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Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity

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Science Advances  15 Jan 2016:
Vol. 2, no. 1, e1500678
DOI: 10.1126/sciadv.1500678
  • Fig. 1 Structure of the EPO glycoforms and sialyloligosaccharide.

    (A) Primary structure of EPO 2 showing the amino acid sequence, glycosylation sites, ligation sites (red circle), and disulfide bonds (red dotted line). The glutamine at position 78 was substituted with alanine. (B) Structure of the asparaginyl sialyloligosaccharide 1 used for the chemical synthesis of a sialylglycopeptide-α-thioester in Boc SPPS. Sialic acid was protected as a phenacyl (Pac) ester, shown in magenta. (C) Acceleration of hydrolysis by an intramolecular acid catalyst.

  • Fig. 2 Scheme for the synthesis of the EPO glycoforms by chemical ligation.

    The suitably glycosylated or nonglycosylated segments of [1–28], [29–49], and [79–97] were selected depending on the synthesis of EPO glycoforms 2 to 6 assembled by peptide ligation reactions. The full-length polypeptides were folded to form a three-dimensional structure through oxidative folding methods. Conditions: (i) NCL; (ii) conversion of thiazolidine into cysteine; (iii) deprotection of the Pac and formyl groups; (iv) deprotection of the Pac group; (v) thioesterification of hydrazide; (vi) desulfurization; (vii) deprotection of the Acm group.

  • Fig. 3 Proposed EPO in vitro folding process.

    All folding intermediates were analyzed by trypsin digestion and subsequent MS/MS analyses. The analysis revealed that the disulfide bond Cys29-Cys33 formed first under redox conditions.

  • Fig. 4 Characterization of EPO glycoforms 2 to 6.

    (A) (a to e) ESI mass spectra of EPO glycoforms 2 to 6. All mass spectra are not of the ESI-MS data derived from the top area of the HPLC profile. All mass spectra were measured with total solution of individual EPO glycoforms isolated. (B) RP-HPLC chromatogram and ESI-MS spectrum of folded 2. (C) CD spectra of EPO glycoforms 2 to 6 in 0.1% TFA aq.

  • Fig. 5 Characterization of the EPO glycoforms.

    (A) RP-HPLC chromatogram of a mixture of EPO glycoforms 2 to 6 to obtain insight into their hydrophobicity assessed by the elution time. Compounds 2, 4, 5, and 6 (3.0 μg each) and 3 (4.5 μg) were mixed, and the resultant solution was injected in RP-HPLC. Retention time: 2, 33.38 min; 3, 33.99 min; 4, 34.52 min; 5, 38.64 min; 6, 35.35 min. (B) Cell proliferation assay. Orange circle, synthetic EPON24, N38, N83 2; gray triangle, EPOGIN. (C) EPO protein surface, with the three N-glycosylation sites highlighted in purple. The hydrophilic amino acids are shown in yellow, and the hydrophobic amino acids are shown in orange [the model was created from the NMR structure of human EPO (Protein Data Bank: 1BUY)]. (D) In vivo hematopoietic activity of the synthesized EPO glycoforms and EPOGIN. The concentrations of 2 to 7 were set at 1.4 μM. Sample 7 was a misfolded form of EPO 4. Sample 8 was a mixture of 2 to 6 (individual EPO glycoform concentration was set at 0.28 μM, but total EPO protein concentration was 1.4 μM). Blue bar, 0 days; red bar, 2 days; green bar, 5 days; purple bar, 7 days.

  • Table 1 Yields of in vitro folding.

    Yields (%) were estimated by RP-HPLC area.

    ConcentrationEPON24, N38, N83 2EPON38, N83 3EPON24, N83 4EPON24, N38 5EPON83 6
    0.1 mg/ml68 (±1)58 (±5)43 (±3)49 (±5)37 (±3)
    0.01 mg/ml86 (±6)90 (±6)66 (±2)76 (±11)63 (±2)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/1/e1500678/DC1

    Fig. S1. Acid stability of sialyloligosaccharide phenacyl ester.

    Fig. S2. General scheme of the synthesis of a sialylglycopeptide-α-thioester by an improved Boc SPPS method.

    Fig. S3. HPLC profile and ESI mass spectrum of H-[Ala1-Gly28]-α-thioester.

    Fig. S4. HPLC profile and ESI mass spectrum of H-[Cys29,33(Acm)-Tyr49]-α-thioester.

    Fig. S5. HPLC profile and ESI mass spectrum of H-[Cys29,33(Acm)-Asn38(glycan)-Tyr49]-α-thioester.

    Fig. S6. HPLC profile and ESI mass spectrum of H-[Cys79(Thz)-Trp88-(formyl)-Lys97]-α-thioester.

    Fig. S7. HPLC profile and ESI mass spectrum of H-[Cys79(Thz)-Asn83(glycan)-Trp88(formyl)-Lys97]-α-thioester.

    Fig. S8. HPLC profile and ESI mass spectrum of H-[Cys98(Thz)-Ala127]-α-thioester.

    Fig. S9. HPLC profile and ESI mass spectrum of H-[Cys50-Ala78]-α-hydrazide.

    Fig. S10. HPLC profile and ESI mass spectrum of H-[Ala1-Asn24(glycan)-Gly28]-α-thioester.

    Fig. S11. Monitoring NCL between H-[Cys29, 33(Acm)-Asn38(glycan)-Tyr49]-α-thioester and H-[Cys50-Ala78]-α-hydrazine.

    Fig. S12. Monitoring NCL between H-[Cys29, 33(Acm)-Asn38(glycan)-Ala78]-α-hydrazide and H-[Cys79-Asn(glycan)-Arg166]-OH.

    Fig. S13. Monitoring the desulfurization reaction of H-[Cys29, 33, 161(Acm)-Cys50, 79, 98, 128-Asn38, 83(glycan)-Arg166]-OH.

    Fig. S14. Monitoring of the removal of Acm group of H-[Cys29, 33, 161(Acm)-Asn38, 83(glycan)2-Arg166]-OH by RP-HPLC and ESI-MS.

    Fig. S15. Monitoring the NCL between H-[Ala1-Asn24(glycan)-Gly28]-α-thioester and H-[Cys29-Asn38, 83(glycan)2-Arg166]-OH.

    Fig. S16. The folding reaction of EPON24, N38, N83 (polypeptide form of H-[Ala1-Asn24, 38, 83(glycan)3-Arg166]-OH.

    Fig. S17. The folding reactions of EPON38, N83 (polypeptide form of H-[Ala1-Asn38, 83(glycan)2-Arg166]-OH) and EPON24, N83 (polypeptide form of H-[Ala1-Asn24, 83(glycan)2-Arg166]-OH).

    Fig. S18. The folding reactions of EPON24, N38 (polypeptide form of H-[Ala1-Asn24, 38(glycan)2-Arg166]-OH) and EPON83 (polypeptide form of H-[Ala1-Asn83(glycan)-Arg166]-OH).

    Results of folding experiments

    Fig. S19. Monitoring of in vitro folding by SDS-PAGE.

    Fig. S20. Analysis of disulfide bond positions of EPON24, N38, N83 2 by trypsin digestion.

    Fig. S21. Analysis of disulfide bond positions of EPON38, N83 3 by trypsin digestion.

    Fig. S22. Analysis of disulfide bond positions of EPON24, N83 4 by trypsin digestion.

    Fig. S23. Analysis of disulfide bond positions of EPON24, N38 5 by trypsin digestion.

    Fig. S24. Analysis of disulfide bond positions of EPON83 6 by trypsin digestion.

    Fig. S25. Characterization of misfolded EPON24, N83 (compound 7).

    High-resolution mass spectra of EPO glycoforms

    Fig. S26. High-resolution mass spectrum of EPON24, N38, N83 2.

    Fig. S27. High-resolution mass spectrum of EPON38, N83 3.

    Fig. S28. High-resolution mass spectrum of EPON24, N38, 4.

    Fig. S29. High-resolution mass spectrum of EPON38, N83 5.

    Fig. S30. High-resolution mass spectrum of EPON83 6.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Acid stability of sialyloligosaccharide phenacyl ester.
    • Fig. S2. General scheme of the synthesis of a sialylglycopeptide-α-thioester by an improved Boc SPPS method.
    • Fig. S3. HPLC profile and ESI mass spectrum of H-Ala1-Gly28-α-thioester.
    • Fig. S4. HPLC profile and ESI mass spectrum of H-Cys29,33 (Acm)-Tyr49-α-thioester.
    • Fig. S5. HPLC profile and ESI mass spectrum of H-Cys(Acm)-Asn38(glycan)-Tyr49-α-thioester.
    • Fig. S6. HPLC profile and ESI mass spectrum of H-Cys79(Thz)-Trp88-(formyl)-Lys97-α-thioester.
    • Fig. S7. HPLC profile and ESI mass spectrum of H-Cys79(Thz)-Asn83(glycan)-Trp88(formyl)-Lys97-α-thioester.
    • Fig. S8. HPLC profile and ESI mass spectrum of H-Cys98(Thz)-Ala127-α-thioester.
    • Fig. S9. HPLC profile and ESI mass spectrum of H-Cys50-Ala78-α-hydrazide.
    • Fig. S10. HPLC profile and ESI mass spectrum of H-Ala1-Asn24(glycan)-Gly28-α-thioester.
    • Fig. S11. Monitoring NCL between H-Cys (Acm)-Asn38(glycan)-Tyr49-α-thioester and H-Cys50-Ala78-α-hydrazine.
    • Fig. S12. Monitoring NCL between H-Cys (Acm)-Asn38(glycan)-Ala78-α-hydrazide and H-Cys79-Asn83(glycan)-Arg166-OH.
    • Fig. S13. Monitoring the desulfurization reaction of H-Cys (Acm)-Cys -Asn (glycan)-Arg166-OH.
    • Fig. S14. Monitoring of the removal of Acm group of H-Cys (Acm)-Asn (glycan)2-Arg166-OH by RP-HPLC and ESI-MS.
    • Fig. S15. Monitoring the NCL between H-Ala1-Asn24(glycan)-Gly28-α-thioester and H-Cys29-Asn (glycan)2-Arg166-OH.
    • Fig. S16. The folding reaction of EPON24, N38, N83 (polypeptide form of H-Ala1-Asn (glycan)3-Arg166-OH.
    • Fig. S17. The folding reactions of EPON38, N83 (polypeptide form of H-Ala1-Asn (glycan)2-Arg166-OH) and EPON24, N83 (polypeptide form of H-Ala1-Asn24, 83(glycan)2-Arg166-OH).
    • Fig. S18. The folding reactions of EPON24, N38 (polypeptide form of H-Ala1-Asn (glycan)2-Arg166-OH) and EPON83 (polypeptide form of H-Ala1-Asn83(glycan)-Arg166-OH).
    • Results of folding experiments
    • Fig. S19. Monitoring of in vitro folding by SDS-PAGE.
    • Fig. S20. Analysis of disulfide bond positions of EPON24, N38, N83 2 by trypsin digestion.
    • Fig. S21. Analysis of disulfide bond positions of EPON38, N83 3 by trypsin digestion.
    • Fig. S22. Analysis of disulfide bond positions of EPON24, N83 by trypsin digestion.
    • Fig. S23. Analysis of disulfide bond positions of EPON24, N38 5 by trypsin digestion.
    • Fig. S24. Analysis of disulfide bond positions of EPON83 6 by trypsin digestion.
    • Fig. S25. Characterization of misfolded EPON24, N83 (compound 7).
    • High-resolution mass spectra of EPO glycoforms
    • Fig. S26. High-resolution mass spectrum of EPON24, N38, N83 2.
    • Fig. S27. High-resolution mass spectrum of EPON38, N83 3.
    • Fig. S28. High-resolution mass spectrum of EPON24, N38, 4.
    • Fig. S29. High-resolution mass spectrum of EPON38, N83 5.
    • Fig. S30. High-resolution mass spectrum of EPON83 6.

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