Erythropoietin: Gene Cloning, Protein Structure, and Biological Properties

Browne, Jeffrey K.; Cohen, Aliza; Egrie, Joan C.; Lai, Por-Hsiung; Fu, Lin; Strickland, Thomas W.; Watson, Eileen L.; Stebbing, Nowell

doi:10.1101/sqb.1986.051.01.082

Cited by 105 publications

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“…For example, it has been reported that Pro at the ϩ3 position relative to Asn or Asp at ϩ1 can inhibit glycosylation (14,17,21,22). Distance from the C terminus may also affect glycosylation (23).Human erythropoietin is a 165-amino acid glycoprotein with three N-linked carbohydrates attached to asparagines at amino acid positions 24, 38, and 83 and one O-linked carbohydrate attached to Ser 126 (24,25). The structure of rHuEPO 1 and the functional importance of the amino acids for activity is well studied (26 -30).…”

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Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin

Elliott

Chang

Delorme

et al. 2004

Journal of Biological Chemistry

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N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn 123 -Thr 125 ) introduced into a position close to the existing O-glycosylation site (Ser 126 ) had an additional O-linked carbohydrate chain and not an additional Nlinked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.Secreted proteins are often glycosylated during transit through the secretory apparatus in eukaryotic cells. These carbohydrates can be attached to the hydroxyl group on a serine or threonine (O-linked glycosylation) or the amine of an asparagine via an N-glycosidic bond (N-linked glycosylation). The addition of carbohydrate chains to the polypeptide backbone of a protein may have an impact on the structure, solubility, antigenicity, folding, secretion, and stability of the protein (1-8). The carbohydrate may also affect the clearance rate and in vivo activity of the protein (9 -12).The nature of the signal for carbohydrate addition is partially understood. N-Linked carbohydrate addition is mediated by oligosaccharide transferase and occurs at asparagine residues that are part of the consensus sequence Asn-Xaa-(Ser/ Thr), where Xaa can be any amino acid, except proline (13-16). The observation that not all consensus sequences are glycosylated suggests that there are additional sequence or conformational requirements essential for efficient carbohydrate attachment (17,18). Although at least 12-14 amino acids must be synthesized and have entered the luminal surface of the endoplasmic reticulum for carbohydrate addition, the synthesis of the protein need not be completed for glycosylation to take place (19,20). This suggests that the structures for carbohydrate addition are recognized in partially folded molecules. The sequence context of the glycosylation site has also been shown to influence the efficiency of glycos...

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“…Human erythropoietin is a 165-amino acid glycoprotein with three N-linked carbohydrates attached to asparagines at amino acid positions 24, 38, and 83 and one O-linked carbohydrate attached to Ser 126 (24,25). The structure of rHuEPO 1 and the functional importance of the amino acids for activity is well studied (26 -30).…”

mentioning

confidence: 99%

Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin

Elliott

Chang

Delorme

et al. 2004

Journal of Biological Chemistry

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“…Human EPO protein has a 79% amino acid homology with mouse EPO, and its administration induces marked erythropoiesis in mice as well as other species of animals including rat and dog (28,29). To avoid immunological consequences engendered by human EPO produced in a different species, we used nude mice and attempted to determine the feasibility of a myoblast gene transfer approach in the treatment of renal failure.…”

Section: Discussionmentioning

confidence: 99%

Myoblast transfer of human erythropoietin gene in a mouse model of renal failure.

Samal¹,

Tian²,

Kedes³

1995

J. Clin. Invest.

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Key words: gene therapy * anemia * renal failure * muscle cell transplantation * nude mouse Anemia is an invariable consequence of end-stage renal failure (ESRF) and recombinant erythropoietin has dramatically improved the quality of life of patients with ESRF.As an alternative approach, we developed a myoblast gene transfer system for the systemic delivery of human erythropoietin (EPO). We recently reported that transplantation of 4 x 107 cells of a C2 myoblast cell clone that stably secretes high level of functional human EPO, increased hematocrit from 44.6±3.0 to 71.2±7.9(%) in 2 wk, and the increase was sustained for at least 12 wk in nude mice. A renal failure model was created by a two-step nephrectomy in nude mice, and myoblasts were transplanted 3 wk after the second nephrectomy, when mean blood urea nitrogen level had increased from 26.3±6.1 to 85.4±24.0 (mg/ dl) and the hematocrit had decreased from 45.2±2.7 to 33.9±3.7(%). After transplantation, the hematocrit markedly increased to 68.6±4.2(%) 2 wk, and to 68.5±4.0(%) 7 wk after the transplantation. Serum human EPO concentration determined by ELISA indicated a persistent steady EPO production from the transplanted muscle cells 8 wk after the transplantation. The fate of transplanted myoblasts in uremic mice was monitored by transplanting the EPOsecreting clone which had also been transduced with BAG retrovirus bearing the fi-galactosidase gene. 8 wk later, Xgal positive myofibers were detected in the entire transplanted area. The results demonstrate that myoblasts can be transplanted in uremic mice, and that myoblast gene transfer can achieve sufficient and sustained delivery of functionally active EPO to correct anemia associated with

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“…The remaining 40% of the native EPO mass consists of carbohydrate covalently attached at three N-glycans sugar chains at Asn 24, 38 and 83, and one O-linkeded sugar chain at Ser126 (Browne et al, 1986;Davis et al, 1987;Miyake et al, 1977). Since the consensus sequences for N-linked glycans was known (Asn-X-Ser/Thr where X is any amino acid except proline), we speculate that the increase in size of this molecular mass is attributed to the attachment of two additional oligosaccharides at asparagine residues positions 30 and 88.…”

Section: Detection Of Rhuepomentioning

confidence: 99%

Expression of Modified Recombinant Human Erythropoietin in Cho-K1 Cells and Its in Vitro Proliferation Assay in Tf-1 Cells

Santoso

2014

Indonesian J. Pharm.

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Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-α and epoetin-β, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIV-AIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-α (DARB) which possesses two additional Nlinked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 Nlinked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation.

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Erythropoietin: Gene Cloning, Protein Structure, and Biological Properties

Cited by 105 publications

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Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin

Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin

Myoblast transfer of human erythropoietin gene in a mouse model of renal failure.

Expression of Modified Recombinant Human Erythropoietin in Cho-K1 Cells and Its in Vitro Proliferation Assay in Tf-1 Cells

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