Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin

Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.     

High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant

The highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild-type and Δ-fuc-t Δ-xyl-t mutant, the latter containing N -glycans lacking the plant-specific, core-bound α1,3-fucose and β1,2-xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene-derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20 °C. In Western blot analysis, the molecular size of moss-produced recombinant human EPO (rhEPO) was identified to be 30 kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5 µg/mL. Transgenic Physcomitrella Δ-fuc-t Δ-xyl-t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5- and 10-L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250 µg/g dry weight of moss material after 6 days. Silver staining of rhEPO on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) taken from the bioreactor culture demonstrated a high purity of the over-expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella Δ-fuc-t Δ-xyl-t mutant indicated correct processing of the plant-derived signal peptide. All three N -glycosylation sites of rhEPO were occupied by complex-type N -glycans completely devoid of the plant-specific core sugar residues fucose and xylose.     

Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin

Abstract

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37?°C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20?mM, 1?mM, 2?mM, and 40?μg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production.

Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle’s medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1     

Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences

MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.      

Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes

Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased ～32%, but ratios of asialo- and mono-sialylated glycans were decreased ～50%, compared with control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases the intracellular CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT and α2,3-ST.     

人红细胞生成素受体胞外区克隆及序列测定

人红细胞生成素受体 (h EPOR)是人红细胞生成素的作用配体 ,其胞外区是 h EPO的作用域 ,它的克隆、表达对两种分子的相互作用机制以及 EPO类似物 (新型造血药物 )的筛选都有十分重要的意义。以人胎肝为材料 ,通过对其总 RNA的提取 ,利用 RT- PCR方法扩增 h EPOR的胞外区基因和跨膜区基因及推导相应的氨基酸残基排列 ,结果与国外文献报道相比较从而检验其正确性。     

The status of half-cystine residues and locations of N-glycosylated asparagine residues in human eosinophil peroxidase

The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.     

Influence of recombinant human erythropoietin on neutrophil function in premature neonates

The in vivo influence of recombinant human erythropoietin (rhEpo) and iron on human neutrophil (PMN) antimicrobial function was assessed. A total of 21 preterm infants were randomized to receive either 200 U/kg/other day of rHuEPO+12 mg/kg/day of iron (EPO+high Fe, seven infants) or 200 U/kg/other day of rhEPO+4 mg/kg/day of iron (EPO+standard Fe, 9 infants) or 4 mg/kg/day of iron only (standard Fe, five infants). PMNs were isolated from blood of these infants 60+/-5 days after birth and from eight healthy adults. No differences between infants and adults were found in PMN random migration and chemotactic activity to N-formylmethionyl leucyl phenylalanine (FMLP), superoxide anion production in response to FMLP and phagocytosis of Staphylococcus aureus. In contrast, percentage phagocytosis was significantly lower in EPO+standard Fe as compared to both EPO+high Fe and standard Fe groups (P<0.01). This modest impairment of phagocytic activity of neonatal PMNs found in association with administration of rhEPO and standard iron may be related to consumption of iron during rhEPO-enhanced erythropoiesis.     

Expression of human erythropoietin cDNA in human lymphoblastoid Namalwa cells: the inconsistency of a stable expression level with transient expression efficiency

Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.     

Development of a chemiluminescent imaging assay for the detection of anti‐erythropoietin antibody in human sera

Measuring low amounts of anti‐erythropoietin antibodies (anti‐EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high‐throughput chemiluminescent (CL) imaging assay was developed for the detection of anti‐EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12–13.91 ng/mL. The limit of detection (LOD, 3σ) for the CL‐imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme‐linked immunosorbent assay (ELISA), the LOD of the CL‐imaging assay is 50‐fold lower. The recoveries of anti‐EPO Abs in the fortified serum were in the range 87.1–116.9% using the present method, which highlighted the validity of the CL‐imaging assay system to accurately determine the anti‐EPO Abs in serum samples. CL‐imaging assay was used to evaluate the presence of anti‐EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti‐EPO Abs. Copyright © 2008 John Wiley & Sons, Ltd.     

胞外唾液酸酶造成工程中国仓鼠卵巢细胞株所产人源重组促红素唾液酸含量降低

为了对工程中国仓鼠卵巢(CHO)细胞所产人源重组促红素(rhEPO)的N-糖基化特点进行考察,静置培养工程细胞后,通过等电聚焦和凝集素共沉淀对培养上清中的rhEPO进行分析,并对无血清培养上清中乳酸脱氢酶(LDH)和唾液酸酶活性进行检测,发现这株CHO细胞可以表达唾液酸含量较高的rhEPO蛋白。但是随着培养时间的延长,细胞的存活率逐渐降低,死亡的细胞将胞内的唾液酸酶释放到胞外,唾液酸酶的降解作用会造成N-糖链分枝末端的唾液酸占有率降低,导致rhEPO蛋白糖基化形态的变化。所使用的方法及得到的结果为进一步对工业过程进行分析提供了参考。     

Regional assignment of the erythropoietin gene to human chromosome region 7pter----q22

The chromosomal location of the human gene for erythropoietin (EPO) was determined by Southern blot hybridization analysis of a panel of human-mouse somatic hybrid cell DNAs. DNAs from cell hybrids containing reduced numbers of human chromosomes were treated with the restriction enzyme PstI and screened with a cloned human EPO cDNA probe. EPO is assigned to human chromosome 7 based on the complete cosegregation of EPO with this chromosome in all 45 cell hybrids tested. A cell hybrid containing a translocated derivative of chromosome 7 localizes EPO to 7pter----q22. A HindIII restriction fragment length polymorphism is detected by hybridization of the EPO cDNA probe to human genomic DNA.     

Recombinant human erythropoietin produced in milk of transgenic pigs

We have developed a line of transgenic swine harboring recombinant human erythropoietin through microinjection into fertilized one cell pig zygotes. Milk from generations F1 and F2 transgenic females was analyzed, and hEPO was detected in milk from all lactating females at concentrations of approximately 877.9+/-92.8 IU/1 ml. The amino acid sequence of rhEPO protein in the transgenic pig milk matched that of commercial rhEPO produced from cultured animal cells. In addition, an F-36 cell line, which proliferates in the presence of hEPO or commercial EPO, was induced to synthesize erythroid by extracts from tg sow milk. This study provides evidence that production of purified rhEPO from transgenic pig milk is a potentially valuable technology, and can be used as a cost-effective alternative in clinical applications as well as providing other clinical advantages.     

Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n?=?16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24?h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3?K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2?h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.     

Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1alpha (HIF-1alpha) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1alpha induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1alpha and EPO for the possible treatment of stroke.     

The role of excessive versus acute administration of erythropoietin in attenuating hepatic ischemia-reperfusion injury

Ischemia-reperfusion injury (I/R) is the main cause of primary graft nonfunction. Our aim was to evaluate the effect of excessive versus acute administration of erythropoietin (EPO) in attenuating the hepatic injury induced by I/R in mice. The effect of segmental (70%) hepatic ischemia was evaluated in a transgenic mouse line with constitutive overexpression of human EPO cDNA and in wild-type (WT) mice. Mice were randomly allocated to 5 main experimental groups: (i) WT-sham, (ii) WT ischemia, (iii) WT ischemia + recombinant human erythropoietin (rhEPO), (iv) transgenic-sham, and (v) transgenic ischemia. The EPO-pretreated mice showed a significant reduction in liver enzyme levels and intrahepatic caspase-3 activity and fewer apoptotic hepatocytes (p < 0.05 for all) compared with the WT untreated I/R group. EPO decreased c-Jun N-terminal kinase (JNK) phosphorylation and nuclear factor-κB (NF-κB) expression during I/R. In transgenic I/R livers, baseline histology showed diffused hepatic injury, and no significant beneficial effect was noted between the WT untreated and the transgenic I/R mice. In conclusion, acute pretreatment with EPO in WT mice attenuated in vivo I/R liver injury. However, in excessive EPO overexpression, the initial liver injury abolished the beneficial effect of EPO. These findings have important implications for the potential use of acute EPO in I/R injury during liver transplantation.     

两个不同启动子在COS7细胞中转录效率的比较

 两个不同启动子在ＣＯＳ７细胞中转录效率的比较陈坚刘小萍曹韫旭陆德如（第二军医大学医学生物技术和分子遗传研究所,上海２００４３３）ＣｏｍｐａｒｉｓｏｎｏｆｔｈｅＴｒａｎｓｃｒｉｐｔｉｏｎａｌＥｆｉｃｉｅｎｃｙｏｆＴｗｏＤｉｆｆｅｒｅｎｔＰｒｏｍｏｔｅｒ...     

The role of erythropoietin and its receptor in growth,survival and therapeutic response of human tumor cells: From clinic to bench — a critical review

Recombinant human erythropoietin (rhEPO) has been used clinically to alleviate cancer- and chemotherapy-related anemia. However, recent clinical trials have reported that rhEPO also may adversely impact disease progression and survival. The expression of functional EPO receptors (EPOR) has been demonstrated in many human cancer cells where, at least in vitro, rhEPO can stimulate cell growth and survival and may induce resistance to selected therapies.     

Phosphorylatable and epitope-tagged human erythropoietins: Utility and purification of native baculovirus-derived forms

The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.     

Cloning of human erythropoietin gene

The 17-mer oligonucleotide probe homologous to the fragment of the gene for human erythrocyte differentiation factor erythropoietin was used to screen the human genomic library for this gene. Restriction analysis and partial sequencing of one of the identified clones have confirmed that the clone does contain the human erythropoietin gene. We are planning to use the cloned human erythropoietin gene for developing a stably transfected mammalian cell line that should secrete erythropoietin.