Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging.

Affiliation

Sales MLM(1), Kratje R(1), Oggero M(1), Ceaglio N(2).
Author information:
(1)UNL, CONICET, FBCB
(School of Biochemistry and Biological Sciences), CBL
(Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168, Km 472.4, C.C. 242, S3000ZAA, Santa Fe, Argentina.
(2)UNL, CONICET, FBCB
(School of Biochemistry and Biological Sciences), CBL
(Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168, Km 472.4, C.C. 242, S3000ZAA, Santa Fe, Argentina. Electronic address: [Email]

Abstract

Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification.