Heterologous expression of rat ST6Gal-I in Pichia pastoris for structural and functional studies

Abstract

ST6Gal-I plays an important role in immune regulation mediated via the lectin CD22. The enzyme catalyses transfer of sialic acid to form a Sia[alpha]2-6Ga[beta]1-4GlcNAc (Sia6LacNAc) tri-saccharide product, which is ligand for CD22. Despite such a critical role in immune function, no structural data is yet available for ST6Gal-I or any other GT29 enzyme. A major limitation in the structural analysis of glycosylation enzymes, including ST6Gal-I, is the large-scale expression and purification of the glycosylated enzymes in forms compatible with structural analysis. We have successfully expressed a functional, soluble form of the recombinant ST6Gal-I catalytic domain using the Pichia pastoris expression system. The enzymatic properties of the ST6Gal-I were comparable to those of rat liver ST6Gal-I with regard to substrate specificity and stability. We have also established conditions for N-glycan removal from the recombinant ST6Gal-I for structural and functional studies. The results suggest that the enzymatic properties of the deglycosylated enzyme were comparable to that of glycosylated enzyme. Conditions for stable isotope labeling for structural characterization by NMR have been established and initial studies on the spectral assignment have been initiated. Our NMR data supports the theory of substrate induced conformational changes for ST6Gal-I. Also, here we have demonstrated that any chemical modification of ST6Gal-I, which are essential for immobilization of enzyme for substrate binding studies using SPR, leads to inactivation of enzyme.