Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

Mutagenesis of human granulocyte colony stimulating factor

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.     

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.     

Biological activity of human granulocyte colony stimulating factor with a modified C-terminus

Granulocyte colony-stimulating factor (G-CSF) undergoes receptor-mediated internalization into target cells which are normally restricted to neutrophilic granulocytes and their committed progenitor cells, suggesting that it may be applicable as a myeloid cell-targeting vehicle. To test this notion, we constructed a cDNA encoding a human G-CSF/murine stem cell factor (mSCF) chimeric molecule in a mammalian expression vector and transfected NIH3T3 cells with this plasmid. The resulting chimeric cytokine consisted of the entire G-CSF sequences fused to Lys148 of mSCF. It can be released from the surface membrane of NIH3T3 transformants through proteolytic cleavage at Ala164 of mSCF. The culture media conditioned by a number of stable transformants, which were confirmed by an enzyme-linked immunosorbent assay (ELISA) to secrete an hG-CSF derivative, were examined for their ability to stimulate CFU-G-derived colony formation as well as the proliferation of G-CSF-dependent NFS-60 cells. The results indicated that this C-terminus modified version of hG-CSF is as potent as recombinant hG-CSF in both assays.     

Human granulocyte colony stimulating factor: effects on human long-term bone marrow cultures

Human granulocyte colony stimulating factor (G-CSF) was previously shown to support the survival and proliferation of early myeloid progenitors (pre-CFU) that are capable of generating more mature CFU-GM progenitor cells. To evaluate the scope of action of G-CSF in the hierarchy of hematopoietic stem cells, we studied the effects of recombinant G-CSF (rhG-CSF) on long-term cultures of normal human bone marrow cells (LTBMC). We found that rhG-CSF predominantly influenced initial cell proliferation and expansion of CFU-GM progenitor cells in LTBMC before establishment of a confluent adherent layer. In rhG-CSF-treated LTBMC, the stromal cell layer was associated with a higher proliferative capacity and progenitor cell content as compared to control cultures. This effect was pronounced early after layer confluence and was gradually lost with culture time. rhG-CSF did not alter the duration of the productive phase of LTBMC, suggesting that it may not be active on the hematopoietic stem cells responsible for LTBMC propagation. Alternatively, stromal cells may exert tight regulatory control over progenitor cells, even in the presence of rhG-CSF.     

Aggregation of recombinant bovine granulocyte colony stimulating factor in solution

Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50°C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50°C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.     

Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50g/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.     

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wild-type form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

Clinicopathological study of involvement of granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor in non-lymphohematopoietic malignant tumors accompanied by leukocytosis

Involvement of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in non-lymphohematopoietic malignant tumors accompanied by leukocytosis was clinicopathologically investigated. Among 1,778 autopsy cases in the last 20 years, 485 lesions of 439 cases with non-lymphohematopoietic malignant tumors accompanied by leukocytosis with a white blood cell count of 10,000/mm3 or greater during the course were immunohistologically examined for G-CSF and GM-CSF. Three (0.7%) and two cases (0.5%) were G-CSF- and GM-CSF-positive, respectively. GM-CSF mRNA was confirmed by using non-fixed cryopreserved tumor tissues in one case positive for GM-CSF. G-CSF-positive cases were large cell carcinoma of the lung, adenocarcinoma of the colon, and adenocarcinoma of the stomach, and GM-CSF-positive cases were spindle cell carcinoma of the lung and malignant thymoma. In the case with stomach carcinoma, the primary lesion showing moderately differentiated adenocarcinoma was negative, but the lung metastatic lesion showing less differentiated adenocarcinoma was G-CSF-positive. The survival period was six months or less in four out of five positive cases. The highest white blood cell count in five CSF-positive cases was markedly elevated: 29,400-103,500/mm3 (mean: 59,700/mm3). In four cases, excluding one case which may have been markedly affected by chemotherapy, the bone marrow showed hyperplasia, and the number of the granulocyte series cells significantly increased. There were three cases (0.7%) negative for both G-CSF and GM-CSF, although they showed marked leukocytosis (60,000/mm3 or higher) which were higher than the mean count of CSF-positive cases and was not observed in autopsy cases with non-tumorous diseases. Other stimulating factors may be involved in the development of leukocytosis in such cases.     

Characterization of the receptor binding determinants of granulocyte colony stimulating factor.

We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.     

Functional characterization of recombinant human granulocyte colony stimulating factor (hGMCSF) immobilized onto silica nanoparticles

Objectives

Granulocyte macrophage colony stimulating factor (GMCSF), an important therapeutic cytokine, was immobilized onto silica nanoparticles. Maintenance of structural integrity and biological performance in immobilized cytokine was assessed to augment its applicability in possible biomedical implications.

Results

Following its cloning and expression in E. coli, the recombinant human GMCSF (hGMCSF) was purified as a GST-tagged protein corresponding to a 42 kDa band on SDS-PAGE. The purified cytokine was immobilized onto biocompatible silica nanoparticles (~129.4 nm) by adsorption and the binding was confirmed by dynamic light scattering and infrared spectroscopy. Maximum binding of hGMCSF was at 6.4 µg mg^?1 silica nanoparticles. Efficient release of the cytokine from the nanoparticles with its structural integrity intact was deduced from circular dichroism spectroscopy. hGMCSF-immobilized silica nanoparticles efficiently increased the proliferation of RAW 264.7 macrophage cells with 50 % increase in proliferation at 600 ng hGMCSF µg^?1 silica nanoparticles.

Conclusions

Silica nanoparticles successfully immobilized hGMCSF maintaining its structural integrity. The release of the immobilized cytokine from silica nanoparticles resulted in the increased proliferation of macrophages indicating the potential of the system in future applications.

     

Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor.

Normal as well as retrovirally transformed avian myeloid precursor cells require the colony stimulating factor cMGF for their survival, proliferation and colony formation in vitro. cMGF has been shown to be a glycoprotein which is active in the picomolar concentration range. Co-expression of kinase type oncogenes in v-myb or v-myc transformed myeloid cells induces cMGF expression and confers factor independence via an autocrine mechanism. Here we describe the molecular cloning of cMGF from a myeloblast cDNA library and show that it is a 201 amino acid residue secretory protein which is modified by signal peptide cleavage and glycosylation during translocation into the lumen of membrane vesicles. A bacterially expressed trpE-cMGF fusion protein induces proliferation of E26 transformed myeloblasts in a cMGF bioassay suggesting that glycosylation is not absolutely necessary for biological activity. Sequence comparison reveals that cMGF is distantly related to G-CSF and IL-6.     

Isolation of colony stimulating factor from human milk

Human milk contains colony stimulating factor (CSF), a polypeptide growth factor, which stimulates in in vitro bone marrow culture proliferation and differentiation of colony forming granulocytic macrophage progenitor cells (CFU-GM) to form colonies. This activity was not found in either bovine milk or colostrum when assayed in human or mouse bone marrow cells. The human milk CSF activity is destroyed by treatment with proteases. However, neither 6M urea, 4M guanidine hydrochloride, 5 mM dithiothreitol, nor exposure to pH 2 will inactivate the milk derived CSF. Gel filtration and isoelectric focusing indicate that human milk CSF differs biochemically from the other CSFs isolated from various sources and has a molecular weight between 250,000 and 240,000 and an isoelectric point between 4.4 and 4.9.     

Modulation of granulocyte colony stimulating factor conformation and receptor binding by methionine oxidation

Biosimilars offer an avenue for potential cost savings and enhanced patient access to various emerging therapies in a budget neutral way. Biosimilars of the granulocyte colony stimulating factor (GCSF) are an excellent example in this regard with as many as 18 versions of the drug being currently approved across globe for treatment of neutropenia. Here, we identified oxidation of the various methionine residues in GCSF as a key heterogeneity that adversely impact its efficacy. In agreement with earlier studies, it was found that oxidation of Met 122 and Met 127 significantly contributes toward reduction of GCSF efficacy, measured using binding affinity to the GCSF receptor. The combination of molecular dynamics simulation along with structural characterization studies established that oxidation of Met 127 and Met 122 brings about a small local conformational change around the B‐C loop in GCSF structure due to slight displacement of Asp113 and Thr117 residues. The simulation studies were validated using fluorescence quenching experiments using acrylamide as quencher and site‐directed mutagenesis by replacing Met 122 and Met 127 residues with alanine. The results of this study lead to an enhanced mechanistic understanding of the oxidation in GCSF and should be useful in protein engineering efforts to design stable, safe, and efficacious GCSF product. In addition, the structure‐function information can provide targets for protein engineering during early drug development and setting specifications of allowable limits of product variants in biosimilar products.     

Partial primary structures of human and murine macrophage colony stimulating factor (CSF-1)

Approximately 40 amino-terminal residues and 20 internal residues of CSF-1 purified from the media of cultured human pancreatic carcinoma (MIA PaCa) cells and of cultured murine L cells have been identified. Results indicated that the two subunits in each molecule of biologically active CSF-1 are identical in their amino-terminal portions. The twelve amino-terminal residues of MIA PaCa CSF-1 were found to be identical to those of human-urinary CSF-1, suggesting that the polypeptide portions of the two human proteins may be identical. Approximately 75% of the amino acids identified in both MIA PaCa CSF-1 and murine CSF-1 were found to be common to both. No homology to other proteins was observed. This study suggests a subunit polypeptide Mr nearer to 17K than to 26K predicted from cDNA.     

Low-resolution structure of recombinant human granulocyte-macrophage colony stimulating factor

A recombinant form of human granulocyte-macrophage colony stimulating factor (GM-CSF) which contains no carbohydrate has been crystallized. Multiple isomorphous replacement analysis using five heavy-atom derivatives has yielded an image of the structure at 6 A resolution that showed two molecules per asymmetric unit and allowed determination of the non-crystallographic symmetry transformation. The 6 A resolution result shows that the core of GM-CSF consists of four helices. The angles at which the helices pack together distinguishes this structure from known antiparallel four-helix bundle proteins. Consideration of the amino acid sequence properties and previous structural characterizations of GM-CSF leads to an assignment of the probable protein segments that form the helices.