FUSING THE CARBOXY-TERMINAL PEPTIDE OF THE CHORIONIC-GONADOTROPIN (CG) BETA-SUBUNIT TO THE COMMON ALPHA-SUBUNIT - RETENTION OF O-LINKED GLYCOSYLATION AND ENHANCED IN-VIVO BIOACTIVITY OF CHIMERIC HUMAN CG
M. Furuhashi et al., FUSING THE CARBOXY-TERMINAL PEPTIDE OF THE CHORIONIC-GONADOTROPIN (CG) BETA-SUBUNIT TO THE COMMON ALPHA-SUBUNIT - RETENTION OF O-LINKED GLYCOSYLATION AND ENHANCED IN-VIVO BIOACTIVITY OF CHIMERIC HUMAN CG, Molecular endocrinology, 9(1), 1995, pp. 54-63
The hCG beta-subunit contains a carboxy-terminal extension bearing fou
r serine-linked oligosaccharides [carboxy-terminal peptide (CTP)], whi
ch is important for maintaining its longer half-life compared with the
other glycoprotein hormones. Previously, we enhanced the in vivo half
-life of FSH by fusing the CTP to the carboxy end of FSH beta coding s
equence. The alpha-subunit is common to the glycoprotein family. We co
nstructed alpha-subunit CTP chimeras, since such analogs with the appr
opriate O-linked glycosylation and conformation would increase the in
vivo stability of the entire glycoprotein hormone family. Two chimeras
were constructed using overlapping polymerase chain reaction mutagene
sis: a variant with CTP at the carboxy end and another analog with the
CTP at the N-terminal region of the subunit, between amino acids 3 an
d 4. The latter design was based on models showing that the amino-term
inal region of alpha is not involved in assembly with the beta-subunit
, nor is it essential for receptor binding and signal transduction. Th
ese chimeras were cotransfected with the hCG beta gene into Chinese ha
mster ovary cells. The chimeras were secreted and combined efficiently
with the CG beta-subunit, comparable to the wild type alpha-subunit.
CG dimers containing the alpha-subunit chimera with CTP at the carboxy
end of the subunit had a much lower binding affinity for the hLH-hCG
receptor in vitro, whereas the binding of the dimer containing the CTP
at the amino-terminal end of the subunit was similar to wild type hCG
. Furthermore, the in vivo activity of this analog was enhanced signif
icantly. Moreover, regardless of the two insertion points in the alpha
-subunit, the CTP sequence was O-glycosylated. These data suggest that
the entire signal for O-glycosylation is primarily contained within t
he CTP sequence and is not dependent on the flanking regions of the re
cipient protein. The transfer of CTP to the alpha-subunit of hCG resul
ts in an agonist with prolonged biological action in vivo. These data
further support the rationale for using the CTP as a general target to
increase the potency of bioactive glycoproteins.