Abstract
Plant glycoproteins display a characteristic type of O-glycosylation where short arabinans or larger arabinogalactans are linked to hydroxyproline. The conversion of proline to 4-hydroxyproline is accomplished by prolyl-hydroxylases (P4Hs). Eleven putative Nicotiana benthamiana P4Hs, which fall in four homology groups, have been identified by homology searches using known Arabidopsis thaliana P4H sequences. One member of each of these groups has been expressed in insect cells using the baculovirus expression system and applied to synthetic peptides representing the O-glycosylated region of erythropoietin (EPO), IgA1, Art v 1 and the Arabidopsis thaliana glycoprotein STRUBBELIG. Unlike the situation in the moss Physcomitrella patens, where one particular P4H was mainly responsible for the oxidation of erythropoietin, the tobacco P4Hs exhibited rather similar activities, albeit with biased substrate preferences and preferred sites of oxidation. From a biotechnological viewpoint, this result means that silencing/knockout of a single P4H in N. benthamiana cannot be expected to result in the abolishment of the plant-specific oxidation of prolyl residues in a recombinant protein.
Highlights
Plant-made pharmaceuticals are gaining more and more attention, the difference between human post-translational modifications (PTMs) and plant PTMs should be addressed
A phylogenetic analysis was carried out including protein sequences of these N. benthamiana prolyl 4-hydroxylase (P4H) candidates and other P4H sequences of different plant origin
Some of the known plant P4Hs displayed very low sequence identity to N. benthamiana P4H candidates, these sequences were omitted from the final alignment
Summary
Plant-made pharmaceuticals are gaining more and more attention, the difference between human post-translational modifications (PTMs) and plant PTMs should be addressed. The first step in the plant-specific O-glycosylation is the generation of Hyp residues. P4Hs belong to the large class of non-heme iron dioxygenases that use α-ketoglutarate (2-oxoglutarate) and O2 as cosubstrates to activate the metal center and form succinate and a high-valent iron(IV)-oxo species (Price et al, 2003; Proshlyakov et al, 2004). These plant-specific modifications are undesired as concerns have been raised for the presence of non-human PTMs on recombinant proteins for therapeutic applications. The responsible enzymes must be discovered and characterized in order to engineer Nicotiana benthamiana into a competitive expression system for recombinant O-glycosylated proteins
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