title: The overlooked difference between human endogenous and recombinant erythropoietins and its implication for sports drug testing and pharmaceutical drug design.
abstract: Sequential deglycosylation by exoglycosidase treatment (Reagent Array Analysis Method, RAAM) and subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a profound structural difference between human endogenous and recombinant erythropoietins. While both proteins behaved similarly upon digestion with Arthrobacter ureafaciens α-sialidase and Steptococcus pneumoniae β-D-galactosidase, the action of N-acetyl-β-D-glucosaminidase from Steptococcus pneumoniae was partly blocked by endogenous but not recombinant erythropoietins. Consequently, further treatment with Jack bean α-D-mannosidase and Helix pomatia β-D-mannosidase led to only very limited additional deglycosylation of endogenous EPO, while rhEPO glycans continued to be degraded. The behaviour was visualized by SDS-PAGE combined with Western blotting. While the apparent molecular masses of most endogenous glycoforms did not further decrease after treatment with the first three enzymes, masses of most rhEPO glycoforms continued to drop after digestion with the two mannosidases. Both, human urinary and serum EPO showed this blocking effect, and all of the tested 28 recombinant epoetins were accessible to further degradation by exo-mannosidases. The majority of EPO pharmaceuticals is produced in Chinese hamster ovary (CHO) cell lines, few in other ones (i.e. baby hamster kidney (BHK) or human fibrosarcoma (HT-1080) cells). Since human endogenous EPO is primarily produced by the kidneys, tissue specific glycosylation might explain the altered deglycosylation behaviour. This difference was overlooked since EPO was first isolated from human urine in 1977. The results might prove useful for anti-doping testing and future EPO drug development.
authors.affiliation: Doping Control Laboratory, AIT Seibersdorf Laboratories, A-2444 Seibersdorf, Austria. christian.reichel@seibersdorf-laboratories.at
authors.forename: Christian
authors.initials: C
authors.lastname: Reichel
mesh_terms: D000818:Animals; D001173:Arthrobacter / Q000201:enzymology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004300:Doping in Sports; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology; D015813:Substance Abuse Detection
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D006026:Glycoside Hydrolases