Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity

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.

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis

“…Membrane-anchored prolyl-4-hydroxylases (P4H) are responsible for modifying proline residues into trans -4-L-proline in N. tabacum (Yuasa et al, 2005 ), which is likely similar to the mechanism in N. benthamiana . Recently, four paralogs of P4H of N. benthamiana have been studied for their substrate specificity and subcellular localization (Mócsai et al, 2021 ). All paralogs were found to localize to the Golgi apparatus with the exception of P4H4, which was also found in the ER.…”

Recombinant neutralizing secretory IgA antibodies for preventing mucosal carriage and transmission of SARS-CoV-2

“…Interestingly, SIgA1 and SIgA2 showed similar rates of degradation, although in humans SIgA1 is more prone to degradation by proteases produced by oral bacteria selectively cleaving sites in the extended hinge-region of IgA1, leaving SIgA2 as the predominant isotype in mucosal secretions 43,44 . Here, the activity of bacterial proteases on the extended IgA1 hinge-region might be reduced due to the conversion of prolines to hydroxyprolines, which lie within the recognition sequence of many bacterial proteases and are partially extended with arabinose chains, thereby potentially masking the cleavage site 45,46 . To date there is only limited knowledge about safety and efficacy of the repeated application of recombinant mAbs let alone plant-produced IgAs to mucosal surfaces or influences of plant-specific modifications.…”

Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects

“…A P4H1 knock-out in Physcomitrella patens prevents proline conversion into hydroxyproline (Hyp) on recombinant EPO (Parsons et al, 2013). In N. benthamiana, P4H1, P4H4, P4H9 and P4H10 have recently been identified as enzymes capable of forming hydroxyproline on recombinant proteins (Mócsai et al, 2021). Unfortunately, transient RNAi of these genes in N. benthamiana was insufficient to reduce P4H activity completely, due to redundancy within the substrate specificity of the P4H genes.…”