Lectin-binding assays for the isoforms of human erythropoietin: comparison of urinary and four recombinant erythropoietins

Abstract

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis. Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Gal beta 1-4GlcNAc (RCA and ECA), NeuAc alpha 2-3Gal beta 1-4GlcNAc (MAL), NeuAc alpha 2-6Gal (SNA), or repeating Gal beta 1-4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAc alpha 2-6GalNAc (SNA) and Gal beta 1-3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding. Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA- and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Gal beta 1-4GlcNAc sequences and of those with O-glycans containing Gal beta 1-3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Gal beta 1-3GalNAc-containing O-glycans but not NeuAc alpha 2-6GalNAc-containing O-glycans or NeuAc alpha 2-6Gal-containing N-glycans. Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

“…The in vitro mouse spleen cell bioassay of Krystal (Krystal, 1983) was carried out with modifications, as described previously (Storring et al, 1996). The in vivo post-hypoxic polycythaemic mouse bioassay (Cotes & Bangham, 1961) was carried out with modifications, as described previously (Storring et al, 1998).…”

“…However, structure-activity studies among the isoforms of EPO have usually used only chargebased separations by isoelectric focusing (IEF) or electrophoresis to distinguish between the isoform compositions of different EPOs, ascribing observed differences between isoforms mainly to differences in sialylation. Lectin binding has been used in some structure-activity studies of EPO isoforms (Fukuda et al, 1989;Storring et al, 1996Storring et al, , 1998. However, only limited data have been reported relating to the detailed N-glycan structures of the isoforms of EPO with their biological activities (Takeuchi et al, 1989).…”

Epoetin alpha and beta in their erythropoetin isoform compositions and biological properties

“…Like other glycoprotein hormones, EPO exists as mixtures of a multitude of isoforms, differing mainly in their glycosylation (Storring, 1992). The isoform composition of EPO preparations varies with their source, differing between samples from serum and urine (Tam et al, 1991;Wide et al, 1995), between samples obtained from subjects under different pathophysiological conditions (Wide & Bengtsson, 1990), and between human urinary EPOs and different recombinant DNA-derived human EPOs (rEPOs) (Storring & Gaines Das, 1992;Storring et al, 1996). This is because glycosylation of the polypeptide moiety of a glycoprotein such as EPO is a post-translational process which is influenced by the type of cell in which the EPO is synthesized and the physiological factors, including culture conditions, acting upon this cell (Rademacher et al, 1988;Cumming, 1991).…”

Treating anemia associated with chronic renal failure with erythropoiesis stimulators: Recombinant human erythropoietin might be the best among the available choices

“…rHu-Epo is a product of the human gene expressed in cultured mammalian cell lines that typically differ from endogenous human Epo-producing cells in respect to glycosilation pattern and its isoform profile could be distinguished from the circulating endogenous Epo [12]. Isoform composition in rHu-Epo is affected by the cell line, cell feeding process, harvesting, purification and other technological procedures [13].…”

“…Hence, their elimination half-life is very short and erythropoietic effect is weak ("naked Epo" is cleared within minutes and has no erythropoietic effect in vivo). Conversely, larger isoforms have longer circulating time and correspondingly more pronounced erythropoietic effect (circulating time compensates for reduced receptor affinity) [2,11,13].…”