Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Abstract

SummaryPlant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.

Human protein paucimannosylation: cues from the eukaryotic kingdoms

“…The above observations indicate that plant cells are capable of secreting PMPs although the secretory mechanism and secretion type (constitutive or regulated/packaged) remain to be described. A study reporting multiple intracellular localisations of plant HEXO1 and HEXO3 (Liebminger et al ., ), both critical enzymes for plant PMP formation, supports that non‐secreted PMPs reside in plant vacuoles and the plasma membrane and possibly in other subcellular compartments (Paul et al ., ; Ocampo et al ., ), including the non‐vacuolar intercellular fluid of N. benthamiana (Castilho et al ., ; Schneider et al ., ) and A. thaliana (Zeng et al ., ). Collectively, these observations advance our understanding of the biosynthesis and subcellular distribution of PMPs in plants, which appear to be multi‐compartmental, dynamic and most likely physiology‐dependent, knowledge that may aid our understanding of human PMPs (see Section IV).…”

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

“…ER-Ab and vac-Abs accumulations levels were 10-15-fold higher than sec-Ab. 33 Although NPIRL motif is typical of lytic vacuole proteins and the short and hydrophobic C terminus are distinctive of storage proteins 39 , no significant differences were found between vac1-Ab and vac2-Abs yields. Another important finding of our work, was the presence of oligomannosidic (Man 7-9) as the major glycoform in vac-Abs (75%), what suggests a direct transport from the ER to vacuoles bypassing the Golgi apparatus.…”

“…Although Nt-VSS and Ct-VSS were supposed to target proteins to lytic and storage vacuoles, respectively, both type of motif targeted proteins to central vacuole of vegetative tissue by a molecular mechanism that is currently unclear. 43 The N-glycosylation pattern of the foreign exemplified differences in vacuolar sorting mechanism, for example glucocerebrosidase-Ct-VSS exhibited paucimannose structures and complex glycan added in the trans Golgi; supporting a Golgi dependent transport 24 while a mouse IgG1 fused to a ssVSS and Ct-VSS of amaranth storage proteins is decorated with Man 7 and Man 8 glycans supporting a direct transport bypassing the Golgi 33 (Fig. 1) These glycosylation patterns maybe adequate for some foreign proteins such as glucocerebrosidase whose vacuolar variant is easily internalized by human cells, but it is no convenient for therapeutic antibodies since effectors' functions are dependent of heavy chain glycosylation.…”

“…Another important finding of our work, was the presence of oligomannosidic (Man 7-9) as the major glycoform in vac-Abs (75%), what suggests a direct transport from the ER to vacuoles bypassing the Golgi apparatus. 33 Furthermore vacAbs have 25% of GlcNAc2Man3XylFucGlcNAc2 therefore removal of terminal GlcNAc residues in the vacuole did not occur. 33 Ability of plants cells to accumulate toxic proteins in vacuoles is not surprising since variety of natural and synthetic chemicals are inactivated and transported to vacuole by different detoxification mechanisms.…”

“…33 Furthermore vacAbs have 25% of GlcNAc2Man3XylFucGlcNAc2 therefore removal of terminal GlcNAc residues in the vacuole did not occur. 33 Ability of plants cells to accumulate toxic proteins in vacuoles is not surprising since variety of natural and synthetic chemicals are inactivated and transported to vacuole by different detoxification mechanisms. 40 For example, some xenobiotic compounds are conjugated to glutathione in the cytosol and then transported to vacuole by an ATP-dependent tonoplast transporter.…”