A designed glycoprotein analogue of Gc-MAF exhibits native-like phagocytic activity.
Rational protein design has been successfully used to create mimics 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 immune system activation that has shown anticancer activity in mice. Gc-MAF is derived in vivo from vitamin D binding protein (VDBP) via enzymatic 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 (alpha3W, 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) form. Circular dichroism spectroscopy confirmed the expected alpha-helical secondary structure. The thermodynamic stability as evaluated from chemical and thermal denaturation is comparable with that of the scaffold protein, alpha3W, indicating that the insertion of the exogenous loop of Gc-MAF did not significantly perturb the overall structure. GalNAc-MM1 retains the macrophage stimulation activity of natural Gc-MAF; in vitro tests show an identical enhancement of Fc-receptor-mediated phagocytosis in primary macrophages. GalNAc-MM1 provides a framework for the development of mutants with increased activity that could be used in place of Gc-MAF as an immunomodulatory agent in therapy.
Bogani
Federica
F
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
McConnell
Elizabeth
E
Joshi
Lokesh
L
Chang
Yung
Y
Ghirlanda
Giovanna
G
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry / Q000494:pharmacology*; D006031:Glycosylation; D016215:Macrophage-Activating Factors / Q000737:chemistry / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010587:Phagocytosis / Q000187:drug effects*; D014809:Vitamin D-Binding Protein / Q000737:chemistry / Q000494:pharmacology*
D006023:Glycoproteins; D016215:Macrophage-Activating Factors; D014809:Vitamin D-Binding Protein; C101908:vitamin D-binding protein-macrophage activating factor