Role of sugar chains in the expression of the biological activity of human erythropoietin.

Various deglycosylated derivatives of recombinant human erythropoietin (hEPO) were prepared and used to determine the role of the sugar chains in the expression of its biological activity in vivo and in vitro. Three N-linked oligosaccharides of hEPO have been partially or fully removed to obtain N-glycan (NG) (2)-, NG(1)-, and NG(0)-hEPO carrying two, one, and no N-linked sugar chains, respectively. The preparation lacking only O-linked sugar chain O O-glycan (OG) (0)-hEPO was also used. As de-N-glycosylation proceeded, the in vivo activity of the hormone decreased drastically, and the activity of these derivatives was correlated with the number of sialic acids bound to them. On the contrary, the in vitro activity was increased by the de-N-glycosylation; NG(0)-hEPO showed a 3-fold higher specific activity than the intact hormone. This was confirmed by binding experiments of the derivatives to target cells. The in vitro activity and the affinity also correlated with the number of sialic acids bound to the deglycosylated hEPO preparations. On the other hand, OG(0)-hEPO was as active as the intact hormone in vivo and in vitro. In conclusion, the N-linked sugar chains are not required for in vitro activity but required for in vivo activity, acting as anchors for the essential terminal sialic acids. The O-linked sugar chain has no essential role in the biological activity of the hormone in vivo or in vitro.     

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.     

鼠粒细胞集落刺激因子受体的纯化及鉴定

粒细胞集落刺激因子受体(G-CSFR)在鼠NFS-60细胞中有较高的含量,通过对NFS-60细胞的大规模培养,用CHAPS及超速离心抽提G-CSFR, 经G-CSF亲和层析纯化获得G-CSFR, 采用ABC-ELISA进行鉴定.     

人血清白蛋白和粒细胞集落刺激因子融合蛋白的克隆表达

构建重组人血清白蛋白粒细胞集落刺激因子（HSA-hG-CSF）表达载体,用毕赤酵母表达该重组蛋白。PCR扩增出人血清白蛋白基因（HSA）和粒细胞集落刺激因子基因（hG-CSF）,GGGGS作为小肽接头,采用重叠PCR的方法将HSA和hG-CSF拼接起来,与质粒载体pPIC9K连接,转化大肠杆菌感受态细胞DH-5α。抽提质粒,用SalI酶切重组质粒,电转化法导入毕赤酵母SMD1168中,通过表型筛选和诱导表达实验得到蛋白表达工程菌。Western-blotting分析表明融合蛋白具有粒细胞集落刺激因子免疫原性。NFS-60细胞测活实验分析表明体外活性达到约4.0×10^7IU/mg。     

Enhanced Heterologous Expression of Biologically Active Human Granulocyte Colony Stimulating Factor in Transgenic Tobacco BY-2 Cells by Localization to Endoplasmic Reticulum

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.     

▽5-人粒系集落刺激因子(▽5-hG-CSF)的cDNA克隆、序列测定与表达

从LPS刺激的正常中国人外周血单核细胞中提取mRNA,经反转录(RT)-PCR扩增出不含信号肽和成熟型N端5氨基酸(V5-hG-CSF)的cDNA片段,酶切后组入大肠杆菌表达载体pJLA602中。序列测定表明,克隆片段与国外报道的高活性hG-CSF cDNA序列一致。重组子经诱导表达、小鼠骨髓细胞体外CFU-G测试表明,表达产物具明显的粒细胞集落刺激活性。     

蛋白质氯胺-T双相碘标法的建立及其应用

常规的蛋白质碘标方法易引起被标细胞因子的失活,是受体配基竞争结合实验失败的原因之一.试用氯胺-T双相碘标法标记rhG-CSF和rhEPO,并应用受体配基竞争结合分析法测定NFS-60细胞G-CSF受体及BET-2细胞EPO受体的特性.结果显示所获 ¹²⁵I-EPO和 ¹²⁵I-G-CSF放射比活度均较高;发现BET-2细胞有高、低两种亲和力的EPO受体,NFS-60细胞只有一种高亲和力的G-CSF受体,所获结果与文献资料相一致.说明氯胺-T双相碘标法是细胞因子同位素碘标记的理想方法之一.     

Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.     

Purification and characterization of the receptor for murine granulocyte colony-stimulating factor.

A receptor for mouse granulocyte colony-stimulating factor (G-CSF) has been found on the cell surface of mouse myeloid leukemia cell line NFS-60. Chemical cross-linking of the receptor with radioiodinated G-CSF, followed by gel electrophoresis in the presence of sodium dodecyl sulfate, has revealed that the G-CSF receptor in the NFS-60 cells is a single polypeptide of Mr approximately 100,000-130,000. The receptor in the membrane fraction of NFS-60 cells were solubilized in an active form with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid. The solubilized receptor was purified approximately 100,000-fold to near homogeneity using a G-CSF affinity gel and gel filtration on a Superose 12 column, as measured by the selective precipitation of the 125I-G-CSF-receptor complex by polyethylene glycol. The purified G-CSF receptor has two classes of binding characteristics, one with an equilibrium dissociation constant (Kd) of 120-360 pM which is comparable with the Kd value for the cell-surface receptor, and the other with a higher Kd value of 2.6-4.2 nM. Analyses of the purified receptor by ligand blotting and sucrose density gradient centrifugation indicated that the low-affinity receptor is the monomer of the Mr 100,000-130,000 protein, whereas the high-affinity receptor consists of oligomers of the protein.     

Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures

Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice alpha-amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was confirmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was confirmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF.     

Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin

The effect of carbohydrate on the conformation and chain dimensions of mucous glycoproteins was investigated by using light-scattering and circular dichroism studies of native, asialo, and deglycosylated (apo) ovine submaxillary gland mucin (OSM). OSM is a large glycoprotein that is extensively O-glycosylated by the disaccharide alpha-NeuNAc(2-6)alpha-GalNAc-O-Ser/Thr. Measurements of root mean square radius of gyration, (Rg2)1/2, and hydrodynamic radius, Rh, for OSM and its derivatives were carried out as a function of molecular weight by using static and dynamic light-scattering techniques. The results were fit to the wormlike chain model for describing the dimensions of extended polymer chains. By use of this model, values of h, the length per amino acid residue, and q, the persistence length, which is a measure of chain stiffness, were obtained. These values were then used to assess the conformation and degree of chain extension of intact OSM and its partially and totally deglycosylated derivatives. Native and asialo mucin are found to be highly extended random coils, with asialo mucin having a somewhat less extended structure than intact mucin. Upon the complete removal of the carbohydrate side chains, the extended structure characteristic of intact and asialo mucin collapses to chain dimensions typical of denatured globular proteins. Conformational analyses based on the rotational isomeric state model were also performed by using the probability maps of N-acetyl-O-(GalNAc)-Thr-N-methylamide as starting conformations for native and asialo mucin. The results suggest that both the glycosylated and nonglycosylated residues in native mucin may occupy a small region of conformational space having -90 degrees less than phi less than -60 degrees and 60 degrees less than psi less than 180 degrees, while a slightly broader range is found to fit asialo mucin. The proposed conformations obtained for these mucins are consistent with their circular dichroism spectra. Significantly larger ranges of phi and psi values were obtained for apo mucin, as would be expected from its circular dichroism spectra and increased flexibility. These results indicate the expanded mucin structure is the direct result of peptide core glycosylation. These observations together with the results of earlier studies indicate that steric interactions of the O-linked GalNAc residue with the peptide core are primarily responsible for the expanded mucin structure and that these perturbations extend to the nonglycosylated amino acid residues. This expanded mucin conformation must be a significant determinant of the viscoelastic properties of these molecules in solution.     

Conformation, and dissociation-recombination of chemically deglycosylated ovine lutropin

The conformations of intact and acid dissociated, as well as the relative rates of conformational repair of reassociating, ovine lutropin, and a chemically deglycosylated form of lutropin, have been studied by circular dichroism, zero-order, and difference absorption spectroscopy. Deglycosylation produces only small conformational changes in the intact or acid dissociated form of the molecule. While conformational repair of reassociating lutropin requires up to 72 h, and goes no further than 75-80%, deglycosylated lutropin reaches greater than 90% conformational repair in less than 4 h. The efficacy of second-order absorption spectra in characterizing conformations and conformational changes in these molecules is demonstrated.     

Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells.

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.     

Conservation of human fibrinogen conformation after cleavage of the B beta chain NH2 terminus

Human fibrinogen exposed to protease III from Crotalus atrox venom is cleaved near the NH2 terminus of the B beta chain yielding a species of Mr 325,000 (Fg325) with impaired thrombin clottability. The derivative was compared with intact fibrinogen in a number of ways to determine whether the functional defect resulted from a conformational change or from the loss of a polymerization site. NH2-terminal amino acid sequencing of isolated A alpha, B beta, and gamma chains showed that Fg325 contained intact A alpha and gamma chains, but differed from fibrinogen by the absence of the first 42 residues of the B beta chain. Fibrinopeptide A was present and was cleaved at the same rate in both fibrinogen and Fg325. The rate and extent of A alpha and gamma cross-linking by factor XIIIa was also indistinguishable. In contrast, the thrombin-catalyzed coagulation of Fg325 was 46% less in extent and 180-fold slower than observed for intact fibrinogen. A conformational comparison of Fg325 and fibrinogen was made using immunochemical and spectroscopic approaches. Antisera specific for different regions of the fibrinogen molecule were used to characterize the epitopes in Fg325. The only significant differences were found in the NH2-terminal region of the B beta chain, probed with antiserum to B beta 1-118. The conformational similarity of Fg325 and fibrinogen was confirmed by the identity of both near and far UV CD spectra of the two proteins. Structural, functional, and immunochemical results imply that cleavage of 42 NH2-terminal residues from the B beta chain is not accompanied by a measurable conformational change. The residues of this B beta chain segment, which are evidently located on the surface of the molecule, in conjunction with the NH2-terminal part of the A alpha chain appear to play an important role in the expression of a fibrin polymerization site.     

Structural characterization of natural and recombinant human granulocyte colony-stimulating factors.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which stimulates predominantly neutrophilic granulocyte colony formation in mammals. Natural human G-CSF (hG-CSF) and recombinant hG-CSF produced in Chinese hamster ovary (CHO) cells transfected with the cDNA clone for hG-CSF have been purified to apparent homogeneity for structural and biological comparison. The amino acid sequence of recombinant hG-CSF, composed of 174 amino acid residues, was identical with that of natural hG-CSF and also with the sequence predicted from the cDNA. Both forms of hG-CSF have a free Cys-17 and two intramolecular disulfide linkages, between Cys-36 and Cys-42, and between Cys-64 and Cys-74. The O-glycosylation occurred at Thr-133 in both hG-CSFs. Similar CD spectra were obtained for both hG-CSFs. Additionally, both forms showed almost the same biological activities determined by in vitro colony-forming assay and in vivo assay. It is thus concluded that the recombinant hG-CSF is indistinguishable from its natural counterpart and that the former is valuable for more detailed characterization and clinical use.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.     

O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.     

Expression cloning of a receptor for murine granulocyte colony-stimulating factor

Two cDNAs encoding the receptor for murine granulocyte colony-stimulating factor (G-CSF) were isolated from a CDM8 expression library of mouse myeloid leukemia NFS-60 cells, and their nucleotide sequences were determined. Murine G-CSF receptor expressed in COS cells could bind G-CSF with an affinity and specificity similar to that of the native receptor expressed by mouse NFS-60 cells. The amino acid sequence encoded by the cDNAs has demonstrated that murine G-CSF receptor is an 812 amino acid polypeptide (Mr, 90,814) with a single transmembrane domain. The extracellular domain consists of 601 amino acids with a region of 220 amino acids that shows a remarkable similarity to rat prolactin receptor. The cytoplasmic domain of the G-CSF receptor shows a significant similarity with parts of the cytoplasmic domain of murine interleukin-4 receptor. A 3.7 kb mRNA coding for the G-CSF receptor could be detected in mouse myeloid leukemia NFS-60 and WEHI-3B D+ cells as well as in bone marrow cells.