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

Elliott, Steve; Chang, David; Delorme, Evelyne; Eris, Tamer; Lorenzini, Tony

doi:10.1074/jbc.m311095200

Cited by 56 publications

(49 citation statements)

References 47 publications

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“…Nevertheless, examples of glycol-engineering for directed glycosylation have been shown successfully to improve the solubility and stability of therapeutic proteins or antibodies and thus to enhance the therapeutic effects. 7,[22][23][24] …”

Section: Discussionmentioning

confidence: 99%

“…To solve this problem, additional potential glycosylation sites have been introduced into recombinant therapeutic molecules such as human erythropoietin (rHuEPO). 7 However, because the relationship between the locations of the N-glycosylation to the functions of the oligosaccharide chain is still not completely understood, the selection of the sites to introduce the N-glycosylation on a recombinant protein has been a relatively random process, especially for proteins without X-ray crystal structures. To simplify the search and design of such N-glycosylation sites, we have tried to introduce additional NXS/T sequons at both the N-and C-terminals of the proteins.…”

Section: Introductionmentioning

confidence: 99%

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Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Liu

Nguyen

Wolfert

et al. 2009

Biotechnology Progress

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N-glycosylation is important for the folding and quality control of membrane and secretory proteins. We used mutagenesis to introduce N-glycosylation sequons in recombinant proteins to improve their secretion in HEK293 cells. Seven recombinant proteins, with or without endogenous N-glycosylation sequons, were tested by this method. Our results indicate that N-glycosylation sequons located at the N-or C-terminal are glycosylated at high rates and thus the N-and C-terminal may be convenient sites for effectively attaching oligosaccharide chains. More importantly, introduction of oligosaccharide chains at such positions has been found to improve the secretion levels for the majority of the recombinant proteins in our studies, regardless of endogenous N-glycosylation, presumably by improving their folding in the endoplasmic reticulum. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1468Prog., 25: -1475Prog., 25: , 2009 Keywords: N-glycosylation, protein expression, secretion, protein folding, mutagenesis, quality control IntroductionAsparagine glycosylation (N-glycosylation) is an important process for the delivery of secretory and membrane proteins to the extracellular space or cell surface. Thus, most secretory and membrane proteins contain asparagine-linked (N-linked) glycans, with one or more oligosaccharide chains attached to the polypeptide backbone. For some proteins, Nglycosylation is absolutely required for their secretion. For others, it may not be necessary. Nevertheless, oligosaccharide chains have been shown to be the important parts of the proteins and may play roles in the folding, transport, degradation, structure, stability, receptor-recognition, cellular localization, or other biological activities of the glycoproteins (reviewed in Refs. 1-3). Protein N-glycosylation takes place at the membrane of the endoplasmic reticulum (ER). The ER plays a primary quality control function to ensure the fidelity and proper folding of proteins before they travel out of the compartment, as incorrectly folded proteins can be toxic to the cell. A presynthesized oligosaccharide chain on a lipid carrier, dolichol, is transferred onto the asparagine residue in the NXS/T sequon of the nascent polypeptide by the oligosaccharyltransferase (OST) complex while translation is in process. 2 The attached oligosaccharide chains continue to be modified along the maturation pathway of the proteins in the ER and the N-glycosylation status will dictate the fate of the synthesized protein. The newly synthesized protein can proceed to secretion, aggregation, or degradation depending on the status and pattern of its N-glycosylation. [4][5][6] In the overexpression and production of recombinant proteins, the efficiency of N-glycosylation will affect the yield of the protein recovery and the circulation life of a pharmaceutical molecule. Although N-glycosylation is important, it is well known that not all potential glycosylation sites are attached with oligosaccharide chains. In our routine expression experi...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Liu

Nguyen

Wolfert

et al. 2009

Biotechnology Progress

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“…N-linked carbohydrate attachment points consist of welldefined three amino acid consensus sequences (Asn-XxxSer/Thr), where Xxx could be any amino acid except proline [43,44]. In order to add additional carbohydrate chains to rHuEPO, the coding region of the EPO gene was altered so that new N-linked consensus sequences were present and that a cell expressing these modified genes Fig.…”

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Kiss¹,

Elliott

Jedynasty³

et al. 2010

Eur J Clin Pharmacol

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Cloning of the human erythropoietin (EPO) gene and development of the first recombinant human erythropoietin (rHuEPO) drug were truly breakthroughs. This allowed a deeper understanding of the structure and pharmacology of rHuEpo, which in turn inspired the discovery and development of additional erythropoiesisstimulating agents (ESAs). In vivo specific activity and serum half-life of rHuEPO are influenced by the amount and structure of the attached carbohydrate. Increased numbers of sialic acids on carbohydrate attached to rHuEPO correlated with a relative increase in in-vivospecific activity and increased serum half-life. The effect of increasing the number of sialic-acid-containing carbohydrates on in-vivo-specific activity was explored. Initial research focused on solving the problem of how the protein backbone could be engineered so a cell would add more carbohydrate to it. Additional work resulted in darbepoetin alfa, a longer-acting molecule with two additional carbohydrate chains.

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“…Biochemical studies showed that EPO contains two binding sites for the EPOR, a high-affinity binding site with a K D of Ϸ1 nM and a low-affinity site of 1 M (20). Mutational analysis suggested that residues 11-15, 44-51, 100-108, and 147-151 on EPO are important for erythropoiesis activity (21). The crystal structure of the EPO-EPOR complex has been determined at a resolution of 1.9 Å [Protein Data Bank (PDB) ID code 1EER] (22).…”

mentioning

confidence: 99%

“…The crystal structure of the EPO-EPOR complex has been determined at a resolution of 1.9 Å [Protein Data Bank (PDB) ID code 1EER] (22). This structure demonstrates that Phe-48, Asn-147, Arg-143, and Arg-150 of EPO are among the key residues for binding to EPOR (20)(21)(22).We have taken the primary EPO-EPOR binding site as our target of protein-protein interface redesign. After scanning the PDB, rat pleckstrin homology (PH) domain of phospholipase C-␦1 (PLC␦ 1 -PH) was found to accommodate well the key interaction residues in EPO and at the same time can form a good interface with EPOR.…”

mentioning

confidence: 99%

Nonnatural protein–protein interaction-pair design by key residues grafting

Liu

Zhu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLC␦ 1-PH (pleckstrin homology domain of phospholipase C-␦1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLC␦ 1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLC␦ 1-PH (ERPH1) was found to bind EPOR with high affinity (K D of 24 nM and an IC50 of 5.7 M) both in vitro and in a cell-based assay, respectively, although the WT PLC␦ 1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLC␦ 1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting proteinprotein interfaces.de novo design of protein-protein interaction pair ͉ erythropoietin ͉ functional site grafting ͉ key residue at interface P rotein-protein interaction is critical in many biological processes ranging from cell differentiation to apoptosis; however, little is known about the general principles governing the specificity and binding affinity of protein-protein interactions. Recent developments in structural bioinformatics have contributed greatly to our understanding of protein-protein interactions (1-6). Several groups have addressed the feasibility of computational redesign of protein-protein interactions by using native or homologous protein-protein interfaces (7-10). Because different protein backbones can perform similar functions (11), in the current study we explored the feasibility of designing nonnatural protein-protein interaction pairs using known protein scaffolds.To reconstruct the function of a protein, one of the common strategies is grafting, i.e., transferring the functional epitopes from one protein to another. The critical step for protein grafting is to find suitable sites for functional epitope transfer. To this purpose, several computational methods have been developed including a geometric hashing paradigm (12), FITSITE (13), GRAFTER (14), and DEZYMER (15, 16). We have developed a strategy for protein-protein interface redesign by grafting discontinuous interaction epitopes to nonhomologous proteins (17-19). The erythropoietin (EPO)-EPO receptor (EPOR) system was used as an example for nonnatural protein-protein interaction-...

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

Cited by 56 publications

References 47 publications

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Nonnatural protein–protein interaction-pair design by key residues grafting

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