N-glycosylation is one of the best studied post-translational modifications and can have a significant impact of the biological activity, targeting and pharmacokinetic behavior of therapeutic glycoproteins. Oligo-mannose type (OMT) N-glycans can be produced by using a C-terminal ER-retreival signal (H/ KDEL) which targets recombinant proteins to accumulate in the ER.
Glycoproteins targeted to lytic or storage vacuoles contain paucimannosidic N-glycans whereas those targeted to the extracellular space contain either terminal Glc-NAc residues in addition to the plant specific core fucose and xylose or contain further extensions such as Lewisa determinants [4, 5].
Although ER targeting is often preferred as it leads to higher protein stability and yield, in some cases the presence of OMT N-glycans is not always desirable for pharmaceutical proteins [6, 7]. Other reports show that plant specific paucimannosidic N-glycans are desirable and have been related to an enhancement of the therapeutical efficiency of the recombinant protein .
Thus, subcellular targeting and deposition sites have become an important consideration for the production of pharmaceuticals. Previous studies done in our group showed that a recombinant fungal phytase carrying a signal peptide and no further targeting signal was deposited within the cell in the protein storage vacuoles of cereal endosperm [9, 10, 11].
Fig. 1: DsRed localization in tobacco. A. Fluorescence microscopy, B, C. Electron microscopy, B’, C’. Confocal microscopy,. A. Leaf epidermis, see the accumulation of DsRed in the apoplast (arrows). B, B’. Seed embryo, significant signal on the cell walls (cw, arrows). C, C’. Seed endosperm. See the labelling within the apoplast (cw, arrows) and also in some structures within the cells (C’, arrowhead). Bars 2 µm (A, B’, C’), 0,25 µm (B, C).
Figure 1 shows the deposition of recombinant DsRed in tobacco cells. Recombinant DsRed carrying a signal peptide and no further targeting signal was constitutively expressed in tobacco. DsRed fluorescence in leaves was observed in vivo in a Leica DM5000 (A) whereas seeds were observed under a Leica SP2confocal microscope (B’, C’).
For electron microscopy (B, C), small pieces of tobacco leaves, as well as decoated tobacco seeds were fixed in 4 % paraformaldehyde and 0,5 % glutaraldehyde. After dehydration through ethanol series, samples were infiltrated and embedded in LRWhite resin and the blocks were allowed to polymerise at 60 °C for 2 days. Ultrathin sections (80 nm) were obtained with a Leica Ultracut UCT and collected on gold grids and observed with a Philips EM-400 electron microscope after immunogold localization of recombinant DsRed.
The fluorescence in the periphery of the leaf epidermical cells (Figure 1A) indicates that recombinant DsRed is efficiently secreted via the vegetative tissue default pathway , similar to other recombinant proteins in tobacco leaves  or in rice leaves . DsRed is also efficiently secreted in tobacco embryo (Figure 1B, B’) and abundant gold probes can be observed decorating the embryo cell walls. No significant signal can be observed within the oil bodies or any other compartment of the cells (Figure 1B, B’). In the endosperm, significant labelling can be also observed within the cell walls indicating that the protein is secreted (Figure 1C, C’). However, some fluorescent structures (most probably protein storage vacuoles) can be observed within the endosperm cells (Figure 1C’).
It is interesting to remark that the endosperm in tobacco, as that of cereal seeds, is a short-lived terminal tissue specialized for food storage that will not survive germination. Cereal starchy endosperm cells are meant to be dead upon germination and hydrolytic enzymes for the mobilization of reserves are secreted by aleurone cells during germination, while the mature endosperm cells of dicotyledonous seeds often comprises living cells secreting hydrolytic enzymes that will be needed to degrade the endosperm cell walls to allow radicle emergence [14, 15, 16, 17].
Our results confirm the physiological differences between the endosperm tissues in monocotyledonous and dicotyledonous plants. While in cereals recombinant phytase and recombinant anti-HIV antibody 2G12 were directed to the PSVs, DsRed in tobacco endosperm was only partially retained, like also reported for the above mentioned recombinant proteins .
The different localization of recombinant proteins in different tissues and species results in different glycoforms  and therefore should be taken into the account when it comes to choosing a platform for the production of pharmaceuticals in plants.
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