Structural analysis of eythropoietin glycans
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Gutiérrez Gallego, Ricardo
Pascual Esteban, José A.
Pascual Esteban, José A.
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Llop Escorihuela, E. Structural analysis of eythropoietin glycans. Universitat Pompeu Fabra; 2008. handle: http://hdl.handle.net/10803/7129
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Programa de doctorat en Biomedicina
Abstract
La eritropoyetina o EPO es una hormona glicoproteica. En los seres humanos adultos, se produce principalmente en el riñón, en respuesta a la reducción de oxígeno en los tejidos (hipoxia tisular). La EPO estimula la eritropoyesis, es decir, estimula a las células madre de la médula ósea para que aumenten la producción de eritrocitos. La molécula de EPO está formada por una cadena peptídica de 165 aminoácidos que contiene dos puentes disulfuro, un O-glicano (Ser-126), y tres N-glicanos (Asn-24, 38, 83). En conjunto, la glicosilación de esta proteína representa aproximadamente el 40% del peso total (29.4 kDa). La EPO sintetizada con tecnología recombinante (rEPO) se administra como fármaco, desde el año 1989, para el tratamiento de anemias, insuficiencia renal, cancer etc. También se ha observado la utilización de rEPO en deportistas con el fin de aumentar el suministro de oxígeno a los tejidos y así incrementar el rendimiento en deportes de resistencia. En el año 2001, se comercializó una "nueva proteína estimuladora de la eritropoyesis" llamada NESP. Esta proteína es un análogo hiperglicosilado de la EPO que posee dos N-glicanos adicionales (Asn-30, 88). El número y composición de los N-glicanos es muy importante para el metabolismo de estas glicoproteínas, ya que el contenido de carbohidratos (número de ácidos siálicos) determina su vida media. Los métodos que se utilizan actualmente para diferenciar la eritropoyetina endógena urinaria (uEPO) de sus análogos recombinantes (rEPO, NESP) están basados en las diferencias que existen entre sus perfiles isoelectroforéticos (IEF). Se cree que estas diferencias provienen de las células y/o especies en las que se expresan estas glicoproteínas. En este estudio, se llevo a cabo la caracterización estructural de diferentes preparaciones de EPO recombinante. Para ello, se utilizaron las técnicas más comunes en el campo de la glicoproteómica además de otras nuevas, desarrolladas para poder analizar los glicanos de muestras procedentes de geles 2-DE. Los perfiles de glicosilación total de cada una de estas glicoproteínas mostraron características estructurales que pueden facilitar la detección de rEPO y NESP en atletas que abusan de estas sustancias. Un ejemplo es la presencia de Neu5Gc únicamente en fármacos que han sido expresados en células CHO. La metodología desarrollada en este trabajo, podría emplearse también, para el control de calidad de estos fármacos y para el diagnóstico de ciertas patologías.
Erythropoietin (EPO) is a glycoprotein hormone secreted primarily by adult kidneys in response to tissue hypoxia. It is involved in the maturation and ultimately regulation of the level of red blood cells. The EPO molecule comprises a single polypeptide chain of 165 aminoacids with two disulfide bonds, 1 O-linked (Ser-126), and 3 N-linked (Asn-24, 38, 83) glycans representing about 40% of the total mass (29.4 kDa). The recombinant analogue (rEPO), available since 1989 has found widespread use in the treatment of anaemia, renal failure, cancer etc. Besides, rEPO is illicitly used by athletes to boost the delivery of oxygen to the tissue and enhance performance in endurance sports. In 2001, a novel erythropoiesis-stimulating protein (NESP) was also marketed. NESP possesses two additional N-glycans (Asn-30, 88). The number and composition of the N-glycans is very important in the metabolism of this glycoprotein because the carbohydrate content (sialylation degree) determines its half-life time. Current tests to differentiate between urinary endogenous (uEPO) and its recombinant analogues (rEPO, NESP) are based on differences in their iso-electric focussing (IEF) profiles. Those differences are believed to stem from the cells/species in which they are expressed. In this study, the structural characterisation of different rEPO preparations was conducted using standard techniques in the field and developing new ones to address glycans from 2-DE sample preparations. Overall glycosylation profiling of each glycoprotein revealed structural features that may pave the way to the unambiguous detection of rEPOs and NESP abuse, such as the presence of Neu5Gc in CHO cell derived drugs only, and the developed methodology may be also employed for maintaining pharmaceutical quality control and for diagnosing pathologic conditions.
Erythropoietin (EPO) is a glycoprotein hormone secreted primarily by adult kidneys in response to tissue hypoxia. It is involved in the maturation and ultimately regulation of the level of red blood cells. The EPO molecule comprises a single polypeptide chain of 165 aminoacids with two disulfide bonds, 1 O-linked (Ser-126), and 3 N-linked (Asn-24, 38, 83) glycans representing about 40% of the total mass (29.4 kDa). The recombinant analogue (rEPO), available since 1989 has found widespread use in the treatment of anaemia, renal failure, cancer etc. Besides, rEPO is illicitly used by athletes to boost the delivery of oxygen to the tissue and enhance performance in endurance sports. In 2001, a novel erythropoiesis-stimulating protein (NESP) was also marketed. NESP possesses two additional N-glycans (Asn-30, 88). The number and composition of the N-glycans is very important in the metabolism of this glycoprotein because the carbohydrate content (sialylation degree) determines its half-life time. Current tests to differentiate between urinary endogenous (uEPO) and its recombinant analogues (rEPO, NESP) are based on differences in their iso-electric focussing (IEF) profiles. Those differences are believed to stem from the cells/species in which they are expressed. In this study, the structural characterisation of different rEPO preparations was conducted using standard techniques in the field and developing new ones to address glycans from 2-DE sample preparations. Overall glycosylation profiling of each glycoprotein revealed structural features that may pave the way to the unambiguous detection of rEPOs and NESP abuse, such as the presence of Neu5Gc in CHO cell derived drugs only, and the developed methodology may be also employed for maintaining pharmaceutical quality control and for diagnosing pathologic conditions.
Keywords
Aminoacids, Alkylation, Acetyllactosamine, Acetylation, 2-DE, 2-aminobenzamide, 5-methylene-dioxybenzene, 2-diamino-4, 1, Aminoácidos, Alquilación, Agencia Mundial Antidopaje, Aclaramiento, Ácido siálico, Ácido neuramínico, Acetillactosamina, Acetilación, 2-DE, 2-aminobenzamida, 5-metileno-dioxybenzeno, 2-diamino-4, 1, Analysis, Antennae, Antibody, Antigen, Asialo-receptor, Band
Subjects
615 - Pharmacology. Therapeutics. Toxicology. Radiology
Publisher
Universitat Pompeu Fabra
