Rational protein design has been successfully used to create
mi
mics of natural proteins that retain native activity. In the present work, de novo protein engineering is explored to develop a
mini-protein analogue of Gc-MAF, a glycoprotein involved in the i
mmune syste
m activation that has shown anticancer activity in
mice. Gc-MAF is derived in vivo fro
m vita
min D binding protein (VDBP) via enzy
matic processing of its glycosaccharide to leave a single GalNAc residue located on an exposed loop. We used
molecular
modeling tools in conjunction with structural analysis to splice the glycosylated loop onto a stable three-helix bundle (
mages/gifchars/alpha.gif" BORDER=0>3W, PDB entry 1LQ7). The resulting 69-residue
model peptide, MM1, has been successfully synthesized by solid-phase synthesis both in the aglycosylated and the glycosylated (GalNAc-MM1) for
m. Circular dichrois
m spectroscopy confir
med the expected
mages/gifchars/alpha.gif" BORDER=0>-helical secondary structure. The ther
modyna
mic stability as evaluated fro
m che
mical and ther
mal denaturation is co
mparable with that of the scaffold protein,
mages/gifchars/alpha.gif" BORDER=0>3W, indicating that the insertion of the exogenous loop of Gc-MAF did not significantly perturb the overall structure. GalNAc-MM1 retains the
macrophage sti
mulation activity of natural Gc-MAF; in vitro tests show an identical enhance
ment of Fc-receptor-
mediated phagocytosis in pri
mary
macrophages. GalNAc-MM1 provides a fra
mework for the develop
ment of
mutants with increased activity that could be used in place of Gc-MAF as an i
mmuno
modulatory agent in therapy.