spliced mRNA, lacking the codons for exon 6, has also been described. Since the amino acids encoded by exon 6 include the proteolytic cleavage site, the form expressed from the alternatively spliced mRNA tends to remain membrane-bound. In the present study, we have begun to explore structure/function relationships within the extracellular domain of SCF. Forms beginning at amino acid 1 (after the leader sequence) and ranging from 127 to 189 at the C-terminus have been recombinantly expressed in Escherichia coli and purified. In addition, forms missing the amino acids encoded by exon 6, forms missing up to 10 amino acids from the N-terminus, and forms with disulfide bond alterations have been expressed and purified. The forms have been characterized structurally, as well as functionally, in quantitative cell proliferation and receptor-binding assays. The results indicate that amino acids 1-141 comprise a structural and functional core and allow conclusions about the necessity of each of the two disulfide bonds for structure and function.
Assuntos
Escherichia coli/genética , Expressão Gênica , Fatores de Crescimento de Células Hematopoéticas/genética , Processamento Alternativo , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Fatores de Crescimento de Células Hematopoéticas/química , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Humanos , Substâncias Macromoleculares , Dobramento de Proteína , RNA Mensageiro/genética , Proteínas Recombinantes/química , Análise de Sequência , Fator de Células-Tronco , Relação Estrutura-AtividadeRESUMO
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
This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies. These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations. They exhibit full in vitro biological activity and have slightly lower pI's. Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively. The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis. These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor.
Assuntos
Fator Estimulador de Colônias de Granulócitos/biossíntese , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Códon , Escherichia coli , Glutamina , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/isolamento & purificação , Histidina , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.
Assuntos
Glicosídeo Hidrolases/metabolismo , Fator Estimulador de Colônias de Granulócitos/análise , Sequência de Aminoácidos , Animais , Células CHO , Sequência de Carboidratos , Carboidratos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cricetinae , Eletroforese em Gel de Poliacrilamida , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Hidrólise , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Proteínas Recombinantes/químicaRESUMO
This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Dissulfetos/química , Glicosilação , Fatores de Crescimento de Células Hematopoéticas/química , Humanos , Técnicas In Vitro , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Células-Tronco , Relação Estrutura-AtividadeRESUMO
Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/isolamento & purificação , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetinae , Relação Dose-Resposta a Droga , Escherichia coli/genética , Glicosilação , Fatores de Crescimento de Células Hematopoéticas/química , Fatores de Crescimento de Células Hematopoéticas/genética , Humanos , Mastócitos/efeitos dos fármacos , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Fator de Células-TroncoRESUMO
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.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cobre/química , Sulfato de Cobre , Cisteína , Escherichia coli/genética , Polarização de Fluorescência , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Cinética , Oxirredução , Conformação Proteica , Receptores de Fator Estimulador de Colônias de Granulócitos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Serina , Espectrofotometria UltravioletaRESUMO
Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.
Assuntos
Fator Estimulador de Colônias de Granulócitos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Focalização Isoelétrica , Mapeamento de Peptídeos , Proteínas Recombinantes/análiseRESUMO
We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow. A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211). The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain. This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated). Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment. Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet. Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion. The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar. The presence or absence of the carbohydrate does not influence the results of the various structural analyses.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/metabolismo , Células-Tronco/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Glicosilação , Fatores de Crescimento de Células Hematopoéticas/química , Humanos , Peso Molecular , Conformação Proteica , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Infravermelho , Fator de Células-TroncoRESUMO
Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N-terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high-performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. C., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/metabolismo , Fígado/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Células Cultivadas , Dissulfetos/química , Glicosilação , Fatores de Crescimento de Células Hematopoéticas/química , Fígado/citologia , Espectrometria de Massas , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos BUF , Fator de Células-TroncoRESUMO
The amino acid sequences of human interleukin-6 and granulocyte colony stimulating factor are approximately 30% homologous in the N-terminal region. The relative positions of four half-cystines in human interleukin-6 (IL-6) match four of the five in human granulocyte colony stimulating factor. Labeling experiments of recombinant interleukin-6 with tritiated iodoacetate confirmed that the molecule forms two intramolecular disulfide bonds and contains no detectable level of free sulfhydryls. By isolation and characterization of tryptic and subtilytic peptides obtained from different proteolytic digestions, the disulfide bonds of the IL-6 molecule were assigned to Cys44-Cys50 and Cys73-Cys83. The two disulfide bridges form two small loops which are separated by 22 amino acids. These structures are similar to those of recombinant granulocyte colony stimulating factor.