Biology Reference
In-Depth Information
ing [18]. Glycoengineered molecules bind and activate EPOR in the same
manner as rHuEPO, resulting in similar biological responses while at the same
time reducing clearance and enhancing activity [19]. Other strategies to
increase duration of action of EPO included chemical modifications, such as
the addition of a polyethylene glycol molecule (pegylation) or gene fusions
between EPO and other proteins. In these cases, the goal is to reduce clearance
rate by increasing hydrodynamic size.
Glycoengineering
rHuEPO is a glycoprotein hormone consisting of approximately 40% carbo-
hydrate [20]. The carbohydrate component consists of three
N
-linked carbo-
N
hydrates attached to Asp at amino acid positions 24, 38, and 83, and an
O
-linked carbohydrate attached to Ser at amino acid position 126 [20] (Fig.1).
Unlike the invariant protein sequence, the carbohydrate is variable in structure,
resulting in glycoforms with modest differences in sizes, structures, and sugar
content [22, 23]. A typical
N
-linked carbohydrate made by mammalian cells is
N
Figure 1. Amino acid sequence of human erythropoietin. Recombinant human erythropoietin
(rHuEPO) is 165 amino acids in length [21]. Disulfide bonds (-S-S-) join Cys7 to Cys161 and Cys29
to Cys33. The 3
N
-linked glycosylation attachment points are at Asn24, Asn38, and Asn83 and the
N
O
-linked carbohydrate is attached to Ser126. Forked structures schematically depict the attached car-
bohydrates.
