Article

Hydrogen Deuterium Exchange Reveals Changes to Protein Dynamics of Recombinant Human Erythropoietin upon N- and O- Desialylation

Authors:
  • Alkermes, Waltham
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Abstract

Recombinant human erythropoietin (EPO) is a therapeutic glycoprotein widely used for treating anemia. EPO glycans carry extensive sialylation and the level of the modification is known to affect receptor binding, protein stability and pharmacokinetics. Nonetheless, a detailed understanding of the effects of sialylation on EPO conformation and dynamics is still lacking. Here we investigate the changes to EPO dynamics following enzymatic trimming of terminal sialic acid by amide hydrogen deuterium exchange mass spectrometry (HDX-MS). The results revealed that desialylation enhances structural flexibility near the glycosylation sites, with greater effects observed around the O-glycosylation site relative to the N-glycosylation sites. The affected regions are surface-exposed loops connecting the helix bundle, which do not appear to reduce the thermostability of the molecule as revealed from melting measurement. Our findings demonstrate the feasibility of HDX-MS technique in deciphering the function of specific type of glycosylation that can provide novel insights into the role of sialylation on protein therapeutics.

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... [73][74][75] More specifically, removal of sialic acid induces conformational changes in human serum amyloid P, a 2 macroglobulin, b 1 integrin, a 1 acid glycoprotein, K + and Na + channels, and erythropoietin. [76][77][78][79][80][81] Even more relevant to this discussion, NEU1-mediated desialylation itself reportedly promotes conformational changes in the insulin receptor in both HEK293T cells and C57BL/6 mice, and b2-integrin in human neutrophils. 82,83 Each MUC1 molecule contains hundreds of bulky, negatively-charged sialic acid molecules. ...
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... 548−551 In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. 541,552 Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal. 553 The available studies have thus far revealed a complex and very context-dependent relationship between glycosylation and structural dynamics. ...
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... Previously, glycan related conformational change has also been reported for erythropoietin following desialylation. 25 Other analytical tools, such as differential scanning calorimetry (DSC), have been evaluated and are less informative ( Figure S-7 and Table S-2). The differences observed by DSC as related to thermal stability are not as easily differentiated as compared to HDX-MS and do not provide any site-specific structural information as does HDX-MS. ...
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Recombinant human erythropoietin has N‐linked sugar [Tsuda et al., (1988) Biochemistry 27 , 5646–5654]. Here we have demonstrated the presence of O‐linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and Galβ(1–3)GalNAc. To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activity of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitro activity. Although erythropoiein from which N‐linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N‐linked sugar abolished the in vivo biological activity completely, indicating that the presence of N‐linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin. Incubation of asialo‐erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 °C for 15 min decreased the biological activity to 35% and 11% of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.
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Controversy exists regarding the functional role of N-linked oligosaccharides in the hormone erythropoietin. We have now examined the role of carbohydrates in the hormone's action using quantitative enzymatic deglycosylation. N-deglycosylated hormone exhibited full biological activity and potency in vitro. Denaturing with 6M urea and renaturing revealed that both the native and N-deglycosylated forms recovered full activity as long as the intrachain disulfide bonds remained intact. Therefore, receptor recognition, subsequent biological activity and maintenance of tertiary structure are intrinsic properties of the polypeptide chain of erythropoietin.
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1. Recombinant human erythropoietin has N-linked sugar [Tsuda et al., (1988) Biochemistry 27, 5646-5654]. Here we have demonstrated the presence of O-linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and Gal beta(1-3)GalNAc. 2. To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activity of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitro activity. Although erythropoietin from which N-linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N-linked sugar abolished the in vivo biological activity completely, indicating that the presence of N-linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin. 3. Incubation of asialo-erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 degrees C for 15 min decreased the biological activity to 35% and 11% of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.
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Recent advances in experimental hematology have provided new insight into the physiology of erythropoiesis. Techniques are now available for cloning in vitro the various hematopoietic progenitor cells of both animals and man. Erythropoietin has been purified and a radioimmunoassay for the hormone has been described. In this paper, we discuss the physiology of erythropoietin and erythropoiesis with special emphasis on data obtained through the employment of new technology.
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Among bacterial, fungal and viral sialidases, the sialidase from Arthrobacter ureafaciens has the unique property of cleaving sialic acids linked to the internal galactose of gangliotetraose. In this study, we examined the ability to cleave the internal sialic acids of GM1 and fucosyl GM1 of sialidases from several bacterial and fungal origins, including Clostridium perfringens and Vibrio cholerae. We found that A. ureafaciens sialidase could liberate the sialic acid of GM1 at the highest rate, and was the only enzyme which could cleave fucosyl GM1 among the sialidases examined. The affinity-purified sialidase derived from the culture medium of A. ureafaciens was comprised of four isoenzymes with different molecular weights and isoelectric points, the isoenzymes that cleaved fucosyl GM1 being L (88 kDa, pI 5.0), M1 (66 kDa, pI 6.2) and M2 (66 kDa, pI 5.5), but not S (52 kDa, pI 6.2) which showed the highest specific activity toward colominic acid among the four isoenzymes. Abbreviations: SA, sialic acid; PBS, phosphate-buffered saline; PVP, polyvinylpyrrolidone; FABMS, fast atom bombardment mass spectrometry; Galβint, internal galactose of Gg4Cer; Galβext, external galactose of Gg4Cer
Article
Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.
Article
Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.
Article
Anemia has an incidence both on the quality of life and the evolution of cancer. Anemia may result in cancer from either a bone marrow infiltration of cancer cells or a cytotoxic effect of chemotherapy and/or radiotherapy, or both. EPO is a glycoprotein which stimulates erythrocyte formation by bone marrow progenitory cells. Recombinant EPO has considerably improved treatment of anemic patients, by increasing hemoglobin serum levels and reducing the need for blood transfusion. The quality of life of cancer patients is thus improved and several studies highlight the beneficial role of EPO on the clinical outcome. A preclinical background and some clinical data suggest however a detrimental role of EPO in cancer by a possible stimulation of tumor growth. There is a need of more clinical trials in order to assess the effects of EPO on tumors and their treatment.
  • C Lacombe
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Structure and role of carbohydrate in human erythropoietin
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Characterization of IgG1 conformation and conformational dynamics by hydrogen/deuterium exchange mass spectrometry
  • Houde