Sialyltransferases are essential enzymes for the biosynthesis of sialyloligosaccharides. However, it is known that sialyltransferases expressed in E. coli show very poor enzymatic activities. We consider that the biosynthesis of these series of sialyltransferases requires glycosylation and the glycoprotein quality control (GQC) system, which promotes the native folding of glycoproteins in the endoplasmic reticulum (ER). In order to study the necessity of glycan and GQC in folding of sialyltransferases, we synthesized human β-galactoside α2,6-sialyltransferase (ST6GAL1) with and without a homogeneous high-mannose type oligosaccharide which is a tag of GQC.
The membrane extracellular domain of ST6GAL1 consists of 318 amino acids. We planned to synthesize ST6GAL1 from three peptide segments: sialyltransferase (1-53)-thioester, (54-95)-glycopeptide-hydrazide, (96-318)-peptide, and these segments were prepared by Boc Solid Phase Peptide Synthesis and E. coli expression. High-mannose type oligosaccharide isolated from egg yolk was used for the preparation of the glycopeptide segment. These peptides were ligated from N-terminus to C-terminus by native chemical ligation. Finally, enzymatic active sialyloligosaccharide could be obtained by folding experiments of chemically synthesized ST6GAL1-polypeptide. Kinetic parameters (Km, Kcat) for the chemically synthesized sialyltransferase are evaluated.
ST6GAL1 could be folded as an enzymatic active form, regardless of whether it had a glycan or not, if protein aggregation could be suppressed. Because an expression system in mammalian cell robustly yields an active ST6GAL1, the glycan and GQC seem to suppress aggregation efficiently and to promote the folding of ST6GAL1.