Nagasawa K et al. (2015), The influence of artificially introduced N-glyc...

XB-ART-50578

PLoS One 2015 Jan 01;104:e0124676. doi: 10.1371/journal.pone.0124676.

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The influence of artificially introduced N-glycosylation sites on the in vitro activity of Xenopus laevis erythropoietin.

Nagasawa K , Meguro M , Sato K , Tanizaki Y , Nogawa-Kosaka N , Kato T .

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Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1-3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR.

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Species referenced: Xenopus laevis
Genes referenced: epo epor tff3.7

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	Fig 2. Expression, secretion, and physicochemical properties of N-glycosylated xlEPO muteins.(A) Western blot analysis of xlEPO muteins secreted from COS-1 cells. Conditioned media were concentrated 10-fold (top panel), and subjected to PNGase F treatment (bottom panel). Wild-type xlEPO and N-glycosylated muteins were separated by SDS-PAGE (reducing conditions) and visualized by Western blot analysis. Equal volumes of samples were loaded into each lane. (B) Western blot analysis of xlEPO muteins remaining in COS-1 cells. Whole-cell lysates were loaded into each lane. xlEPO (top panel) and EGFP (bottom panel) were visualized by Western blot analysis. Co-expressed nonsecretory EGFP was used as a loading control. (A, B) Standard proteins are shown at the far left, and the letters above each lane represent the names of the muteins shown in Fig 1. (C) Effects of N-glycosylation on the secretion of xlEPO muteins. The relative amounts of xlEPO muteins in the culture media of COS-1 cells were measured by densitometry analysis of Western blots (filled bars). The relative expression levels were calculated based on the fluorescence intensity of co-expressed EGFP (open bars). The relative efficiency of secretion was obtained by the division of relative amount by relative expression level (open circles). Values were normalized to that of the wild type, which was set at 1. p < 0.05 and *p < 0.01 compared to the wild type. (D) Physicochemical properties of xlEPO muteins. Cation-exchange chromatography (CIEC, top panel) and the reverse-phase high-pressure liquid chromatography (RP-HPLC, bottom panel) of wild-type xlEPO and N-glycosylated muteins were performed as described in the Materials and methods section. The collected fractions were subjected to Western blot analysis. ‘N-CHO’ indicates the number of N-linked carbohydrates. Abbreviations are: EB, elution buffer; Fr., fraction.
	Fig 3. Effects of wild-type xlEPO and the glycosylated muteins on the proliferation of X. laevis and human EPOR-expressing cells.(A, B) The proliferative responses of xlEPOR-FDC/P2 cells (A) and UT-7/EPO cells (B) to the wild-type xlEPO and the N-glycosylated muteins were assessed using the MTS assay. Incubations were performed in triplicate, and the results are presented as the mean (± SD) from 1 of 4 independent experiments. (C) The proliferative responses of xlEPOR-FDC/P2 cells to wild-type xlEPO and the N-glycosylated muteins treated with (dashed line) and without (solid line) PNGase F were also assessed. The PNGase F-treated samples are the same fractions displayed in Fig 2A, bottom panel. Incubations were performed in triplicate, and the results are presented as the mean (± SD) from 1 of 3 independent experiments. **p < 0.01 for the comparison. Symbols are: multiple marks, wild-type xlEPO; open squares, xlEPO-1; open triangles, xlEPO-2; open circles, xlEPO-3; closed squares, xlEPO-12; closed triangles, xlEPO-13; closed circles, xlEPO-23; closed rhombuses, xlEPO-123; plus sign, Mock (control culture supernatant without xlEPO).
	Fig 4. Relationship between carbohydrate content and biological activity.The COS-1 supernatants containing the wild-type xlEPO and the N-glycosylated muteins were assayed by proliferation of xlEPOR-FDC/P2 cells (A, C) and UT-7/EPO cells (B, D). EC50-derived relative potency (A, B) and Emax-derived relative efficacy (C, D) are shown as a percentage relative to the wild-type xlEPO (Table 2) and are plotted against the number of added N-glycosylation sites. Each point represents different xlEPO muteins.
	Fig 1. Design of N-glycosylated xlEPO muteins.(A) Schematic drawings of Xenopus laevis erythropoietin (xlEPO) muteins. N- and O-glycosylation sites are indicated by closed and opened rhombuses, respectively. The terms to the left of the bars represent the names of the muteins. “X” in xlEPO-XXX indicates N-glycosylation positions (1, Asn24; 2, Asn38; 3, Asn83). (B) Amino acid sequences around the introduced N-glycosylation sites in the xlEPO muteins. The parallel sequences of wild-type xlEPO, human EPO, and mouse EPO are also shown. Consensus sequences for N-glycosylation (Asn-Xxx-Ser/Thr) are underlined, with the amino acid substitutions in bold.

References [+] :

Aizawa, Expression of erythropoietin receptor-like molecule in Xenopus laevis and erythrocytopenia upon administration of its recombinant soluble form. 2005, Pubmed, Xenbase