In Planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway

Heribert Quendler,Johannes Stadlmann,Friedrich Altmann,Richard Strasser,Pia Gattinger,Alexandra Castilho,Josephine Grass,Herta Steinkellner,Renate Kunert,Renaud Leonard,Martin Pabst,Jakub Jez

doi:10.1074/jbc.m109.088401

Abstract

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

Highlights

The outstanding specificity of therapeutic glycoproteins places them among the fastest growing class of pharmaceutical products
Initial attempts to introduce Neu5Ac residues into plant N-glycans have resulted in the expression of some of these proteins in plants [11, 12], and recent progress in our laboratory allowed the synthesis of the sugar nucleotide CMP-Neu5Ac from endogenous metabolites by the simultaneous overexpression of three mammalian enzymes in Arabidopsis thaliana [13]
In Planta Synthesis of CMP-Neu5Ac—The initial step in the mammalian sialylation pathway is the biosynthesis of the activated sugar nucleotide precursor cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac)

Summary

Introduction

The outstanding specificity of therapeutic glycoproteins places them among the fastest growing class of pharmaceutical products. We demonstrated recently that by overexpressing a modified version of the human ␤1,4-galactosyltransferase (GalT), plant N-glycans can be extended with ␤1,4-linked galactose at great uniformity [10] These structures serve as acceptor substrates for subsequent terminal sialylation. Sialylation is difficult to accomplish even in the presence of ␤1,4-galactosylated structures because plants lack some further essential prerequisites: (i) the biosynthetic capability to produce the sugar nucleotide precursor CMPsialic acid, CMP-N-acetylneuraminic acid (CMPNeu5Ac); (ii) a transporter that delivers CMP-sialic acid into the Golgi in sufficient amounts; and (iii) a sialyltransferase (ST) to transfer sialic acid from CMP-Neu5Ac to terminal galactose on the nascent glycoprotein These proteins and their substrates must work in a highly coordinated fashion at different stages of the pathway, and organelle-specific targeting of several components is required to enable proper protein sialylation (see Fig. 1). In planta protein sialylation has not yet been achieved

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Conclusion