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

Abstract

The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.

Keywords: Biotechnology; chemical glycoprotein synthesis; chemical protein synthesis; glycoprotein; glycosylation; oligosaccharide.

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 EPO^{N24, 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.