N-linked glycan analysis of glycoproteins secreted from rice cell suspension cultures under sugar starvation

Abstract

Recently, the rice α-amylase 3D promoter system activated under sucrose-starved conditions has emerged as an alternative system for the production of useful recombinant proteins. However, the influence of N-linked glycan structures related to sugar starvation conditions has not yet been investigated. In this study, we determined the N-linked glycans of glycoproteins and human granulocyte-macrophage colony stimulating factor secreted from rice cell suspension cultures under sugar starvation conditions. The N-glycan patterns obtained from glycoproteins grown in growth medium with sucrose (+S) were different from those obtained under sucrose starvation (−S). The mass spectra of +S glycans exhibited eleven N-glycan patterns. Among these, the most abundant glycans were GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc (27.4% of total), followed by Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, GlcNAcβ1-2Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, and Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, each of which represented 15–17% of the total. Except for Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, all +S N-glycans were complex-types containing β1,3-galactose or α1,4-fucose. The five N-glycans that disappeared under −S conditions were GlcNAcβ1-2Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, Galβ1-3GlcNAcβ1-2Manα1-6(Galβ1-3GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, and Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc. The N-glycan pattern of glycoproteins included hGM-CSF derived from rice suspension cell culture, and all N-glycans contained plant-specific β1,2-xylose, core α1,3-fucose, and some Lewis a epitopes. High-mannose type N-glycans were not detected under either condition (−S or +S) among the secreted glycoproteins.