Rec. ST6Gal-I variants to control enzymatic activity in processes of in vitro glycoengineering

Abstract

Background Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety (sialic acid, 5-N-acetylneuramic acid) is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, e.g. IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life. The biosynthesis of sialyl conjugates is controlled by a set of sugar-active enzymes including sialyltransferases which are classified as ST3, ST6 and ST8 based on the hydroxyl position of the glycosyl acceptor the Neu5Ac is transferred to [1]. The ST6 family consists of 2 subfamilies, ST6Gal and ST6GalNAc. ST6Gal catalyzes the transfer of Neu5Ac residues to the hydroxyl group in C6 of a terminal galactose residue of type 2 disaccharide (Galb1-4GlcNAc). To our knowledge, the access to recombinant ST6GalI for therapeutic applications is still limited due to low expression and/or poor activity in various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli). The present study describes the high-yield expression of two variants of human beta-galactoside alpha-2,6 sialyltransferase 1 (ST6Gal-I, EC 2.4.99.1; data base entry P15907) by transient gene expression in HEK293 cells with yields >100 mg/L featuring distinct mono(G2 +1SA) as well as bi(G2+2SA) sialylation activity. Materials and methods Two N-terminally truncated fragments of human ST6Gal-I (delta89, residues 89-406, and delta108, residues 109-406) were designed for transient gene expression (TGE): Instead of the natural leader sequence and N-terminal residues, both ST6Gal-I coding regions harbor the Erythropoietin (EPO) signal sequence in order to ensure correct processing of the polypeptides by the secretion machinery. Following cloning into pM1MT, expression of the ST6Gal-I coding sequences is under control of a hCMV promoter followed by an intron A. Sialyltransferase assays: 1. Asialofetuin was used as acceptor and CMP-9F-NANA as donor substrate. Enzymatic activity was determined by measuring the transfer of 9F-NANA to asialofetuin. 2. Recombinant humanized IgG1 and IgG4 monoclonal antibodies (mabs), characterized as G2+0SA, as well as desialylated EPO were used as targets in sialylation experiments (30 μg enzyme/300 μg target protein). Both enzyme variants of ST6Gal-I (delta89 and delta108) were used under identical reaction conditions and the sialylation status was analyzed by mass spectrometry.