BACKGROUND
plants emerged as effective systems for production of pharmaceutically important mammalian proteins, and a number of transgenic plant-based products are currently available in the market  <cit> .
work in the field of plant molecular farming revealed that, while there are obvious advantages to using plant bioreactors, including low cost, fast scalability, biological safety, proper protein folding and assembly, there are also some limitations.
glycosylation differences between plants and humans is one of the major drawbacks that can negatively influence properties of plant-derived recombinant proteins  <cit> .
significant efforts have been made to "humanize" plant n-glycan structures, either by retaining the recombinant proteins in the endoplasmic reticulum   <cit>  or by modifying the enzyme machinery in the golgi apparatus to eliminate plant-specific β-xylose and α-fucose residues  <cit>  and extend plant n-glycans with terminal β-galactose residues  <cit> .
however, little attention has been paid to the structural differences between plant and mammalian o-glycans.
o-glycans are attached primarily to hydroxyproline  and serine residues in plant glycoproteins.
typically, non-contiguous hyp residues are modified with arabinogalactan polysaccharides, whereas contiguous hyp are exclusively arabinosylated  <cit> .
the extent of proline hydroxylation and hyp glycosylation depends on the side chains of flanking amino acids  <cit> .
a specific motif  has been defined, based on examination of amino acid sequence requirements, for efficient hydroxylation of proline and arabinogalactosylation of hyp  <cit> .
no rules for selection of serine residues for o-glycosylation have been defined so far, probably because the number of identified proteins with glycosylated serines is still limited to a few plant lectins.
usually, plant o-glycans attached to serine residues are short, composed of galactopyranosides present either as monosaccharides or as 1→3-linked disaccharides  <cit> .
in general, information about plant o-glycosylation enzyme machinery is lacking.
in contrast to plants, mammalian o-glycosylation is a well understood posttranslational modification.
substitutions of serine and threonine residues with sugars such as fucose, galactose and n-acetylglucosamine are common modification on mammalian proteins  <cit> , but the most abundant o-glycans in mammalian cells are mucin type o-linked glycans.
they are represented by structurally diverse oligosaccharides attached through n-acetylgalactosamine  to serine and/or threonine residues.
besides the initial galnacα-ser/thr, known as tn antigen, other structures, including core  <dig> or t antigen , core  <dig> , core  <dig> , core  <dig> galnacα-ser/thr), core  <dig> , core  <dig> , and core  <dig> , are also commonly linked to human proteins.
these core structures can undergo further extension and branching to form more highly complex o-glycans  <cit> .
mucin type o-linked glycans are synthesized in the golgi apparatus  <cit>  in a stepwise and consensus motif-independent manner.
a family of udp-galnac polypeptide:n-acetyl-α-galactosaminyltransferases  initiates the process by transferring n-acetylgalactosamine  from the charged sugar donor, udp-galnac, to the hydroxyl group of serine and threonine amino acid residues .
abundance of galnac-t isoforms , distinct spatial and temporal gene expression patterns  <cit>  and unique substrate specificities  <cit>  ensure precise selection of attachment sites and fine regulation of galnac glycosylation of fully folded proteins.
depending on the final o-glycan structure, different sets of glycosyltransferases participate in elongation of the initial galnac residue.
for example, udp-gal:galnacα-ser/thr β1- <dig> galactosyltransferase and udp-glcnac:galnacα-ser/thr β1- <dig> n-acetylglucosaminyltransferase are required for synthesis of core  <dig> o-glycans and core  <dig> o-glycans, respectively.
a previous study reported attachment of core  <dig> galβ1-3galnac disaccharide to rice glutelin basic subunits  <cit> , however our in silico analysis and more recent experimental data  <cit>  suggest that plants do not have the enzyme machinery to support mucin type o-glycosylation.
in light of this, we reasoned that genetic manipulations could potentially modify plants for production of mammalian glycoproteins with homogenous o-glycan structures.
the present study tested, for the first time, the feasibility of generating a plant-based platform for production of tn antigen-containing proteins/peptides.
many valuable pharmaceuticals, such as immunomodulators and tumor-associated antigens essential for the development of anticancer therapeutic vaccines, are galnac -glycosylated  <cit> .
despite a growing demand for these products, manufacturing remains difficult and costly.
in our study, we first addressed the question of whether the initiation of mucin type o-glycosylation in plants could be performed with the precision typical of mammalian cells.
therefore, one of the well characterized isoforms of the human galnac-ts family, galnac-t <dig>  was stably expressed in n. benthamiana and used to demonstrate that mammalian galnac-ts retain their functionality, specificity and localization in plant cells.
we further tested the engineered galnac-t <dig> transgenic plants for their ability to produce galnac-glycosylated proteins.
a model glycoprotein, mucosally targeted mucin  <dig>  tandem repeat-derived peptide, was transiently expressed using the magnicon deconstructed viral vector system to ensure rapid accumulation and high yield  <cit> .
glycosylation efficiency was monitored by lectin chromatography.
our results suggest that co-expression of genes for udp-galnac polypeptide:n-acetyl-α-galactosaminyltransferase, udp-glcnac 4-epimerase, and udp-galnac transporter are required to achieve efficient plant-based production of galnac-glycosylated proteins.
RESULTS
generation and molecular analyses of n. benthamiana l. plants expressing human galnac-t2
agrobacterium-mediated transformation of n. benthamiana l. with ph7wg2:gnt <dig> binary vector  was performed to generate plants stably expressing human galnac-t <dig> gene.
the putative transgenic plants were subjected to routine molecular analyses, including pcr, rt-pcr and western blotting, to select for the best producers.
plants that exhibited the expected pcr product of  <dig>  kb in size , rt-pcr band of  <dig>  kb in size , and a polypeptide of ~  <dig> kda after probing with monoclonal antibodies raised against the full length galnac-t <dig> protein  were selected for further experiments.
none of the transgenic plants had apparent morphological abnormalities .
localization of human galnac-t <dig> expressed in n. benthamiana l
a fusion with aequorea victoria green fluorescent protein , positioned at the c terminus of galnac-t <dig> to avoid interfering with the transmembrane domain , was used to monitor the localization of mammalian enzyme in tobacco cells.
a discrete punctuate pattern was observed by confocal microscopy .
this pattern could be indicative of golgi localization or for targeting to the plant prevacuolar or late endosomal compartments , as shown by experiments with tobacco bright yellow - <dig> cells expressing fluorescent markers  <cit> .
to better define the localization of the expressed human galnac-t <dig>  plant leaves were treated with brefeldin a. brefeldin a is a macrocyclic lactone isolated from alternaria carthami known to block protein er to golgi transport.
in plants, the drug causes loss of golgi-based vesicle formation and fusion of the individual golgi cisternae with the er  <cit> .
it also affects pvcs morphology leading to extensive aggregation and tubulation.
however, in contrast to the high sensitivity of the golgi apparatus, pvcs respond to brefeldin a treatment only at drug concentrations greater than  <dig> μg/ml  <cit> .
tobacco plant leaves expressing galnac-t2:gfp fusion protein were incubated for  <dig> h with  <dig> μg/ml brefeldin a. the treatment caused loss of the bright green spots and replacement of the punctuate pattern with a network-type fluorescence .
these results strongly suggest that mammalian galnac-t <dig> is retained in the golgi in plant cells.
functionality of human galnac-t <dig> expressed in n. benthamiana l
to assess the functionality of the plant-expressed human galnac-t <dig>  we used an in vitro assay that included plant microsomal fraction as an enzyme source, udp-galnac as the donor, and the 113- <dig> aa fragment of human chorionic gonadotropin β-subunit  as an acceptor peptide.
under the chosen hplc conditions, the retention time for udp-galnac was  <dig>  min, while hcg-β peptide eluted at  <dig> / <dig>  min as two peaks , as confirmed by maldi .
hplc separation of an aliquot of the enzymatic reaction with the microsomal fraction from control tobacco plants showed the presence of only the non-glycosylated hcg-β peptide .
the observation was further confirmed by maldi analysis .
in contrast, when microsomal fraction of galnac-t <dig> transgenic plants was used as the enzyme source, two additional peaks  with slightly shorter retention times were visible on the chromatogram .
correlation between the accumulated radioactive label in the fractions corresponding to peaks "b" and "c" and the amount of enzyme added to the reaction mixture suggested that these peaks represent products of the enzymatic reaction .
further maldi-tof ms analysis of the combined "b" and "c" fractions revealed three major + ions at m/z  <dig> ,  <dig> , and  <dig>  .
the ion at m/z  <dig>  indicated that the analyzed sample contained the initial  acceptor peptide.
this was not surprising since the peak of the acceptor hcg-β peptide  partially overlapped peaks "b" and "c".
the ions at m/z  <dig>  and m/z  <dig>  were suggestive of two glycosylated forms of hcg-β peptide with attached one or two galnac residues.
maldi-tof-tof spectra analysis further supported the assumption.
the major fragmentation product resulting from loss of one hexnac residue from the glycosylated peptide containing a single galnac was evidenced by the abundant ion at m/z  <dig>  .
similarly, during the fragmentation of the ion at m/z  <dig>  corresponding to the hcg-β peptide with two galnac residues, the massive peak envelope centered at m/z  <dig>  and smaller peak envelope at m/z  <dig>  are indicative of the loss of one and two hexnac residues, respectively .
de-novo peptide sequencing on maldi-tof/tof ms spectrometer was employed to determine the position of the attached galnac residues.
based on b- and y-ions generated during the sequencing, the attachment of galnac for the mono- and diglycosylated peptides most likely occurred at ser- <dig> , and at ser- <dig>  and ser- <dig> , respectively.
effect of the expression of human galnac-t <dig> on glycosylation status of n. benthamiana l. endogenous proteins
the substrate preferences of galnac-t <dig> have been extensively studied and the most favorable amino acids at positions - <dig>  - <dig>  - <dig>   <dig>   <dig>  and  <dig> relative to the site of glycosylation , have been determined  <cit> .
the first three amino acids with the highest enhancement values for each of these positions were used to design a series of peptides consisting of  <dig> amino acids.
in silico analyses using the peptides to search ncbi plant databases  indicated that some annotated plant proteins known to transport through the er-golgi pathway do contain potential sites for galnac-glycosylation .
therefore, we tested for possible attachment of galnac to endogenous proteins of n. benthamiana l plants engineered to express human galnac-t <dig>  equal amounts of soluble proteins from the control and transgenic plants were subjected to affinity chromatography on vicia villosa agglutinin -agarose.
vva is a lectin that strongly recognizes terminal galnac residues attached to serine or threonine in a polypeptide.
the binding buffer was supplemented with  <dig>  m nacl to prevent non-specific interactions, and the resin was thoroughly washed before the elution.
eluted proteins were further subjected to lectin blot analysis with alkaline phosphatase-conjugated vva.
several positive bands were detected in the transgenic plant sample, but not in the negative control .
following silver staining, the strongest band, corresponding to a  <dig> kda polypeptide , was digested with chymotrypsin.
analysis of the recovered peptides by nano lc-ms/ms using a linear quadrupole ion trap mass spectrometer and a database search identified the band with 99% probability as nicotiana endochitinase .
modification of threonine at position  <dig> by  <dig> amu was also detected , as evidenced by a series of y ions  containing the modification, thus strongly suggesting attachment of a n-acetylhexosamine residue to the polypeptide chain.
it is of interest that cleavage on the carboxy-terminal side of the potentially modified thr- <dig> was absent , possibly as a result of the sugar moiety blocking access of the ionizing mobile proton at that position.
loss of the sugar moiety from glycopeptides is often observed during ms/ms, but in certain cases of low-energy-collision induced dissociation the sugar moiety may remain attached, resulting in fragment ions carrying the modification  <cit> .
the threonine residue  modified by  <dig> amu is in boldface type, carboxymethylated cysteine residues are denoted by asterisks.
the amino acids in parenthesis at the beginning and end of each peptide show the flanking residue in the entire protein sequence.
a homology model of tobacco endochitinase , built based on the three dimensional structure of o. sativa l. japonica class i chitinase , suggests that thr- <dig> should be located on the surface of the catalytic domain, therefore accessible for glycosylation.
previous study has shown that thr- <dig> is not directly involved in the catalytic act  <cit> .
efficiency of attachment of galnac to the model protein, e. coli enterotoxin b subunit:human muc <dig> tandem repeat-derived peptide fusion protein expressed transiently in galnac-t <dig> plants by magnicon deconstructed viral vector system
as the transgenic galnac-t <dig> plants demonstrated capability for attaching galnac to the endogenous endochitinase, we further examined their potential to glycosylate a model protein expressed at high levels.
a synthetic gene coding for a c-terminal his-tagged fusion between the e. coli enterotoxin b subunit and human muc <dig> tandem repeat -derived peptide  was used for this purpose .
mucins are heavily glycosylated membrane-bound proteins containing serine and threonine-rich trs that are often polymorphic and vary in number  <cit> .
in most cancers of glandular epithelial origin, muc <dig> is overexpressed and aberrantly glycosylated, leading to exposure of highly immunogenic truncated carbohydrate structures  <cit> .
tn antigen is one of the most common among them, and some studies have already demonstrated the potential of muc- <dig> tr-derived galnac-glycopeptides for active immunotherapy of cancer patients  <cit> .
the muc <dig> tr-derived peptide used as a part of the model ltbmuc <dig> protein has seven potential o-glycosylation sites predicted in silico by netoglyc , but only serine and threonine in gstap motif  and threonine in gvtsa motif  have been verified experimentally as target sites of galnac-t <dig>  <cit> , .
therefore, upon a complete galnac-glycosylation by galnac-t <dig>  the molecular mass of the recombinant protein should increase by ~  <dig> kda, which is difficult to resolve during electrophoresis to differentiate the glycosylated and non-glycosylated forms.
however, the forms can be distinguished by exploiting differences in their affinity to a galnac-specific lectin.
accordingly, the strategy to verify ltbmuc <dig> glycosylation was designed as follows:  transient expression of the model protein using the magnicon deconstructed viral vector system to ensure rapid accumulation and high yield  <cit> ;  purification by ni2+ sepharose fast flow chromatography combined with a his tag-based screen to detect the fractions containing the recombinant protein;  affinity chromatography on vva agarose to capture the galnac-glycosylated population of ltbmuc <dig>  assisted by a his-tag-based and vva-lectin blot detection to verify the molecular mass of the captured protein;  lc-ms/ms analysis to confirm the identity of ltbmuc <dig> and determine the sites of glycosylation.
leaves of the galnac-t <dig> transgenic tobacco plants were transiently transformed with the magnicon 3' vector module pich11599:ltbmuc <dig> co-delivered with the 5' module pich <dig> and the integrase module pich <dig>  <cit> .
because of rapidly developed necrosis leaves were harvested relatively early at  <dig> day post-infiltration.
the levels of ltbmuc <dig> at that time were 3% of the total soluble proteins at most .
following protein extraction and ammonium sulfate precipitation, the his-tagged recombinant protein was purified by chromatography on ni2+ fast flow sepharose.
aliquots of the fractions eluted from the column were analyzed for purity by ponceau s staining and by western blotting to detect the his-tag on ltbmuc <dig>  the recombinant protein was not visible on the ponceau s-stained pvdf membranes, probably because its amount was below the detection limit of the stain .
it should be noted that the ltbmuc1-positive fractions contained co-eluted endogenous tobacco proteins .
the main contaminant appears to be the enzyme ribulose- <dig> -bisphosphate carboxylase/oxygenase represented under denatured conditions by its large  and small  subunits.
the protein is known to be a major obstacle in achieving homogenous preparations of plant-expressed biopharmaceuticals  <cit> .
the theoretical molecular mass of ltbmuc <dig> is  <dig>  kda, but on the his-tag blots the recombinant protein migrated as a single ~ <dig> kda band .
an ~ <dig> kda band was also detected upon expression of ltbmuc <dig> in control, nontransgenic n. benthamiana l. plants; this band was positively stained with roche diagnostics dig glycan detection kit .
these data suggest that plant-expressed ltbmuc <dig> undergoes glycosylation by endogenous enzyme activities.
the amino acid sequence of ltbmuc <dig> lacks consensus motifs for n-glycosylation , but there are several proline residues in a context favoring their hydroxylation and subsequent glycosylation  <cit> .
therefore, it is likely that plant-expressed ltbmuc <dig> is decorated by plant-specific o-glycans, arabinogalactans and/or arabinans.
lectin chromatography on vva agarose coupled with vva lectin blot and his-tag-assisted detection further demonstrated that trace amounts of ltbmuc <dig> expressed in galnac-t <dig> transgenic plants were modified by galnac-glycosylation .
co-expression of galnac-t <dig>  udp-glcnac- <dig> epimerase and udp-galnac transporter is required for galnac-glycosylation of recombinant proteins expressed in n. benthamiana l. at high levels
the activity of galnac-t is not the only factor affecting the rate of galnac-transfer to serine/threonine residues on acceptor proteins.
the concentration of the udp-galnac donor and the efficiency of transfer to the golgi lumen also contribute to the final outcome.
thus, we further tested whether a boost in the endogenous udp-galnac substrate pool or the translocation of udp-galnac to the golgi lumen could improve the efficiency of ltbmuc <dig> galnac-glycosylation.
galnac-t <dig> plants were subjected to another round of transformation with either the y. enterocolitica gne gene coding for the enzyme udp-glcnac 4-epimerase that catalyzes the epimerization of udp-glcnac to udp-galnac, or the c. elegans c03h <dig>  gene encoding a protein capable of simultaneous and independent transport of udp-glcnac and udp-galnac  <cit> .
the double transgenics were further used for production of ltbmuc <dig> as described earlier, and in both cases the efficiency of glycosylation of the recombinant protein showed no improvement .
next, y. enterocolitica gne gene and c. elegans c03h <dig>  gene were delivered simultaneously by pbyr:gne.gt plant binary vector  and expressed successfully in galnac-t <dig> plants as evidenced by the rt-pcr analysis .
the model ltbmuc <dig> protein expressed in the triple transgenics and purified on ni2+ fast flow sepharose , was completely retained on the vva agarose column, which indicated that it was present exclusively as a galnac-glycosylated form.
an outline of the analyses of ltbmuc <dig> expressed in galnac-t <dig> plants  and galnac-t2//udp-glcnac 4-epimerase//udp-glcnac/udp-galnac transporter plants  after ni2+ fast flow sepharose chromatography is presented in fig.
6c.
identical aliquots of volume-equalized samples were run in parallel and assayed  before vva chromatography: by his-tag blotting to demonstrate that comparable amounts of ltbmuc <dig>  were loaded on the columns , and  after elution of the bound proteins from vva agarose resin: by vva lectin blotting to confirm the attachment of galnac to the polypeptide acceptors , and by his-tag blotting to determine the portion of the loaded ltbmuc <dig> with affinity to the vva lectin .
in contrast to ltbmuc <dig> expressed in galnac-t <dig> plants, the recombinant protein produced in the triple transgenics showed his-tag signals of comparable intensity before and after passage through the lectin column and a strong band upon detection by vva lectin blotting.
in addition, a slight delay in its electrophoretic mobility was detected .
these results clearly indicate that efficient galnac-glycosylation of ltbmuc <dig> was achieved only upon expression of the model protein in the galnac-t2//udp-glcnac 4-epimerase//udp-glcnac/udp-galnac transporter plants.
bound proteins that eluted from the vva-agarose column from both ltbmuc <dig> samples were analyzed by sds-page and subsequent silver staining.
a strong band with a molecular mass of ~20/ <dig> kda was visible only in the sample from the triple transgenics .
the band was identified as ltbmuc <dig> by chymotryptic digest coupled with nano lc-ms/ms analysis .
peptides from the muc <dig> region of the recombinant protein were not recovered, even after alternative in-gel digestion with other proteinases .
thus, this prevented identification of the galnac attachment sites.
the amino acids in parenthesis at the beginning and end of each peptide show the flanking residue in the entire protein sequence.
discussion
galnac-t <dig> is one of the best characterized members of the galnac-ts family that exhibits a very low level of udp-galnac hydrolase activity  <cit> .
thus, it was chosen for a proof of concept study to determine if mammalian enzymes could be engineered into plants capable of initiating mucin type o-glycosylation.
our work provides experimental evidence for several conclusions.
based on the detected single, sharply defined ~  <dig> kda band after western blot analysis of the galnac-t <dig> transgenics, it appears that the human enzyme is stably expressed in plants and similar to expression in mammalian cells the consensus asn-516/xaa-517/ser- <dig> sequence remains unoccupied by n-glycans  <cit> .
the human galnac-t <dig> appears to retain its golgi localization in tobacco cells as evidenced by the gfp-tagging and alteration of the fluorescence pattern after treatment with brefeldin a. the latter result corroborates the idea that some targeting signals and/or targeting mechanisms might be conserved between plants and animals  <cit> .
the plant-expressed human galnac-t <dig> enzyme is functional and retains its specificity, as demonstrated by its ability to glycosylate the 113- <dig> aa fragment of hcg-β defined as a galnac-t2-specific acceptor  <cit>  at sites identical to the glycosylation sites of hcg hormone produced by placental trophoblasts in late pregnancy  <cit> .
specifically, the attachment of galnac to the hcg-β peptide occurs at serine residues corresponding to ser- <dig> and ser- <dig> of the hcg-β subunit.
our results also suggest that attachment of galnac to ser- <dig> precedes the attachment of galnac to ser- <dig>  to our knowledge, there are no previous studies addressing the pathway of incorporation of galnac in hcg or hcg-β-derived peptides.
however, the work of sugahara et al.
<cit>  supports our conclusion, having demonstrated that:  ser- <dig> defines the carboxy border of ser- <dig> recognition site;  the context requirements for ser- <dig> to act as an acceptor site are restricted to the presence of pro- <dig> and ser- <dig>  and do not include ser-121;  elimination of ser- <dig> by site-directed mutagenesis does not impair the acceptor potential of ser- <dig>  in addition, in contrast to ser- <dig>  ser- <dig> is n-terminally flanked by an adjacent proline residue which, according to a model study with a library of oriented random peptides, defines ser- <dig> as the more favorable site for glycosylation by galnac-t <dig>  <cit> .
in silico analysis suggests that plants do not have orthologs of human galnac-ts.
by a combination of in vitro radioactive enzyme assay and mass-spectrometry analysis, our work provides direct experimental evidence for a lack of endogenous galnac-t <dig> activity in n. benthamiana l. plants.
a similar conclusion was conveyed indirectly by a study showing absence of mucin type o-glycans in the hinge region of maize-produced human iga <dig>  <cit> .
our work further demonstrates that ectopic expression of human galnac-t is sufficient to "arm" plant cells with the ability to initiate mucin type o-glycosylation, as evidenced by the appearance of several endogenous proteins with affinity to the galnac-specific lectin, vva.
furthermore, we provide ms evidence for the attachment of galnac to one of those proteins, the vacuole-targeted enzyme endochitinase that travels through the er-golgi route  <cit> .
these data suggest that besides the enzyme entity, all other elements necessary for initiation of mucin type o-linked glycosylation, including a udp-galnac donor and protein capable of translocating udp-galnac from the cytosol into the golgi lumen, exist in plant cells.
the presence of free udp-galnac in plants has not been documented so far, but a cytosolic enzyme, udp-glucose 4-epimerase capable of converting udp-glcnac, an abundant plant compound, to udp-galnac, in addition to its primary substrate, udp-glucose, has been identified  <cit> .
a nucleotide sugar transporter with broader substrate specificity could deliver the activated galnac to the golgi lumen.
such function is likely to be performed by still unknown or one  of the identified plant udp-galactose transporters  <cit> .
similarly to h. sapiens and d. melanogaster udp-galactose transporters  <cit> , the latter might have the ability to transport udp-galnac in addition to their primary substrate udp-galactose.
however, the endogenous plant udp-galnac pool and udp-galnac transporter activity are apparently low and could be easily "exhausted" by endogenous substrates, as only a very small portion of the recombinant ltbmuc <dig> was subjected to glycosylation.
in corroboration of this conclusion, co-expression of human galnac-t <dig> with y. enterocolitica udp-glcnac 4-epimerase and c. elegans udp-glcnac/udp-galnac transporter resulted in the production of ltbmuc <dig> exclusively as a glycoform.
altered electrophoretic mobility of the model protein, and especially its strong binding to vva-agarose, are unequivocal proofs for attachment of galnac residue to the polypeptide backbone.
similar to previous observations with maize-expressed human iga <dig>  <cit> , tobacco-expressed ltbmuc <dig> also appears to be decorated by plant-specific glycans attached to hydroxyproline residues .
this modification explains the discrepancy between the calculated  and the apparent  molecular mass of the model protein.
galnac-glycosylation of ltbmuc <dig> upon expression in galnac-t2//udp-glcnac 4-epimerase//udp-glcnac/udp-galnac transporter plants decreases further the electrophoretic mobility of ltbmuc <dig>  although the shift appears to reflect the expected contribution of the galnac residues  to the protein mass, it is difficult to relate that shift to exact numbers of attached galnac residues.
our attempts to gain more information about the occupancy of the glycosylation sites failed because of the resistance of muc <dig> to lc-ms/ms-compatible proteolytic cleavage.
therefore, it remains unclear if the presence of plant specific o-glycans "shielded" some of the galnac-attachment sites of the model protein used in this study.
it is also unclear if these o-glycans might become a source of immunogenicity or affect the stability and/or the activity of plant-produced ltbmuc <dig>  these general issues should be addressed for any plant-produced o-glycosylated protein therapeutic.
a possible approach to eliminate the attachment of the most common plant-specific o-glycans, arabinans and arabinogalactans, to the recombinant pharmaceuticals, would be to prevent the conversion of proline to 4-hydroxyproline.
the expansion of our knowledge about plant prolyl-4-hydroxylase family would allow targeted knockout to eliminate the activity of a certain member of the family, thereby preventing/minimizing detrimental effects on the plant development.
alteration of the amino acid sequence of the recombinant proteins targeted for plant-based expression that aims at elimination of motifs favoring prolyl hydroxylation  <cit>  could serve as an alternative approach..
CONCLUSIONS
our work provides the first experimental evidence that plants can be engineered for production of tn-antigen-containing therapeutics by "updating" their enzyme machinery with udp-glcnac 4-epimerase, udp-galnac transporter and galnac-t. further genetic manipulations could potentially lead to the synthesis of more complex o-glycans.
thus, plants can become an attractive alternative platform for production of homogenous populations of mucin type o-glycosylated recombinant therapeutics with predictable o-glycan structures.
endogenous plant enzyme activities that target directly the actual sites of o-glycan attachment, serine and threonine residues, are not as abundant as in other heterologous production systems .
however, proline residues are often in proximity to the attachment sites, and some of these prolines might be in a sequence context favoring their plant-specific hydroxylation and subsequent glycosylation.
the possibility of "collision" between the plant o-glycosylation process and the engineered galnac-glycosylation process requires careful examination of the amino acid sequences of mammalian proteins targeted for plant-based expression, and if necessary and possible, appropriate alterations to eliminate the risky motifs.

 plants emerged as effective systems for production of pharmaceutically important mammalian proteins, and a number of transgenic plant-based products are currently available in the market. work in the field of plant molecular farming revealed that, while there are obvious advantages to using plant bioreactors, including low cost, fast scalability, biological safety, proper protein folding and assembly, there are also some limitations. glycosylation differences between plants and humans is one of the major drawbacks that can negatively influence properties of plant-derived recombinant proteins. significant efforts have been made to "humanize" plant n-glycan structures, either by retaining the recombinant proteins in the endoplasmic reticulum or by modifying the enzyme machinery in the golgi apparatus to eliminate plant-specific β-xylose and α-fucose residues and extend plant n-glycans with terminal β-galactose residues. however, little attention has been paid to the structural differences between plant and mammalian o-glycans. o-glycans are attached primarily to hydroxyproline and serine residues in plant glycoproteins. typically, non-contiguous hyp residues are modified with arabinogalactan polysaccharides, whereas contiguous hyp are exclusively arabinosylated. the extent of proline hydroxylation and hyp glycosylation depends on the side chains of flanking amino acids. a specific motif has been defined, based on examination of amino acid sequence requirements, for efficient hydroxylation of proline and arabinogalactosylation of hyp. no rules for selection of serine residues for o-glycosylation have been defined so far, probably because the number of identified proteins with glycosylated serines is still limited to a few plant lectins. usually, plant o-glycans attached to serine residues are short, composed of galactopyranosides present either as monosaccharides or as 1→3-linked disaccharides. in general, information about plant o-glycosylation enzyme machinery is lacking. in contrast to plants, mammalian o-glycosylation is a well understood posttranslational modification. substitutions of serine and threonine residues with sugars such as fucose, galactose and n-acetylglucosamine are common modification on mammalian proteins, but the most abundant o-glycans in mammalian cells are mucin type o-linked glycans. they are represented by structurally diverse oligosaccharides attached through n-acetylgalactosamine to serine and/or threonine residues. besides the initial galnacα-ser/thr, known as tn antigen, other structures, including core or t antigen, core, core, core galnacα-ser/thr), core, core, and core, are also commonly linked to human proteins. these core structures can undergo further extension and branching to form more highly complex o-glycans. mucin type o-linked glycans are synthesized in the golgi apparatus in a stepwise and consensus motif-independent manner. a family of udp-galnac polypeptide:n-acetyl-α-galactosaminyltransferases initiates the process by transferring n-acetylgalactosamine from the charged sugar donor, udp-galnac, to the hydroxyl group of serine and threonine amino acid residues. abundance of galnac-t isoforms, distinct spatial and temporal gene expression patterns and unique substrate specificities ensure precise selection of attachment sites and fine regulation of galnac glycosylation of fully folded proteins. depending on the final o-glycan structure, different sets of glycosyltransferases participate in elongation of the initial galnac residue. for example, udp-gal:galnacα-ser/thr β1- galactosyltransferase and udp-glcnac:galnacα-ser/thr β1- n-acetylglucosaminyltransferase are required for synthesis of core o-glycans and core o-glycans, respectively. a previous study reported attachment of core galβ1-3galnac disaccharide to rice glutelin basic subunits, however our in silico analysis and more recent experimental data suggest that plants do not have the enzyme machinery to support mucin type o-glycosylation. in light of this, we reasoned that genetic manipulations could potentially modify plants for production of mammalian glycoproteins with homogenous o-glycan structures. the present study tested, for the first time, the feasibility of generating a plant-based platform for production of tn antigen-containing proteins/peptides. many valuable pharmaceuticals, such as immunomodulators and tumor-associated antigens essential for the development of anticancer therapeutic vaccines, are galnac -glycosylated. despite a growing demand for these products, manufacturing remains difficult and costly. in our study, we first addressed the question of whether the initiation of mucin type o-glycosylation in plants could be performed with the precision typical of mammalian cells. therefore, one of the well characterized isoforms of the human galnac-ts family, galnac-t was stably expressed in n. benthamiana and used to demonstrate that mammalian galnac-ts retain their functionality, specificity and localization in plant cells. we further tested the engineered galnac-t transgenic plants for their ability to produce galnac-glycosylated proteins. a model glycoprotein, mucosally targeted mucin tandem repeat-derived peptide, was transiently expressed using the magnicon deconstructed viral vector system to ensure rapid accumulation and high yield. glycosylation efficiency was monitored by lectin chromatography. our results suggest that co-expression of genes for udp-galnac polypeptide:n-acetyl-α-galactosaminyltransferase, udp-glcnac 4-epimerase, and udp-galnac transporter are required to achieve efficient plant-based production of galnac-glycosylated proteins.  generation and molecular analyses of n. benthamiana l. plants expressing human galnac-t2 agrobacterium-mediated transformation of n. benthamiana l. with ph7wg2:gnt binary vector was performed to generate plants stably expressing human galnac-t gene. the putative transgenic plants were subjected to routine molecular analyses, including pcr, rt-pcr and western blotting, to select for the best producers. plants that exhibited the expected pcr product of kb in size, rt-pcr band of kb in size, and a polypeptide of ~ kda after probing with monoclonal antibodies raised against the full length galnac-t protein were selected for further experiments. none of the transgenic plants had apparent morphological abnormalities. localization of human galnac-t expressed in n. benthamiana l a fusion with aequorea victoria green fluorescent protein, positioned at the c terminus of galnac-t to avoid interfering with the transmembrane domain, was used to monitor the localization of mammalian enzyme in tobacco cells. a discrete punctuate pattern was observed by confocal microscopy. this pattern could be indicative of golgi localization or for targeting to the plant prevacuolar or late endosomal compartments, as shown by experiments with tobacco bright yellow - cells expressing fluorescent markers. to better define the localization of the expressed human galnac-t plant leaves were treated with brefeldin a. brefeldin a is a macrocyclic lactone isolated from alternaria carthami known to block protein er to golgi transport. in plants, the drug causes loss of golgi-based vesicle formation and fusion of the individual golgi cisternae with the er. it also affects pvcs morphology leading to extensive aggregation and tubulation. however, in contrast to the high sensitivity of the golgi apparatus, pvcs respond to brefeldin a treatment only at drug concentrations greater than μg/ml. tobacco plant leaves expressing galnac-t2:gfp fusion protein were incubated for h with μg/ml brefeldin a. the treatment caused loss of the bright green spots and replacement of the punctuate pattern with a network-type fluorescence. these results strongly suggest that mammalian galnac-t is retained in the golgi in plant cells. functionality of human galnac-t expressed in n. benthamiana l to assess the functionality of the plant-expressed human galnac-t we used an in vitro assay that included plant microsomal fraction as an enzyme source, udp-galnac as the donor, and the 113- aa fragment of human chorionic gonadotropin β-subunit as an acceptor peptide. under the chosen hplc conditions, the retention time for udp-galnac was min, while hcg-β peptide eluted at / min as two peaks, as confirmed by maldi. hplc separation of an aliquot of the enzymatic reaction with the microsomal fraction from control tobacco plants showed the presence of only the non-glycosylated hcg-β peptide. the observation was further confirmed by maldi analysis. in contrast, when microsomal fraction of galnac-t transgenic plants was used as the enzyme source, two additional peaks with slightly shorter retention times were visible on the chromatogram. correlation between the accumulated radioactive label in the fractions corresponding to peaks "b" and "c" and the amount of enzyme added to the reaction mixture suggested that these peaks represent products of the enzymatic reaction. further maldi-tof ms analysis of the combined "b" and "c" fractions revealed three major + ions at m/z,, and. the ion at m/z indicated that the analyzed sample contained the initial acceptor peptide. this was not surprising since the peak of the acceptor hcg-β peptide partially overlapped peaks "b" and "c". the ions at m/z and m/z were suggestive of two glycosylated forms of hcg-β peptide with attached one or two galnac residues. maldi-tof-tof spectra analysis further supported the assumption. the major fragmentation product resulting from loss of one hexnac residue from the glycosylated peptide containing a single galnac was evidenced by the abundant ion at m/z. similarly, during the fragmentation of the ion at m/z corresponding to the hcg-β peptide with two galnac residues, the massive peak envelope centered at m/z and smaller peak envelope at m/z are indicative of the loss of one and two hexnac residues, respectively. de-novo peptide sequencing on maldi-tof/tof ms spectrometer was employed to determine the position of the attached galnac residues. based on b- and y-ions generated during the sequencing, the attachment of galnac for the mono- and diglycosylated peptides most likely occurred at ser-, and at ser- and ser-, respectively. effect of the expression of human galnac-t on glycosylation status of n. benthamiana l. endogenous proteins the substrate preferences of galnac-t have been extensively studied and the most favorable amino acids at positions - - - and relative to the site of glycosylation, have been determined. the first three amino acids with the highest enhancement values for each of these positions were used to design a series of peptides consisting of amino acids. in silico analyses using the peptides to search ncbi plant databases indicated that some annotated plant proteins known to transport through the er-golgi pathway do contain potential sites for galnac-glycosylation. therefore, we tested for possible attachment of galnac to endogenous proteins of n. benthamiana l plants engineered to express human galnac-t equal amounts of soluble proteins from the control and transgenic plants were subjected to affinity chromatography on vicia villosa agglutinin -agarose. vva is a lectin that strongly recognizes terminal galnac residues attached to serine or threonine in a polypeptide. the binding buffer was supplemented with m nacl to prevent non-specific interactions, and the resin was thoroughly washed before the elution. eluted proteins were further subjected to lectin blot analysis with alkaline phosphatase-conjugated vva. several positive bands were detected in the transgenic plant sample, but not in the negative control. following silver staining, the strongest band, corresponding to a kda polypeptide, was digested with chymotrypsin. analysis of the recovered peptides by nano lc-ms/ms using a linear quadrupole ion trap mass spectrometer and a database search identified the band with 99% probability as nicotiana endochitinase. modification of threonine at position by amu was also detected, as evidenced by a series of y ions containing the modification, thus strongly suggesting attachment of a n-acetylhexosamine residue to the polypeptide chain. it is of interest that cleavage on the carboxy-terminal side of the potentially modified thr- was absent, possibly as a result of the sugar moiety blocking access of the ionizing mobile proton at that position. loss of the sugar moiety from glycopeptides is often observed during ms/ms, but in certain cases of low-energy-collision induced dissociation the sugar moiety may remain attached, resulting in fragment ions carrying the modification. the threonine residue modified by amu is in boldface type, carboxymethylated cysteine residues are denoted by asterisks. the amino acids in parenthesis at the beginning and end of each peptide show the flanking residue in the entire protein sequence. a homology model of tobacco endochitinase, built based on the three dimensional structure of o. sativa l. japonica class i chitinase, suggests that thr- should be located on the surface of the catalytic domain, therefore accessible for glycosylation. previous study has shown that thr- is not directly involved in the catalytic act. efficiency of attachment of galnac to the model protein, e. coli enterotoxin b subunit:human muc tandem repeat-derived peptide fusion protein expressed transiently in galnac-t plants by magnicon deconstructed viral vector system as the transgenic galnac-t plants demonstrated capability for attaching galnac to the endogenous endochitinase, we further examined their potential to glycosylate a model protein expressed at high levels. a synthetic gene coding for a c-terminal his-tagged fusion between the e. coli enterotoxin b subunit and human muc tandem repeat -derived peptide was used for this purpose. mucins are heavily glycosylated membrane-bound proteins containing serine and threonine-rich trs that are often polymorphic and vary in number. in most cancers of glandular epithelial origin, muc is overexpressed and aberrantly glycosylated, leading to exposure of highly immunogenic truncated carbohydrate structures. tn antigen is one of the most common among them, and some studies have already demonstrated the potential of muc- tr-derived galnac-glycopeptides for active immunotherapy of cancer patients. the muc tr-derived peptide used as a part of the model ltbmuc protein has seven potential o-glycosylation sites predicted in silico by netoglyc, but only serine and threonine in gstap motif and threonine in gvtsa motif have been verified experimentally as target sites of galnac-t,. therefore, upon a complete galnac-glycosylation by galnac-t the molecular mass of the recombinant protein should increase by ~ kda, which is difficult to resolve during electrophoresis to differentiate the glycosylated and non-glycosylated forms. however, the forms can be distinguished by exploiting differences in their affinity to a galnac-specific lectin. accordingly, the strategy to verify ltbmuc glycosylation was designed as follows: transient expression of the model protein using the magnicon deconstructed viral vector system to ensure rapid accumulation and high yield; purification by ni2+ sepharose fast flow chromatography combined with a his tag-based screen to detect the fractions containing the recombinant protein; affinity chromatography on vva agarose to capture the galnac-glycosylated population of ltbmuc assisted by a his-tag-based and vva-lectin blot detection to verify the molecular mass of the captured protein; lc-ms/ms analysis to confirm the identity of ltbmuc and determine the sites of glycosylation. leaves of the galnac-t transgenic tobacco plants were transiently transformed with the magnicon 3' vector module pich11599:ltbmuc co-delivered with the 5' module pich and the integrase module pich. because of rapidly developed necrosis leaves were harvested relatively early at day post-infiltration. the levels of ltbmuc at that time were 3% of the total soluble proteins at most. following protein extraction and ammonium sulfate precipitation, the his-tagged recombinant protein was purified by chromatography on ni2+ fast flow sepharose. aliquots of the fractions eluted from the column were analyzed for purity by ponceau s staining and by western blotting to detect the his-tag on ltbmuc the recombinant protein was not visible on the ponceau s-stained pvdf membranes, probably because its amount was below the detection limit of the stain. it should be noted that the ltbmuc1-positive fractions contained co-eluted endogenous tobacco proteins. the main contaminant appears to be the enzyme ribulose- -bisphosphate carboxylase/oxygenase represented under denatured conditions by its large and small subunits. the protein is known to be a major obstacle in achieving homogenous preparations of plant-expressed biopharmaceuticals. the theoretical molecular mass of ltbmuc is kda, but on the his-tag blots the recombinant protein migrated as a single ~ kda band. an ~ kda band was also detected upon expression of ltbmuc in control, nontransgenic n. benthamiana l. plants; this band was positively stained with roche diagnostics dig glycan detection kit. these data suggest that plant-expressed ltbmuc undergoes glycosylation by endogenous enzyme activities. the amino acid sequence of ltbmuc lacks consensus motifs for n-glycosylation, but there are several proline residues in a context favoring their hydroxylation and subsequent glycosylation. therefore, it is likely that plant-expressed ltbmuc is decorated by plant-specific o-glycans, arabinogalactans and/or arabinans. lectin chromatography on vva agarose coupled with vva lectin blot and his-tag-assisted detection further demonstrated that trace amounts of ltbmuc expressed in galnac-t transgenic plants were modified by galnac-glycosylation. co-expression of galnac-t udp-glcnac- epimerase and udp-galnac transporter is required for galnac-glycosylation of recombinant proteins expressed in n. benthamiana l. at high levels the activity of galnac-t is not the only factor affecting the rate of galnac-transfer to serine/threonine residues on acceptor proteins. the concentration of the udp-galnac donor and the efficiency of transfer to the golgi lumen also contribute to the final outcome. thus, we further tested whether a boost in the endogenous udp-galnac substrate pool or the translocation of udp-galnac to the golgi lumen could improve the efficiency of ltbmuc galnac-glycosylation. galnac-t plants were subjected to another round of transformation with either the y. enterocolitica gne gene coding for the enzyme udp-glcnac 4-epimerase that catalyzes the epimerization of udp-glcnac to udp-galnac, or the c. elegans c03h gene encoding a protein capable of simultaneous and independent transport of udp-glcnac and udp-galnac. the double transgenics were further used for production of ltbmuc as described earlier, and in both cases the efficiency of glycosylation of the recombinant protein showed no improvement. next, y. enterocolitica gne gene and c. elegans c03h gene were delivered simultaneously by pbyr:gne.gt plant binary vector and expressed successfully in galnac-t plants as evidenced by the rt-pcr analysis. the model ltbmuc protein expressed in the triple transgenics and purified on ni2+ fast flow sepharose, was completely retained on the vva agarose column, which indicated that it was present exclusively as a galnac-glycosylated form. an outline of the analyses of ltbmuc expressed in galnac-t plants and galnac-t2//udp-glcnac 4-epimerase//udp-glcnac/udp-galnac transporter plants after ni2+ fast flow sepharose chromatography is presented in fig. 6c. identical aliquots of volume-equalized samples were run in parallel and assayed before vva chromatography: by his-tag blotting to demonstrate that comparable amounts of ltbmuc were loaded on the columns, and after elution of the bound proteins from vva agarose resin: by vva lectin blotting to confirm the attachment of galnac to the polypeptide acceptors, and by his-tag blotting to determine the portion of the loaded ltbmuc with affinity to the vva lectin. in contrast to ltbmuc expressed in galnac-t plants, the recombinant protein produced in the triple transgenics showed his-tag signals of comparable intensity before and after passage through the lectin column and a strong band upon detection by vva lectin blotting. in addition, a slight delay in its electrophoretic mobility was detected. these results clearly indicate that efficient galnac-glycosylation of ltbmuc was achieved only upon expression of the model protein in the galnac-t2//udp-glcnac 4-epimerase//udp-glcnac/udp-galnac transporter plants. bound proteins that eluted from the vva-agarose column from both ltbmuc samples were analyzed by sds-page and subsequent silver staining. a strong band with a molecular mass of ~20/ kda was visible only in the sample from the triple transgenics. the band was identified as ltbmuc by chymotryptic digest coupled with nano lc-ms/ms analysis. peptides from the muc region of the recombinant protein were not recovered, even after alternative in-gel digestion with other proteinases. thus, this prevented identification of the galnac attachment sites. the amino acids in parenthesis at the beginning and end of each peptide show the flanking residue in the entire protein sequence. discussion galnac-t is one of the best characterized members of the galnac-ts family that exhibits a very low level of udp-galnac hydrolase activity. thus, it was chosen for a proof of concept study to determine if mammalian enzymes could be engineered into plants capable of initiating mucin type o-glycosylation. our work provides experimental evidence for several conclusions. based on the detected single, sharply defined ~ kda band after western blot analysis of the galnac-t transgenics, it appears that the human enzyme is stably expressed in plants and similar to expression in mammalian cells the consensus asn-516/xaa-517/ser- sequence remains unoccupied by n-glycans. the human galnac-t appears to retain its golgi localization in tobacco cells as evidenced by the gfp-tagging and alteration of the fluorescence pattern after treatment with brefeldin a. the latter result corroborates the idea that some targeting signals and/or targeting mechanisms might be conserved between plants and animals. the plant-expressed human galnac-t enzyme is functional and retains its specificity, as demonstrated by its ability to glycosylate the 113- aa fragment of hcg-β defined as a galnac-t2-specific acceptor at sites identical to the glycosylation sites of hcg hormone produced by placental trophoblasts in late pregnancy. specifically, the attachment of galnac to the hcg-β peptide occurs at serine residues corresponding to ser- and ser- of the hcg-β subunit. our results also suggest that attachment of galnac to ser- precedes the attachment of galnac to ser- to our knowledge, there are no previous studies addressing the pathway of incorporation of galnac in hcg or hcg-β-derived peptides. however, the work of sugahara et al. supports our conclusion, having demonstrated that: ser- defines the carboxy border of ser- recognition site; the context requirements for ser- to act as an acceptor site are restricted to the presence of pro- and ser- and do not include ser-121; elimination of ser- by site-directed mutagenesis does not impair the acceptor potential of ser- in addition, in contrast to ser- ser- is n-terminally flanked by an adjacent proline residue which, according to a model study with a library of oriented random peptides, defines ser- as the more favorable site for glycosylation by galnac-t. in silico analysis suggests that plants do not have orthologs of human galnac-ts. by a combination of in vitro radioactive enzyme assay and mass-spectrometry analysis, our work provides direct experimental evidence for a lack of endogenous galnac-t activity in n. benthamiana l. plants. a similar conclusion was conveyed indirectly by a study showing absence of mucin type o-glycans in the hinge region of maize-produced human iga. our work further demonstrates that ectopic expression of human galnac-t is sufficient to "arm" plant cells with the ability to initiate mucin type o-glycosylation, as evidenced by the appearance of several endogenous proteins with affinity to the galnac-specific lectin, vva. furthermore, we provide ms evidence for the attachment of galnac to one of those proteins, the vacuole-targeted enzyme endochitinase that travels through the er-golgi route. these data suggest that besides the enzyme entity, all other elements necessary for initiation of mucin type o-linked glycosylation, including a udp-galnac donor and protein capable of translocating udp-galnac from the cytosol into the golgi lumen, exist in plant cells. the presence of free udp-galnac in plants has not been documented so far, but a cytosolic enzyme, udp-glucose 4-epimerase capable of converting udp-glcnac, an abundant plant compound, to udp-galnac, in addition to its primary substrate, udp-glucose, has been identified. a nucleotide sugar transporter with broader substrate specificity could deliver the activated galnac to the golgi lumen. such function is likely to be performed by still unknown or one of the identified plant udp-galactose transporters. similarly to h. sapiens and d. melanogaster udp-galactose transporters, the latter might have the ability to transport udp-galnac in addition to their primary substrate udp-galactose. however, the endogenous plant udp-galnac pool and udp-galnac transporter activity are apparently low and could be easily "exhausted" by endogenous substrates, as only a very small portion of the recombinant ltbmuc was subjected to glycosylation. in corroboration of this conclusion, co-expression of human galnac-t with y. enterocolitica udp-glcnac 4-epimerase and c. elegans udp-glcnac/udp-galnac transporter resulted in the production of ltbmuc exclusively as a glycoform. altered electrophoretic mobility of the model protein, and especially its strong binding to vva-agarose, are unequivocal proofs for attachment of galnac residue to the polypeptide backbone. similar to previous observations with maize-expressed human iga, tobacco-expressed ltbmuc also appears to be decorated by plant-specific glycans attached to hydroxyproline residues. this modification explains the discrepancy between the calculated and the apparent molecular mass of the model protein. galnac-glycosylation of ltbmuc upon expression in galnac-t2//udp-glcnac 4-epimerase//udp-glcnac/udp-galnac transporter plants decreases further the electrophoretic mobility of ltbmuc although the shift appears to reflect the expected contribution of the galnac residues to the protein mass, it is difficult to relate that shift to exact numbers of attached galnac residues. our attempts to gain more information about the occupancy of the glycosylation sites failed because of the resistance of muc to lc-ms/ms-compatible proteolytic cleavage. therefore, it remains unclear if the presence of plant specific o-glycans "shielded" some of the galnac-attachment sites of the model protein used in this study. it is also unclear if these o-glycans might become a source of immunogenicity or affect the stability and/or the activity of plant-produced ltbmuc these general issues should be addressed for any plant-produced o-glycosylated protein therapeutic. a possible approach to eliminate the attachment of the most common plant-specific o-glycans, arabinans and arabinogalactans, to the recombinant pharmaceuticals, would be to prevent the conversion of proline to 4-hydroxyproline. the expansion of our knowledge about plant prolyl-4-hydroxylase family would allow targeted knockout to eliminate the activity of a certain member of the family, thereby preventing/minimizing detrimental effects on the plant development. alteration of the amino acid sequence of the recombinant proteins targeted for plant-based expression that aims at elimination of motifs favoring prolyl hydroxylation could serve as an alternative approach..  our work provides the first experimental evidence that plants can be engineered for production of tn-antigen-containing therapeutics by "updating" their enzyme machinery with udp-glcnac 4-epimerase, udp-galnac transporter and galnac-t. further genetic manipulations could potentially lead to the synthesis of more complex o-glycans. thus, plants can become an attractive alternative platform for production of homogenous populations of mucin type o-glycosylated recombinant therapeutics with predictable o-glycan structures. endogenous plant enzyme activities that target directly the actual sites of o-glycan attachment, serine and threonine residues, are not as abundant as in other heterologous production systems. however, proline residues are often in proximity to the attachment sites, and some of these prolines might be in a sequence context favoring their plant-specific hydroxylation and subsequent glycosylation. the possibility of "collision" between the plant o-glycosylation process and the engineered galnac-glycosylation process requires careful examination of the amino acid sequences of mammalian proteins targeted for plant-based expression, and if necessary and possible, appropriate alterations to eliminate the risky motifs. 
mucin type o-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. however, glycosylation of highly expressed recombinant glycoproteins, like magnicon-expressed e. coli enterotoxin b subunit:h. sapiens mucin tandem repeat-derived peptide fusion protein, is limited by the low endogenous udp-galnac substrate pool and the insufficient translocation of udp-galnac to the golgi lumen. a large family of udp-galnac polypeptide:n-acetyl-α-galactosaminyltransferases catalyzes the first step of mucin type o-glycosylation by transferring galnac to serine and/or threonine residues of acceptor polypeptides. the recombinant enzyme resides in the golgi as evidenced by the fluorescence distribution pattern of the galnac-t2:gfp fusion and alteration of the fluorescence signature upon treatment with brefeldin a. a galnac-t2-specific acceptor peptide, the 113- aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" galnac attachment sites, ser- and ser- ectopic expression of galnac-t is sufficient to "arm" tobacco cells with the ability to perform galnac-glycosylation, as evidenced by the attachment of galnac to thr- of the endogenous enzyme endochitinase. plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. the present study demonstrates that an isoform of the human galnac-ts family, galnac-t retains its localization and functionality upon expression in n. benthamiana l. plants. plant bioreactors can be engineered that are capable of producing tn antigen-containing recombinant therapeutics.
 mucin type o-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. a large family of udp-galnac polypeptide:n-acetyl-α-galactosaminyltransferases catalyzes the first step of mucin type o-glycosylation by transferring galnac to serine and/or threonine residues of acceptor polypeptides. plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.  the present study demonstrates that an isoform of the human galnac-ts family, galnac-t retains its localization and functionality upon expression in n. benthamiana l. plants. the recombinant enzyme resides in the golgi as evidenced by the fluorescence distribution pattern of the galnac-t2:gfp fusion and alteration of the fluorescence signature upon treatment with brefeldin a. a galnac-t2-specific acceptor peptide, the 113- aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" galnac attachment sites, ser- and ser- ectopic expression of galnac-t is sufficient to "arm" tobacco cells with the ability to perform galnac-glycosylation, as evidenced by the attachment of galnac to thr- of the endogenous enzyme endochitinase. however, glycosylation of highly expressed recombinant glycoproteins, like magnicon-expressed e. coli enterotoxin b subunit:h. sapiens mucin tandem repeat-derived peptide fusion protein, is limited by the low endogenous udp-galnac substrate pool and the insufficient translocation of udp-galnac to the golgi lumen. further genetic engineering of the galnac-t plants by co-expressing y. enterocolitica udp-glcnac 4-epimerase gene and c. elegans udp-glcnac/udp-galnac transporter gene overcomes these limitations as indicated by the expression of the model ltbmuc protein exclusively as a glycoform.  plant bioreactors can be engineered that are capable of producing tn antigen-containing recombinant therapeutics. 