Patent Application
20110195452
The present invention relates to the field of recombinant proteins production and is more particularly related to methods for producing recombinant polypeptides that are post-translationally modified in the endoplasmic reticulum (ER) and/or the Golgi apparatus (GA). The present invention provides tools useful for controlling the post-translational modifications of recombinant polypeptides and more generally DNA manipulation tools for plant genetic modification. The present invention also provides processes for producing a recombinant polypeptide involving these tools.
The tools of the present invention include targeting signals allowing the sorting of recombinant polypeptides during their synthesis in a host cell to specific sub-cellular compartments and allowing also a specific designing of said recombinant polypeptides within said sub-cellular compartments. The present invention allows advantageously, for example, an increase of the yield of production of recombinant polypeptides, a limitation or prevention of immunogenicity of the recombinant polypeptides and obtaining therapeutically active recombinant polypeptides that are the exact copy of their natural counterpart.
The present invention relates particularly to the field of reorientation of plants made pharmaceuticals (PMP).
The present invention relates also to the field of immune targeting of plant made pharmaceutical using protein interaction with Single Chain variable fragment (ScFv) fused with targeting signals.
The present invention relates also to the field of reorientation of plant enzymes involved in the post-translational modifications of plant made pharmaceuticals
The present invention relates also to the field of reorientation of heterologous enzymes involved in the post-translational modifications of plant made pharmaceuticals
Recombinant DNA technology has enabled the production of heterologous recombinant proteins in host systems. The majority of the early work was directed toward the expression of recombinant therapeutic proteins in prokaryote hosts, mainly in Escherichia coli. The advantages of prokaryotes as a production system are the ease with which they can be manipulated genetically, their rapid growth and high expression level of recombinant proteins and the possibility of a large-scale fermentation.
However several post-translational modifications (PTMs), including signal peptide cleavage, propeptide processing, protein folding, disulfide bond formation and glycosylation, might not be carried out in prokaryotes. As a result, complex therapeutic proteins produced in prokaryotes are not always properly folded or processed to provide the desired degree of biological activity. Consequently, microbial expression systems have generally been used for the expression of relatively simple therapeutic proteins, such as insulin, interferon or human growth hormone, which do not require folding or extensive posttranslational processing to be biologically active.
Owing to the limitations of prokaryotes for the production of therapeutic proteins, the biotechnology industry has directed its efforts toward eukaryote hosts, such as mammalian cell cultures, yeast, fungi, insect cells, and transgenic animals. These production systems may suffer, however, of different disadvantages: such as expensive fermentation, low yields, secretion problems, high operating costs, difficulties in scaling up to large volumes and potential contamination by viruses or prions. (Gomord et al. 2004 (ref. 23)):
Plant cells and plant suspension-cultured cells can represent a good alternative: advantageous costs, no pathogenic contamination against human. (Gomord et al. 2004 (ref 23))
But one of the major problems of all eukaryotic cells is inappropriate Post Transcriptional Modifications (PTMs).
Indeed, the vast majority of therapeutic proteins undergo several PTMs, which are the final steps in which genetic information from a gene directs the formation of a functional gene product.
In the present specification, the term “PTM” covers covalent modifications that yield derivatives of individual amino-acid residues for example glycosylation, phosphorylation, methylation, ADP-ribosylation, oxidation and glycation; proteolytic processing by reactions involving the polypeptide backbone; and non-enzymatic modifications, such as deamidation, racemization and spontaneous changes in protein conformation.
Most of the PTMs depend on the presence of the endo-membrane system in the eukaryotic cells. The secretory pathway is composed of the endoplasmic reticulum (ER), the Golgi apparatus (GA), the tonoplast, the lysosomal compartments, the plasma membrane and the extracellular medium as represented in annexed
Most therapeutic proteins, including blood proteins, cytokines, immunoglobulins, structural proteins, (growth) hormones, vaccines, enzymes and lysosomal proteins, are co-translationally inserted in the lumen of the ER, and then transported via the GA to the lysosomal compartment, the extracellular matrix or the blood stream. Most modifications of therapeutic proteins occur in the secretory pathway but in particular in its early compartments (ER and GA, see Gomord and Faye, 2004 (ref 21).
For example, coagulation factor IX is a vitamin-K-dependent glycoprotein synthesized as a precursor molecule of 461 amino acids in the ER. To get a biologically active factor IX, this precursor undergoes extensive posttranslational modifications in the ER and in the GA, including the cleavage of a signal peptide and a propeptide, disulfide bridge formation, γ-carboxylation of the first 12 glutamic acid residues, partial β-hydroxylation of aspartate 64, N-linked glycosylation at Asparagine (Asn) 157 and Asn 167, O-linked glycosylation at Serine (Ser) 63, Ser 61, Threonine (Thr) 159, Thr 169, Thr 172 and Thr 179, sulfation of Tyrosine (Tyr) 155, and phosphorylation of Ser 158. This is one the most complex maturations of a therapeutic protein ever observed.
More generally, most therapeutic proteins require at least proteolytic cleavage(s) and glycosylation for their bioactivity, pharmacokinetics, stability and solubility.
Eukaryotic cells are able to realize most of these modifications. However, these maturations are more generally specific of the host systems. Moreover, post-translational modifications are different from mammalian cell to plant cell. In plants, as in other eukaryotic cells, N-glycosylation starts in the ER, with the cotranslational addition of an oligosaccharide precursor (Glc3Man9GlcNAc2) to specific asparagine residue constitutive of potential N-glycosylation sequences, Asn-X-Ser/Thr. Once transferred on the nascent protein, and while the secreted glycoprotein is transported along the secretory pathway, the oligosaccharide precursor undergoes several maturations involving the removal or addition of sugar residues in the ER and the GA as shown schematically in annexed
In this context, it is very important to be able to control the co- and post-translational maturations in the heterologous expression system.
The structural analysis of plant ER-resident proteins has shown that they bear mainly high-mannose-type N-glycans (Navazio et al., 1997 (ref 41), 1998 (ref 42); Pagny et al., 2000 (ref 49). These oligosaccharide structures are common to plants and mammals, and therefore are not immunogenic. This observation has suggested a strategy to prevent the association of immunogenic residues such beta-(1,2)-xylose or alpha-(1,3)-fucose to plant-made pharmaceuticals (PMPs) N-glycans. This strategy consists in the storage of recombinant proteins within the ER, i.e., upstream of Golgi cisternae, where immunogenic glyco-epitopes are added to complex plant N-glycans. It was first shown that the addition of H/KDEL sequences at the C-terminal end of a recombinant soluble protein is sufficient for its retention in the plant ER (Gomord et al., 1997 (ref. 24), 1999 (ref. 22), Saint-Jore-Dupas et al., et 2004 (ref. 57)).
Using the same strategy, we have fused a H/KDEL-ER retention sequence to both heavy and light chains of the antibody of two different antibodies. These antibodies present exclusively non immunogenic high-mannose-type N-glycans (Sriraman et al., 2004 (ref. 59); Petrucelli et al., 2006 (ref. 51)), indicating a very efficient recycling based on glycan maturation limited to enzymes located in the ER and cis-Golgi, such as alpha-mannosidase I (Nebenfuhr et al., 1999 (ref. 43)). Therefore, preventing the association of immunogenic N-glycans to PMPs through the fusion to ER retention signals is possible.
In contrast, very little is known about the molecular signals responsible for maintaining membrane-bound proteins in the plant ER.
Indeed, only few studies have provided a role of C-terminal di-lysine motif (Barrieu and Chrispeels, 1999 (ref. 3); Benghezal et al, 2000 (ref. 4); McCartney et al., 2004 (ref. 35); Reyes et al, 2006 (ref. 53)), the length of the Trans-Membrane Domain (TMD) (Brandizzi et al., 2002a (ref. 9) or an aromatic amino-acid-enriched ER retrieval signal (McCartney et al., 2004 (ref. 35)). The alpha-glucosidase 1 is the first enzyme involved in the maturation of the N-linked oligosaccharide precursor by removing specifically the distal alpha-(1,2)-linked glucose residue from the oligosaccharide precursor just after its transfer “en block” on the nascent glycoprotein (see
Besides glycosylation, proteins travelling downstream the secretory pathway typically undergo specific proteolytic processing, such as targeting signal and regulatory peptides cleavage. As for N-glycosylation, recent evidence in the literature suggests that proteolytic maturations in the secretory pathway is similar in plant and mammalian cells. These maturations are essential for processing of both endogenous and recombinant proteins, but they also make high-yield production of stable, integral polypeptides a challenging task. These proteolytic maturations also depend on the subcellular compartment where the protein is accumulated (Faye et al., 2005 (ref. 17)).
Many plant proteins transported through the secretory pathway are first synthesized as pre-proproteins (also called “not post-translationally modified” in the following description), including an N-terminal cleavable signal peptide—or pre-region—directing the nascent polypeptide chain to the endoplasmic reticulum, and a regulatory pro(poly)peptide—or pro-region—involved in the stabilization, targeting, inhibition and/or folding of the mature protein before its translocation to and processing at the final cellular destination. After removal of their signal peptide by a signal peptidase, proteins are released into the ER lumen to be properly assembled and folded, and then translocated to the GA, and eventually further downstream to the different compartments of the secretory system.
Many plant proteins leave the ER as proproteins, with the proregion being proteolytically cleaved downstream along their route through the secretory pathway. It is the case of many vacuolar proteins bearing a C- or N-terminal cleavable sorting signal, which are removed during or after their transport to the vacuole by specific proteases. Asn-specific cysteine proteinases found in vacuoles and cell walls, in particular, would be involved in the processing of several secreted proteins, including seed storage proteins such as 2S albumins and 11S globulins, and proteins with antimicrobial or antifeedant/antidigestive activity such as pathogenesis-related proteins, chitinases, glucanases, lectins and wound-inducible proteinase inhibitors. In mammalian cells, numerous secretory proteins are first synthesized as inactive proprotein precursors, which are then processed post-translationally by soluble- or membrane bound proteases while moving through the secretory pathway.
In many cases, activation of protein precursors by limited proteolysis is carried out by subtilisin-like proprotein convertases, a family of enzymes structurally similar to bacterial subtilisins and yeast kexin. Mammalian proprotein convertases, including notably furin and dibasic-specific kexin-like proteases, typically cleave protein precursors at the consensus sequence (Arg/Lys)-Xn-(Arg/Lys), where X is any amino acid except Cys, and n=0, 2, 4 or 6. In vivo, these enzymes usually found in the trans-Golgi network convert a variety of protein precursors to mature proteins, thereby directly or indirectly contributing to the fine control of important processes such as zymogen activation, gene expression, cell cycle, programmed cell death, intracellular protein targeting, and endocrine/neural functions.
In practice, several examples have been provided illustrating the correct processing of animal proproteins to their biologically active form in plants. An interesting example was provided for human procollagen, shown to be converted to the mature protein after cleavage of its C- and N-terminal propeptidein tobacco cells Lienard et al., 2006 (ref. 32).
As shown by recent studies on the protein processing enzymes of Solanaceae, functional homologues of mammalian processing convertases are implicated in protein maturation along the plant cell's secretory pathway. A kex2p-like protease activity was shown to occur in transgenic tobacco lines able to correctly process the viral antifungal, KP6 killer protoxin. This Golgi-resident, kex2p-like convertase was then shown to exhibit substrate specificity characteristic of yeast kex2p, in contrast with an extracellular proprotein convertase from tomato, LeSBT1, exhibiting distinct specificity, like the mammalian convertase SKI-1 belonging to the same subfamily of processing enzymes (Jansik et al, 2000 (ref. 31); Rautengarden et al.; 2005 (ref. 52)).
More recently, functional homologues of yeast and mammalian CAAX proteases processing the C-terminal tetrapeptidic, CAAX motif of proteins undergoing lipid modification were found in Arabidopsis, again confirming the occurrence of well conserved proteolytic processes along the secretory pathway of eukaryotic cells (Bracha et al, 2002 (ref. 7)).
From a practical viewpoint, these conserved processes at the cellular level, along with the overall conserved nature of N-glycosylation, strongly point out the potential of plant systems for the expression and correct processing of various proteins of biological or therapeutic relevance requiring complex post-translational modifications.
In plant, alpha-glucosidase I is primordial in the accumulation of storage proteins, the formation of protein bodies, cell differentiation and cell wall disruptions during Arabidopsis thaliana embryo development. Without alpha-glucosidase I activity Arabidopsis thaliana seed development is blocked at the heart stage (Boisson et al., 2001 (ref. 6)) and cell wall biosynthesis is strongly affected (Gillmor et al., 2002 (ref. 19)).
There is still a need in the art of means for producing proteins by genetic recombination, in particular in eukaryotic host, particularly in plant cells, in order to produce designed or engineered post-transcriptionally modified recombinant proteins, for example to express in hosts heterologous proteins substantially identical to their natural counterpart and having as less a possible immunogenic properties so as to be usable as therapeutic substances, in particular for humans.
The inventors of the present invention have identified two independent types of signals conferring ER residency on Arabidopsis thaliana glucosidase I. Using various deletions or mutants of this glucosidase fused to GFP, they have shown that full length of A. thaliana alpha-glucosidase I (hereafter named GCSI) is strictly accumulated in the ER, and contains Arg-based motifs located in the cytosolic tail sufficient for targeting reporter to the ER. However, these functional Arg-based signals are not required to localize the full-length GCSI in this compartments and a second signal has been identified in the stem of this membrane bound ER enzyme.
The inventors of the present invention have identified three independent types of signals conferring GA residency on Arabidopsis thaliana glycosyltransferases.
The purpose of the present invention is precisely to provide efficient tools responding to this need. The tools of the present invention are in the form of targeting or retention signals and processes involving these targeting signals. The present invention is generally directed to the modification of the post-translational maturation of recombinant polypeptides by different ways using these targeting or retention signals.
Based on their numerous studies on transport and localization of plant or human enzymes, for example of glycosidases and glycosyltransferases, the inventors have identified different peptidic signals specifically involved in the distribution of the membrane proteins between the ER and the GA, in particular in plant cells.
Accordingly, a first aspect of the present invention is to provide particular peptidic signals that allow retention of recombinant polypeptides in specific cell sub-compartment, in particular in plant cells. The sequences and structures of these peptidic signals are described below. The peptidic signals of the present invention are also listed in the annexed sequence listing with references SEQ ID no 1 to SEQ ID no 31. Hereafter, these peptidic signals may be referred to as “retention signal sequence” or “signal sequence” or “targeting signals” or “peptidic signal”. Preferred sequences in the scope of the invention are sequences disclosed in the Table I.
The retention signal sequences of the present invention specifically target a recombinant polypeptide to the ER and/or the GA compartment membranes, in particular of plant cells. These sequences allow retention of the recombinant polypeptides in the ER and/or in different sub-compartments of the GA or under a membrane bound form in the ER and/or different sub-compartments of the GA. By “target” or “targeting” it is meant that a polypeptide fused with a peptidic signal of the present invention will be localized, i.e. confined, in the ER and/or GA because of this peptidic signal.
Maturation and stability of a recombinant polypeptide depend directly on the compartment where the polypeptide is accumulated. For instance, the present inventors have shown that retention of recombinant polypeptides in the ER increases their stability and prevents their N-glycan maturation. They have also shown that expression of a soluble recombinant polypeptide as a membrane polypeptide also increases its stability. Finally, they show that retention of a recombinant polypeptide in the early compartment of the secretory pathway increases the yield of production of said recombinant polypeptide and may prevents immunogenicity due to glycan maturation.
The present inventors fused appropriate signals of the present invention to different recombinant polypeptides for different goals achieved and exposed hereafter. Accordingly, another aspect of the present invention is to provide recombinant polypeptides comprising a peptidic signal according to the present invention and a polypeptide, said peptidic signal being fused to the polypeptide. The fusion of the peptidic signal of the present invention may be at the C-terminal or N-terminal extremity of the polypeptide. In other words, said peptidic signal may be linked to the C-terminal or N-terminal end of the polypeptide or protein.
According to the present invention, the recombinant polypeptide may be any recombinant polypeptide having an interest in pharmaceutical or agri-food industry. The “recombinant polypeptide” may also be named “recombinant protein” or “peptide X” or “target protein” in the present specification. However, when the methods of the present invention are disclosed, it is preferred to use “recombinant polypeptide” for designating the recombinant polypeptide to be produced, for example for a pharmaceutical use; and to use “recombinant protein” for designating the a protein involved (see explanations below in the methods description) in the maturation process, i.e. post-translational modification, of the recombinant polypeptide.
According to the present invention, the polypeptide, also named herein “target polypeptide” may be all membrane therapeutical polypeptide, all soluble therapeutical polypeptide that may be expressed as a membrane protein or not, all antibodies and fragments thereof.
For example, the recombinant polypeptide may be selected from the group comprising an enzyme, an antibody or part thereof, a reporter protein, a nucleotide transporter and a therapeutically active polypeptide. Preferably, the recombinant polypeptide is a soluble polypeptide or protein.
Examples of therapeutically active proteins that may be produced with the present invention are: vaccines, allergens, enzymes, blood proteins, hormone, antibodies, antibody-derived fragments. Preferably, the therapeutically active polypeptide is soluble. “Therapeutical polypeptide” or “therapeutically active polypeptide” have the same meaning in the present description and claims [or the soluble part of a membrane bound proteins].
According to the present invention, when the recombinant polypeptide is an enzyme, said enzyme may be a plant or an animal enzyme. According to the present invention, said enzyme may be selected for example from the group comprising glycosidase, glycosyltransferases, protease, kinase, decarboxylase, epimerase, nucleotide-sugar transporter, for example UDP-sugar transporter, GDP-sugar transporter or CMP-Sugar transporter, amidation enzymes and more generally any maturation enzyme present or not in the ER and/or GA of an host cell, for example a plant cell. Glycosidase may for example be those involved in the N- or O-glycosylation. Glycosyltransferases may for example be those involved in the N- or O-glycosylation. Examples of enzymes are cited in the following table
By “maturation enzyme” it is referred to any enzyme that participate to the maturation of a recombinant protein in a host cell.
According to the present invention, whatever the protein is, it may be fused with a storage protein or proteins stored in the protein bodies, for example a protein fused to the ZERA®. This is interesting for the recovering of the recombinant polypeptide after production in a host cell. This point is discussed below in the light of the description of the processes of the present invention.
A further aspect of the present invention is to provide the nucleic acid sequences encoding the peptidic signals of the present invention. Examples of nucleic acid sequences encoding SEQ ID NOs: 1-31 are given in the annexed listing sequences under references SEQ ID NOs: 102-132. According to the degenerescence of the genetic code, the skill person will easily deduce other suitable nucleic acid sequences that encode also SEQ ID NOs: 1-31.
A further aspect of the present invention is to provide the nucleic acid sequences encoding the recombinant polypeptide according to the present invention.
A further aspect of the present invention is to provide nucleic acid vectors comprising a nucleic acid sequence according the present invention. The nucleic acid vector of the present invention comprises a nucleic acid sequence coding for a peptidic signal of the present invention, wherein said nucleic acid is introduced in the vector in frame with the nucleic acid sequence coding for a polypeptide or protein to produce a recombinant polypeptide or protein containing the “retention signal sequence” at one (or both) extremity (extremities) of said polypeptide.
Any known and suitable method may be use to construct these nucleic acids and nucleic acid vectors. For example, the methods disclosed in (Gomord et al., 1997 (ref. 24) and 1998 (ref. 25), Pagny et al, 2000 (ref. 49) and 2003 (ref. 50), Saint-Jore-Dupas et al 2006 (ref. 58)) may advantageously be used.
A further aspect of the present invention is to provide a plant cell comprising at least one peptidic signal of the present invention and/or at least one recombinant polypeptide of the present invention (i.e. including a peptidic signal of the present invention) and/or at least one nucleic acid sequences encoding a recombinant polypeptide of the present invention and/or at least one nucleic acid vector of the present invention. According to the present invention, said recombinant polypeptide may be a homologous or a heterologous polypeptide. Recombinant polypeptides may be as defined above.
Any known and suitable method may be use to obtain said plant cells. For example, the methods disclosed in Gomord et al., 1997 (ref. 24), 1998 (ref. 25), Pagny et al, 2000 (ref. 49) and 2003 (ref. 50), Saint-Jore-Dupas et al 2006 (ref. 58), Saint-Jore et al., 2002 (ref. 56) may advantageously be used.
A further aspect of the present invention is to provide a plant comprising at least one peptidic signal of the present invention and/or at least one recombinant protein of the present invention (i.e. including a peptidic signal of the present invention) and/or at least one nucleic acid sequences encoding a recombinant protein of the present invention and/or at least one nucleic acid vector of the present invention. According to the present invention, said recombinant protein may be a homologous or a heterologous protein. Recombinant proteins are as above-defined.
Any known and suitable method may be use to obtain said plant. For example, the methods disclosed in Saint-Jore-Dupas et al 2006 (Ref 58), Saint-Jore et al, 2002 (ref. 56) may advantageously be used.
According to the present invention, the plant may be any suitable plant that allows the production of a recombinant protein. For example, the plant may selected from the group comprising Alfalfa, Arabidopsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum tuberosum, oriza sativa, zea maize, moss (physcomitrella patens), Lemna minor, Algae (ostreococcus tauri, phaelodactylum), chlamydomonas reinhardtii.
According to the present invention, the plant cell may be issued or derived from any of these plants.
The present inventors provide further a first method for producing a post-translationally modified heterologous polypeptide in host cells that have been transformed with a vector design for the targeted expression of said recombinant polypeptide. This first method is represented schematically in annexed
This first method for producing a post-translationally modified recombinant polypeptide comprises the steps of:
According to the present invention, the process may further comprise a step of screening the cells before the step of growing the transfected or transformed cells. Any method of screening known by the skilled person may be used. For example, by direct selection with a microscope, by electrophoresis SDS page, by immunodetection, by membrane transfer, etc. An example of a method useful for carrying out the screening is disclosed for example in document Gomord et al. 1997 (Ref 24). This step of screening allows selecting the cells that have been transfected or transformed according to the present invention. This step leads to a better yield relating to the modified polypeptide.
For the present disclosure of the process of the invention, as mentioned above, it is preferred to use “recombinant polypeptide” for designating the recombinant polypeptide to be produced, for example for a pharmaceutical use; and to use “recombinant protein” for designating a protein involved (see explanations below in the second method description) in the maturation process, i.e. post-translational modification, of the recombinant polypeptide.
By this method, the use of the nucleic acid vector encoding the peptidic signal of the present invention fused with the recombinant polypeptide allows the retention of the recombinant polypeptide freely or under a membrane bound form in the ER and/or different sub-compartments of the GA. Part of the heterogeneity observed on recombinant polypeptides with the prior art methods occurs during the transport and maturation through the Golgi. The retention of the recombinant polypeptides in the ER or in the ER-derived protein bodies or in the early Golgi compartments by the use of the peptidic signal of the present invention may reduce this heterogeneity.
A major difficulty is to produce a recombinant protein, which is the exact copy of its natural counterpart. The polypeptide post-translational maturations differ not only from an expression system to the other but also from an organ to the other in the same expression system and from one sub-cellular compartment to the other in a cell constitutive of the same organ.
The addition of a peptidic signal to target a PMP to a specific compartment according to the first method of the present invention may solve this difficulty but this structural modification of the recombinant protein leads of course to a non-native protein. Advantageously, the peptidic signals of the present invention is a real tool allowing further to control the maturation of the recombinant polypeptide, by engineering or designing the ER and GA medium of the host cells. This control allows producing recombinant polypeptides that are more stable within the host cell, that are less immunogenic and that tend to exact copies of their natural counterparts. This is very important for the production of therapeutically active proteins, in particular by plants, usable in human therapy.
The present invention provides several solutions to design the ER and GA environment. These solutions may be used alone or together (combination). Examples of these solutions are schematically represented in annexed
Accordingly, a further aspect of the present invention is to provide a second method for producing a post-translationally modified recombinant polypeptide comprising the steps of:
According to the present invention, the process may further comprise a step of screening the cells before the step of growing the transfected or transformed cells. Any method of screening known by the skilled person may be used. Screening methods may be those disclosed above. This step of screening allows selecting the cells that have been transfected or transformed according to the present invention. This step leads to a better yield relating to the modified polypeptide.
In this second method, the recombinant protein translated in the host cells comprises the peptidic signal of the present invention and this recombinant protein is different to polypeptide to be produced.
In this second method, the recombinant protein play a role in the modulation of the post-translational modification of the recombinant polypeptide to be produced, i.e. it is involved in the maturation process, i.e. post-translational modification, of the recombinant polypeptide.
This role depends on the nature of the selected recombinant protein. The skilled person will easily understand how to select the recombinant protein in order to achieve what he wishes to achieve by using the present invention through the reading of the present description and examples.
According to the present invention, this recombinant protein may be, for example, an enzyme and/or an antibody or part thereof.
According to this second method of the present invention, when the recombinant protein is an antibody or part thereof, it may be such as recognizing and binding specifically the polypeptide to be produced and/or an antibodies or part thereof recognizing and binding specifically with an enzyme involved in the post-translational modification of said polypeptide.
By this method, the antibody or part thereof fused with the peptide signal of the present invention will be localized in the ER and/or GA of the host cell. The role of the recombinant protein is here to capture the recombinant polypeptide in the RE and/or GA. So the instant invention provides a method for producing a post-translationally modified heterologous polypeptide by expressing antibody or antibody fragment in the host cells that have been transformed with an expression vector comprising nucleic acid sequences encoding:
In the context of an antibody or part thereof recognizing and binding specifically said polypeptide to be produced, the present invention allows advantageously to retain a native (not modified by a tag addition consisting of the peptidic signal of the present invention) recombinant protein in the ER and/or the GA of host cells via its binding to a specific antibody or antibody fragment retained in one of these compartments by fusing said antibody or fragment thereof with a peptide signal according to the present invention. For this purpose, ER and/or GA retention peptide signals according to the present invention have been fused to antibodies with specific affinity for the recombinant polypeptide with the ultimate goal to control or to modulate the maturation of recombinant protein.
In the context of an antibody or part thereof recognizing and binding specifically with an enzyme involved in the post-translational modification of said polypeptide, the antibody or part thereof preferably modulates said enzyme. Here, an application of the present invention is to specifically use inactivating antibodies to the ER and/or GA to inactivate specific enzymes located in these compartments. For this purpose, ER and/or GA retention peptide signals according to the present invention have been fused to antibodies with specific affinity for the catalytic domain of ER or GA enzymes with the ultimate goal to inactivate endogenous enzyme to control or to modulate the maturation of recombinant protein. The role of the recombinant protein is here to capture an enzyme involved in the maturation of the recombinant polypeptide. The present invention allows therefore to “immuno-modulate” endogeneous enzymes within the host cell.
According to the present invention, the second method may further be used in order to:
The capacity of a heterologous or homologous enzyme to modify the maturation of recombinant protein will depend on its localization in the cell. Then, in order to modify the enzymatic equipment of a host cell, it is preferable to target the appropriate enzyme in the “good” compartment. So the present invention provides a method for producing a post-translationally modified heterologous polypeptide by expressing maturation enzymes in the host cells that have been transformed with (an) expression vector(s) containing nucleic acid sequences encoding:
Accordingly, said recombinant protein may be an endogenous or heterologous enzyme involved in the post-translational modification of said polypeptide. In this example, the present invention allows modifying the enzymatic equipment of the ER and/or the GA in the host cell, in particular in plant cell, according to two separate but juxtaposable strategies: (a) by reorientation of an endogeneous enzyme to ER and/or GA, and (b) by targeting an heterologous enzyme to ER/GA which can be located in different sub-cellular compartments. This modification of the enzymatic equipment of the ER and/or the GA in the host cell is a tool allowing modifying (i.e. designing) the post-translational maturation of the recombinant polypeptide. For example, this modification may allow to improve the stability and/or to control the immunogenicity of the produced recombinant polypeptide. The role of the recombinant protein is here to capture homologous or heterologous enzymes involved in the maturation of the recombinant polypeptide in the ER and/or GA.
The present invention allows therefore advantageously getting a recombinant polypeptide that is a non-immunogenic glycoprotein but also a recombinant polypeptide that is a homogeneous glycoproteins.
According to the present invention, the polypeptide may be any polypeptide needed to be produced by genetic recombination, in particular in plant cells or whole plants. The polypeptide that may be produced with the present invention are for example those cited above in the disclosure of the recombinant polypeptide of the present invention.
According to the present invention, the first and second method may be used simultaneously. In this case, in the second method, the vector nucleic acid vector encoding said polypeptide is also a nucleic acid vector according to the present invention, i.e. encoding the polypeptide fused with the peptidic signal of the present invention. With this embodiment of the present invention, one may target the recombinant polypeptide for maturation for localization in the ER and/or GA and, in the same time design the ER and/or GA for the maturation, i.e. post-translational modification of the recombinant polypeptide. The polypeptide may be as defined above. For example, it may be a therapeutically active protein.
As disclosed herein, according to the present invention, the post-translational modification of the polypeptide is advantageously carried out in the ER and/or GA compartment membranes.
The present inventors are the very first ones to provide such powerful tools for producing designed recombinant polypeptides in a host cell, particularly in a plant cell.
Whatever the method of the present invention that is used (first and/or second method), the polypeptide may be co-expressed with a storage protein. This storage protein may be for example a protein fused to ZERA® or any other suitable protein. Annexed
Usable methods for obtaining the vectors used in the methods of the present invention are disclosed above.
Usable methods for transfecting or transforming cells, in particular plant cells are disclosed above.
According to the present invention, the cells are preferably plant cells, for example as defined above, e.g. plant cells are cells issued from a plant selected from the group comprising Medicago sativa, Arabidopsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum tuberosum Oriza sativa, Zea mays, Physcomitrella patens, Lemna minor, Ostreococcus tauri, Phaelodactylum.
Usable methods for growing the transfected cell, or plants obtained there from, and harvesting the post-translationally modified polypeptide are those known by the skilled person in the art. For example, the methods disclosed in Iienard et al., 2006 (Ref 32) may advantageously be used.
Others advantages may appear to one skill in the art from the followings non-limiting examples illustrated by annexed figures.
Inserts: magnification of selected Golgi stacks (×2,2).
Note that stacks often appear “tri-coloured” (arrows in F and I) with the GFP fusions on one side (green), the ST52-RFP on the other side (red) and a region of overlap between them (yellow).
A-I: Bars=8 μm; J-L: Bar=16 μm.
For each membrane protein, the position and the size of the transmembrane domain were estimated from the TmHMM—2 software (http://www.cbs.dtu.dk/services/TMHMM/).
For some of proteins, the probability to define the position of the TMD is below to 50% (//).
Boxes outlined in bold correspond to N-glycan processing enzymes whose intracellular localization has been studied to date by confocal and/or electronic microscopy.
GCSI: glucosidase I, α1,2 ManI: α1,2-mannosidase I, β1,2 GNTI: β1,2-N-acetylglucosaminyltransferase I, α-1,3 ManII: α1,3-mannosidase II, β1,2 GNTII: β1,2-acetylglucosaminyltransferase II, β1,2 XylosylT: β1,2-xylosyltransferase, α1,3 FucT: α1,3-fucosyltransferase, α1,4 FucT: α1,4-fucosyltransferase, α2,6 sialylT: α2,6-sialyltransferase.
A-C: Bars=16 μm; D, E: Bars=8 μm
Inserts: magnification (×2,2)
All bars=8 μm.
SV=secretory vesicle.
All bars=16 μm.
Note in all cases, the ER network often turns into fenestrated sheets of fluorescence.
All bars=8 μm.
Bars=8 μm
Bars=8 μm
Bars=8 μm
Bars=8 μm
Bars=8 μm
Bars=8 μm
To better understand the mechanisms allowing the selective retention of N-glycan processing enzymes in the early Golgi compartments, the localization of a series of GFP fusions to four different members of the N-glycan processing machinery (α-glucosidase I, mannosidase I, N-acetylglucosaminyltransferase I and b-1,2-xylosyltransferase, annexed
The construct for expressing the GFP fusion protein was made as disclosed in Saint-Jore-Dupas et al, 2006 (ref. 58). All Mannosidase I fusion constructs were derived from the full-length GFP fusion (here called ManI-GFP) originally described by Nebenführ et al. (1999) (ref. 43).
First, a linker containing an AatII restriction site was introduced between the ManI and the GFP coding regions. In combination with the native AatII site near the end of the predicted stem region this allowed for simple removal of the catalytic domain to yield Man99-GFP.
Second, to facilitate the removal of specific segments of the N-terminal region three new restriction sites were introduced by PCR mutagenesis: One NheI site immediately behind the start codon, one SpeI site at codons 21 and 22, and another AatII site at codons 50 and 51. The integrity of the modified construct was confirmed by sequencing. In this new construct, the cytoplasmic tail could be removed with NheI and SpeI to give Δ19CTManI-GFP, while an AatII digest would remove the entire lumenal part of ManI to yield Man49-GFP. Combination of the two procedures resulted in Δ19CTMan49-GFP. Finally, forward (5′-GATCCTTGGGAATGCTTGCTCTGCTCTTCATCGTTTTCGTTTGTGTCTCTTTCGTMCTGGGACCGTCAAA-3′ (SEQ ID No 40)) and reverse (5′-CTAGTTTGACGGTCCCAGAAAACGAAAGAGACACAAACGAAAACGATGAAGAGCAGAGCAAGCATTCCCAAG-3′ (SEQ ID No 41)) oligonucleotides encoding the 18 aa of the TMD domain with a start codon were synthesized, fused and subcloned into the pBLTI121 binary vector containing the GFP without any Start codon (Kiefer-Meyer et al., unpublished data) to give the DCTMan49-GFP. The same strategy has been used to generated the MAAMan49-GFP using forward (5′-GATCCTTGGGAATGGCTGCTGCTCTTGCTCTGCTCTTCATCGTTTTCGTTTGTGTCTCTTTCGTTTTCTGGGACCGTCAAA-3′ (SEQ ID No 42) and reverse (5′-CTAGTTTGACGGTCCCAGAAAACGAAAGAGACACAAACGAAAACGATGAAGAGCAGAGCAAGAGCAGCAGCCATTCCCAAG-3′ SEQ ID No 43) primers.
Third, a longer TMD region was introduced in a two-step PCR mutagenesis of the modified ManI described above. In the first step, the AatII site following the TMD was replaced with a BspEI site. In the second step a long PCR primer was used to duplicate the last seven aa of the predicted TMD to yield ManTMD23-GFP. Finally, the catalytic domain of this construct was removed with AatII to give Man99TMD23-GFP.
All cloning steps described above were carried out in pBluescript. The finished expression cassettes (including a double 35S promoter and a Nos terminator) were then moved to pBIN20.
To obtain the plant binary vector encoding ST-mRFP, GFP is replaced with monomeric RFP (provided by Roger Tsien) in pVKH18En6 ST-GFP (Saint-Jore et al., 2002 (ref. 56). ST-mRFP expression is under control of 6× tandemly-repeated CaMV 35S promoters.
The GNTI-GFP, the GNT38-GFP were amplified by PCR using the N. tabacum cDNA encoding N-acetylglucosaminyltransferase as template (Strasser et al, 1999 (ref. 60)). Reverse primers 5′-GGTCACTAGTATCTTCATTTCCGAGTTG-3′ (SEQ ID No 44) and 5′-GGTCACTAGTGCGATCTGCATATTCTGACTG-3′ (SEQ ID No 45), were used for PCR with forward 5-AACGTCTAGAATGAGAGGGTACAAGTTTTGC-3′ (SEQ ID No 46) primer to amplify the GNTI and the N-terminal 38aa end of the GNTI to obtain GNTI-GFP and GNT38-GFP, respectively. To express GCSI-GFP, the total cDNA was amplified by PCR using A. thaliana cDNA cloned in Boisson et al, 2001, fused at the N-terminal end of GFP and sub-cloned in pBLTI121 (Pagny et al., 2003 (ref. 50)). Then, the first 90aa were amplified by PCR with forward (5′-CGGGGTACCCCATGACCGGAGCTAGCCGT-3′ (SEQ ID No 48)) and reverse (5′-GACTAGAAAAGGAGTGATAACCCT-3′ (SEQ ID No 49)) primers and subcloned in the Spe I restriction site located at the 5′ end of GFP contained in the pBLTI121 binary vector to give the GCS90-GFP. In the same way, the 90 aa deleted of the first 13 aa were amplified by PCR with forward (5′-CGGGGTACCCCATGAAATCATCATCATTATCTCCC-3′ (SEQ ID No 49)) and the same reverse primers as above to give D13GCS90-GFP.
Fluorescence of a full-length ManI-GFP fusion construct was detected by confocal laser scanning microscopy in small bodies (annexed
In addition, a substantial fluorescence signal was observed in a reticulate network throughout the cytoplasm that was indistinguishable from the ER network stained by a GFP-HDEL construct (annexed
To confirm that fluorescent spots were Golgi stacks, the cells were treated for 2 h with 50 mg·mL−1 of brefeldin A (BFA). This BFA treatment caused the green spots to disappear and the cortical and transvascular ER became more fluorescent (compare annexed
The comparison of the location of ManI to the one of other plant N-glycosylation enzymes in the secretory pathway was analyzed under the same conditions the sub-cellular localization of N-glycan maturation enzymes acting before, just after ManI or much later. The first enzyme studied was α-glucosidase I from Arabidopsis (GCSI). This type II membrane protein trims the first sugar residue from the precursor oligosaccharide in the ER immediately after its attachment to the nascent glycoprotein (see a schematic representation of plant N-glycan maturation in annexed
The second candidate investigated was (GNTI) from Nicotiana tabacum (Strasser et al., 1999 (ref. 60)). This glycosyltransferase adds the first N-acetylglucosamine residue on N-glycans soon after ManI has removed an a-1,2-mannose (annexed
Finally, the third candidate, b-1,2-xylosyltransferase (XylT) from Arabidopsis was located in the Golgi only (annexed
To ascertain whether protein expression levels might alter localization of our fusion proteins, these results were confirmed in different stable independent cell lines expressing the fusion proteins. Imaging of cells was always performed on the third or fourth day after sub-culturing which corresponds to the optimal growth phase under our culture conditions. Nevertheless to further validate that ER labelling was not due to over-expression of the fusion, the labelling pattern for each fusion was controlled by verifying that it was unchanged after a 2 h treatment with cycloheximide.
Western-blots revealed with anti-GFP antibodies and the ECL staining have shown a very low signal over background for the recombinant proteins, this indicating a low level of expression for all fusion proteins in this study (data not shown). Further evidence for a level of fusion protein expression compatible with a functional non saturated secretory pathway was obtained from co-expression experiments when in same cell ManI-GFP is located both in the ER and Golgi, while a Golgi marker (ST52-mRFP) is found exclusively in the Golgi (annexed
All together, the results obtained under these carefully controlled conditions clearly show that N-glycosylation enzymes are targeted specifically to the ER (GCSI) or to the Golgi (XylT) exclusively, but some enzymes have a dual steady state location in both organelles as it is the case for the ManI and the GNTI and other membrane proteins such as prolyl 4-hydroxylase (Yuasa et al., 2005 (ref. 65) and ERD2 (Boevink et al., 1998 (ref. 5); Saint-Jore et al., 2002 (ref. 56)).
In a next step, the signals which were responsible for the targeting of the population of glycosylation enzymes showing a dual steady state distribution Golgi/ER was investigate.
2. Experiments Showing that the Luminal Domain is not Necessary for Golgi and ER Targeting of ManI and GNTI
In the regarding the specific Golgi retention of the three plant glycosylation enzymes GNTI, XylT and Arabidopsis ManI (annexed
In order to determine if the portion of ManI located in the Golgi lumen plays a role in the targeting of this glycosidase to the Golgi and the ER membranes, the first 99 aa (CT+TMD+S) or the first 49 aa (CT+TMD) of ManI were fused to GFP and the corresponding chimeric proteins were named Man99-GFP and Man49-GFP, respectively (annexed
It is important to note that when these truncated fusions were transiently expressed in tobacco leaves, the ER labeling was still observed 5 days after transformation when the overall expression levels are already strongly declining (annexed
To get a better understanding of where the fusion proteins are localized within the Golgi stacks, ManI-GFP was co-expressed with the trans Golgi marker ST52-mRFP which is derived from ST52-GFP (Saint-Jore et al., 2002 (ref. 56); Runions et al., 2006 (ref. 55) by replacing GFP with the monomeric red fluorescent protein (mRFP, Campbell et al., 2002 (ref. 11). When the two chimeric proteins were expressed simultaneously in BY2 cells, in contrast to ManI-GFP, ST52-mRFP was not detected in the ER and both fluorescence signals were observed in Golgi bodies but did not perfectly co-localize (annexed
1) the ManI-GFP fusion is located in the cis-half of the Golgi in BY-2 cells (Nebenführ et al., 1999 (ref. 43)) and
2) the 52 amino-terminal amino acids of rat a-2,6-sialyltransferase are sufficient to target a reporter protein predominantly to the trans-half of Golgi stacks (Boevink et al., 1998 (ref. 5)).
Thus, confocal microscopy was sufficient to illustrate that ManI-GFP and ST52-mRFP accumulate in a different subset of cisternae in the Golgi apparatus. Finally, when, Man99-GFP or Man49-GFP were co-expressed with the trans-Golgi marker ST52-mRFP, the two fluorophores only partially overlapped (annexed
The first 77 N-terminal aa of the tobacco GNTI, including the CT, the TMD and the stem, were previously described to contain the information required to maintain Golgi retention of this glycosyltransferase (Essl at al. 1999 (ref. 16)). This polypeptide domain fused to GFP has been shown to be preferentially located in the Golgi but the chimeric protein was also detected in the ER as observed here for the full length construct (annexed
In both expression systems, GNT38-GFP was located in the Golgi and in the ER (annexed
It is clear from these results that the cytosolic tail and TMD of both ManI and GNTI are sufficient to target these glycosylation enzymes to their steady state location: the ER and the early Golgi compartments. In contrast, the same domain (CT+TMD) targets XylT35-GFP to the Golgi only both in BY-2 cells (Pagny et al., 2003 (ref. 50)) and nicotiana leaf epidermal cells (annexed
3. Experiments Showing that the Cytoplasmic Tail is not Necessary for the Retention of ManI in the Early Compartments of the Secretory Pathway.
The N-terminal cytosolic region of many membrane bound proteins residing in the mammalian and yeast ER and/or in the Golgi apparatus contains signals which facilitate either their retrieval from the Golgi back to the ER (Teasdale and Jackson, 1996 (ref. 62); Zerangue et al., 1999 (ref. 66)) or their export from the ER to the Golgi (Giraudo and Maccioni, 2003 (ref. 20). In plants, the length of cytoplasmic tails can vary widely between the different glycosidases and glycosyltransferases (annexed
To define more precisely the targeting signal of ManI and to investigate the role of the relatively long cytoplasmic domain (29 aa) of this glycosidase in this targeting, two fusion proteins were generated D19Man-GFP and D19Man49-GFP. In the latter two proteins, 19 amino acids were removed so that the CT was shortened down to 10 amino acids, alike the CTs of XylT and GNTI. This truncation removed a potential dibasic motif (KxR) that might function in ER-to Golgi transport (Giraudo and Maccioni, 2003 (ref: 20)); although another potential ER-export signal remained. A complete removal of the CT was attempted (DCTMan49-GFP, annexed
The three constructs, MAAAMan49-GFP, D19Man-GFP and D19Man49-GFP have been expressed in tobacco cells. The two latter labelled the ER and the Golgi (annexed
4. Experiments Showing that the Trans Membrane Domain (TDM) Length Plays the Key Role in Golgi Targeting and Sub-Compartmentation of ManI
For mammalian cells, several models have been proposed to explain how type II membrane proteins are retained at different levels within the Golgi.
According to a first model, the “kin recognition” model (Nilsson et al., 1993b (ref. 45)), aggregation of N-glycan maturation enzymes by homo/hetero-oligomerization would prevent the resulting large complexes from being delivered to secretory vesicles and ongoing forward transport downstream in the secretory pathway.
One of the first reported cases of this type of association involved a-mannosidase II, and GNTI, two glycosylation enzymes located in the medial-Golgi and acting sequentially in mammalian N-glycan maturation (Nilsson et al., 1994 (ref. 46)). It should be noted that this model originally assumes the presence of stable Golgi cisternae and the anterograde flow of secretory cargo via vesicular shuttles (Nilsson et al., 1993b (ref. 45)). To fit with the cisternal progression/maturation concept, the “kin recognition” model would have to be modified to allow for the oligomeric complexes to be preferentially packaged into retrograde vesicles (Füllekrug and Nilsson, 1998 (ref. 18)).
A second model, the lipid bilayer model (Bretscher and Munro, 1993 (ref. 10)) proposes that the fit between the length of TMD of glycan maturation enzymes and the thickness of the lipid bilayer of each organelle membrane determines the localization because each organelle has its specific membrane lipid composition and consequently its own thickness (Hartmann and Benveniste, 1987 (ref. 29); Lynch, 1993 (ref. 33); Moreau et al., 1998 (ref. 36); Morré and Mollenhauer, 1974 (ref. 37)).
According to the membrane thickness model, the distribution of N-glycan maturation enzymes in the secretory pathway is based on the length of their TMDs (Bretscher and Munro, 1993 (ref. 10)). The membranes of the secretory pathway organelles increase in thickness from the ER to the plasma membrane. The ER and the cis-Golgi membranes are only 4-5 nm thick whereas the membranes of the trans-Golgi, the secretory vesicles and the plasma membrane are 8-9.5 nm thick (Grove et al., 1968 (ref. 26); Moue and Mollenhauer, 1974 (ref. 37)). Moreover, targeting related to TMD length was previously illustrated by studying the location of reporter proteins after varying the length of their TMD, in animal systems (Munro 1991, 1995a, 1995b (ref. 38)) and, for type I proteins, also in plant cells (Brandizzi et al., 2002a (ref. 9)). This implies, the membrane of a specific compartment can only accommodate hydrophobic TMDs of the matching length
Comparisons revealed that the length of Golgi protein TMD were on average 5 aa shorter than those of plasma membrane proteins (Masibay et al., 1993 (ref. 41); Munro, 1995a (ref. 48)). Several examples are in favour of this model. An increase of the length of the TMD of rat a-2,6-sialyltransferase and bovine b-1,4-galactosyltransferase reduced the Golgi retention of these glycosyltransferase. In addition, a synthetic type I TMD made of 17 leucines resulted in Golgi retention of the lymphocyte surface antigen CD8 extracellular domain whereas a 23 leucine TMD was found in increased amounts at the cell surface (Masibay et al., 1993 (ref. 41); Munro, 1991 (ref. 47), 1995b (ref. 49)). Furthermore, incremental increases in the length of the 18 amino acids a-2,6-sialyltransferase TMD by insertion of 1-9 hydrophobic amino acids also resulted in increased cell surface expression of similar a-2,6-sialyltransferase-lysosyme chimeras, while the decrease in the length of a plasma membrane protein TMD led to its increased retention in the Golgi (Munro, 1995b (ref. 49)).
In plant cells, preliminary data in favor of a subcompartmentation of membrane proteins along the endomembrane system related to the TMD length was obtained by varying the length of TMDs in two type I membrane proteins fused to GFP (Brandizzi et al., 2002a (ref. 9)).
First, the human lysosomal membrane protein LAMP1 containing a 23 aa TMD was fused to GFP and was expressed in tobacco leaves. The fusion was located in the plasma membrane. In contrast, when the TMD was shortened to 20 and 17 aa, the GFP chimeras were localized to the Golgi and ER membranes, respectively. Secondly the 19 aa long TMD of the vacuolar sorting receptor BP80 targeted GFP to the Golgi whereas a lengthened TMD of 22 aa targeted GFP to the plasma membrane.
The TMD length of the type II membrane protein ManI was investigated in order to know how it could affect its sub-compartmentation in the Golgi. In particular, the TMD length was increased from 16 to 23 aa by duplicating the seven last aa of this domain. In contrast with their homologues containing a 16 aa TMD, which were located in the ER and the cis-half of the Golgi apparatus, chimeric proteins with a 23 aa TMD were localized exclusively to the Golgi and more precisely in the trans-half of the Golgi stacks
In the present example, the information required for ManI targeting is contained within a 20 amino acid (aa) sequence including the 16 aa TMD. To investigate whether the length of the TMD could play a key role in the targeting of this type II membrane protein in the early plant secretory pathway, two fusion proteins, ManTMD23-GFP and Man99TMD23-GFP were designed, where the TMD of ManI was lengthened from 16 to 23 aa by duplication of its last seven aa (annexed
To further investigate the sub-compartmentation of ManTMD23-GFP and Man99TMD23-GFP, stable BY-2 suspension cultured cells co-expressing one or the other of these GFP fusions and ST52-mRFP were established. In the merged images, it was impossible to separate green spots from red spots, suggesting that the GFP-fusions containing a 23 aa TMD have moved forward within the Golgi toward the trans-face so that they co-localize with ST52-mRFP at the confocal level (compare annexed
Electron microscopy coupled to immunogold-labeling with polyclonal anti-GFP antibodies allowed us to determine more precisely the intra-Golgi localization of these fusion proteins. As illustrated in annexed
Similar results were obtained with ManI-GFP and ManTMD23-GFP (data not shown). Control experiments using the pre-immune serum or wild-type tobacco BY-2 suspension-cultured cells showed no or very little non specific Golgi labeling (annexed
This results demonstrating that the TMD is sufficient to confer an identical localization as the full length protein ManI, the data suggest that the length of the TMD is a crucial factor for precise positioning of this type II membrane protein within the Golgi stacks and the ER. Thus, protein-lipid interactions are expected to play a key role in ManI targeting within the secretory system.
Interestingly, these results also clearly point out differences in TMD length requirements in the targeting of type I and type II membrane protein in the plant secretory system. Indeed, the 23 aa TMD of XylT (Dirnberger et al., 2002 (ref. 15); Pagny et al., 2003 (ref. 50)) or the 22 aa TMD of ManII (Strasser et al., 2006 (ref. 60)) targets GFP to the Golgi only. In addition, in the present experiment, ManI with a lengthened TMD (23 aa) was also detected exclusively in the Golgi. In contrast the 23 aa TMD of a type I membrane protein and the lengthened 22 aa TMD of BP80 target GFP to the plasma membrane in (Brandizzi et al., 2002a (ref. 8)).
The TMD length requirements for a membrane protein to stay in a membrane with a given thickness might depend on the topology of the protein (type I or type II).
While these experiments clearly demonstrate the role of TMD length in ManI protein targeting, but others experiment show that other enzymes require other signals for proper localization. For example, contradicting the trend to longer TMDs in the later parts of the Golgi, the ST52-GFP fusion with an 18 aa TMD is found further downstream in the trans Golgi (Boevink et al., 1998 (ref. 5); Wee et al., 1999 (ref. 63)) than the XylT35-GFP fusion with a 23 aa TMD (Pagny et al., 2003 (ref. 50)).
Similar results were obtained in other plant systems used for transient expression. Indeed, Man99-GFP was located in the Golgi and ER in soybean (annexed
Even in a situation illustrated here with GCSI, whose TMD is one of the shortest identified so far for a plant glycosylation enzyme and allows for a localization in the ER, the experiment shown that additional information contained in the CT are required for proper targeting. Thus, in silico analyses and mutagenesis studies performed on GCSI are not consistent with TMD length as the only signal for compartmentation of glycosylation enzymes in the plant secretory system.
In other words, while the TMD length has a key role for ManI targeting in the ER and the cis-Golgi, results obtained with GCSI illustrates that specific localization of some membrane proteins in the ER or Golgi membranes could also depend on both protein-lipid (via the TMD) and protein-protein (via special sorting motifs) interactions. The identification of cytosolic partners such as Golgi matrix proteins or cytoplasmic regulators permit to explain mechanisms involved in this second model for partitioning the N-glycan maturation enzymes along the plant secretory pathway.
The large collection of enzymes localizing to different levels in the Golgi has allowed testing the question whether all cisternae within the Golgi stack fuse with the ER in response to treatment with the fungal toxin brefeldin A (BFA). Indeed the Man-GFP fusions containing either a 16 or 23 aa TMD and ST52-mRFP all moved back to the ER or in Golgi clusters over a 2 h time-course experiment with BFA. These conclusions are consistent with previously published results (annexed in
In conclusion, together these results indicate that the TMD length plays a key role in the targeting of ManI to the ER and the cis-Golgi compartments and an increase in the length of the TMD from 16 to 23 aa relocates this type II membrane protein further downstream toward the trans-face of the Golgi (annexed
5. Experiments Showing that the Late and Early Golgi Proteins Redistribute in the ER in Presence of Brefeldin a (BFA).
Taking advantage of the large panel of Golgi marker generated during this study, the possibility that Golgi proteins located in different Golgi subcompartments may behave differently after BFA treatment was investigated.
Cells expressing ER soluble or membrane markers (GFP-HDEL or Glu90-GFP, annexed
In cells co-expressing, ER/early Golgi or late Golgi proteins with ST52-mRFP, BFA induces the redistribution of both markers into the ER and into Golgi aggregates (annexed
6. Experiments Showing that the TMD Length Model does not Apply to all Type II Membrane Proteins
To determine whether if the TMD length could be the only Golgi sorting determinant allowing the subcompartmentation of all glycosidases and glycosyltransferases along the plant secretory system, the N-terminal sequences of characterized glycosylation enzymes were compared (annexed
This analysis was hampered by the small number of sequences of different enzymes cloned and functionally characterized from a single species as well as a still smaller number of electron microscopy data to correlate TMD lengths and membrane thickness in a single plant system. In silico analysis of the N-terminal sequence (CT+TMD) of all plant glycosylation enzymes cloned so far clearly shows a trend for longer TMDs in proteins with the most downstream location in the Golgi stacks (annexed
Based on its short 18 aa TMD that could perfectly fits with the lipid bilayer model to explain its localization in the ER membrane, GluI was selected to check for general applicability of this model. In order to define whether the TMD of GluI was sufficient for its targeting and retention in the ER, most of the luminal part of this glycosidase was deleted (containing the catalytic domain) and fused its first N-terminal 90 aa (CT+TMD+S) to GFP to get the fusion protein Glu90-GFP (annexed
This result clearly shows that GluI targeting to the ER depends on signals located within the CT, the TMD and/or the 21 luminal aa remaining in this truncated protein.
In a further attempt at defining the minimal protein sequence required for localization of GluI in the ER, the first N-terminal 13 aa from the Glu90-GFP construct have been deleted to obtain D13Glu90-GFP (annexed
In conclusion, the 18 aa long TMD of GluI is not sufficient to target this glycosidase in the ER membrane and additional information contained in the first 13 aa of the CT is required for the normal localization of this glycosylation enzyme in the secretory system.
This result provides experimental proof that factors other than TMD length influence the positioning of glycosylation enzymes in the early secretory pathway.
7. Localisation of Arabidopsis thaliana GCSI Type II Membrane Protein
GCSI accumulates strictly in the tobacco endoplasmic reticulum
Arabidopsis thaliana GCSI is a type II membrane protein, consisting of a 51 amino acid cytosolic tail, an about 18 residues transmembrane domain and a large catalytic domain directed toward the lumen (Boisson et al., 2001 (ref. 6)). To investigate the location of this glycosidase, the localization of a GFP fusion to a full length GCSI (annexed
These results are consistent with the trimming of the first sugar residue from the precursor oligosaccharide in the ER immediately after its attachment to the nascent glycoprotein, and with what was shown for human GluI in COS cells (Hardt et al., 2003 (ref. 28)). Furthermore, no significative difference was observed in the fluorescent patterns of cell expressing either a soluble (GFP-HDEL) or a membrane protein (GCSI-GFP) marker observed without (annexed
8. Experiment Showing that the First 90 Amino Acids of GCSI are Sufficient to Retain GFP in the ER.
To understand the mechanisms allowing the selective retention of GCSI in the ER, the role of the luminal domain in GCSI targeting was first investigated. In order to determine if the portion of GCSI located in the ER lumen plays a role in the targeting of this glycosidase to the ER, the first 150 amino acids (aa) (CT+TMD+stem81aa) or the first 90 aa (CT+TMD+stem21aa) of GCSI were fused to GFP and the corresponding chimeric proteins were named GCS150 and GCS90, respectively (annexed in
9. Experiments Showing that the Cytoplasmic Tail Contains an ER Retention Sequence
The N-terminal cytosolic region of many membrane proteins residing in the mammalian and yeast ER contains signals which facilitate either the strict retention (Nilsson et al., 1994 (ref. 46); Opat et al., 2000 (ref. 48), Hardt et al., 2003 (ref. 28); Ciczora et al., 2005(ref. 12)), or their retrieval from the Golgi back to the ER (Teasdale and Jackson, 1996 (ref. 62); Zerangue et al., 1999 (ref. 66)) whereas some others promote the export from the ER to the Golgi (Giraudo and Maccioni, 2003 (ref. 20)). In plants, only few studies demonstrate the presence of ER export sequence in the CT of membrane proteins (Contreras et al., 2004 (ref. 4613); Yuasa et al., 2005 (ref. 65); Hanton et al., 2005b (ref. 27)) and refer to the characterization of cytosolic motifs responsible of membrane protein ER retention (Benghezal et al., 2000 (ref. 4); McCartney et al., 2004 (ref. 35)).
To define more precisely the sequence containing the ER targeting information in GCSI N-terminus, the first 13 amino acids located at the N-terminal end of GCS90 was first deleted and the resulting chimeric protein was named D13GCS90 (annexed in
The two constructs (D13GCS90 and 13GCS-XYT35) were also stably expressed in BY-2 cells. The deletion of the first 13 amino acid of the cytosolic tail relocated the GCS90 protein into bright spots (
In conclusion, the first 13 amino acid of the GCSI are necessary to retain the GCS90 fusion protein in the ER and are sufficient to relocate a Golgi marker in the ER.
10. Experiments Showing that a Cytosolic Arginine-Rich Sequence is an ER Retention Signal in Plants
In order to further investigate whether arginine residues are essential for ER retention in the 13 N-terminal amino acids of AtGCSI, the peptide for either and arginine rich domain located in the N-terminal end of the human homologue of AtGCSI or an arginine rich domain located in the cytosolic C-terminal end of a type I plant ER resident membrane protein, A. thaliana calnexin was changed.
To investigate the capability of the arginine-rich peptide located at the N-terminal end of human GCSI to be recognized in a plant cell, the first N-terminal 13 amino acids of GCS90 was substituted by the first N-terminal 10 amino acids of human GCSI (Hs10-GCS90,
In a second time, to investigate if a similar arginine-rich motif carried out by a type I membrane protein, could mediate the targeting of a type II protein in the ER, the last 11 amino acids located at the C-terminal end of A. thaliana calnexin was fused to the N-terminal end of the Golgi marker XYLT35 (CNX11-XYLT35,
11 Experiments Showing that the Arginine Residues in the Cytosolic Tail of GCSI Contain ER Localization Information.
In order to define more precisely the role of the four arginine residues within the 13 first amino acids of GCSI, these residues were replaced by either leucine or alanine residues using PCR site-directed mutagenesis (see Table 1 for the construct details) and the resulting fusion proteins were expressed in tobacco cells.
While GCS90 was exclusively located in the ER and perfectly co-localized with the ER marker mRFP-HDEL (
In a second step, to define whether (RR) and (RXR) act as two independent signals, Arg-6 and Arg-7 or Arg-10 and Arg-12 have been substituted with leucine residues. The presence of either an RR motif (Arg-6 and Arg-7 in construct R/L10-12GCS90) or an RXR motif (Arg-10 and Arg-12, in construct R/L6-7GCS90) but also the RXXR motif (Arg-7 and Arg-10 in construct R/L6-12GSC90) was sufficient for ER retention of the fusion protein (
12. Experiments Showing that Fusion Proteins Harboring RR, RXR or RXXR Motifs Accumulate in the ER and in Fluorescent Spots Associated with the Golgi
The next step was to identify the structure labeled as fluorescent spots by the R/L6-7GCS90, R/L10-12GCS90 and R/L6-12GSC90 chimeric fusion protein. As illustrated with the R/L6/7GCS90 proteins, GFP fluorescence accumulated in the ER and in fluorescent structures which are smaller that Golgi stacks (
The results are illustrated in
Considering these results, the R/L6-7GCS90, R/L10-12GCS90 and R/L6-12GSC90 fusion proteins were accumulated in the ER in small intermediate domains located between the ER and the Golgi, from which ER resident soluble proteins are excluded (
Unfortunately, no recruitment of Sar1p-mRFP at the fluorescent spots was shown (
In contrast, GCS90 did not recruit Sar1p-mRFP, as Sar1p-mRFP expressed alone is in the ER (data not shown) and the co-expression of GCS90 with Sar1p-RFP did not modify GCS90 labeling (
In conclusion, the transport of R/LGCS90 is regulated by the cytosolic protein Sar1p. However, the small domains labeled with fusion proteins harboring one RR, RXR or RXXR motif are not associated with the Sar1p-mRFP although they are located between the ER and the Golgi compartments.
13. Experiments Showing that ER Retention of AtGCSI does not Depend on the N-Terminal Arginine Motifs Only
In the present example the GCSI arginine-motifs fused to a Golgi reporter protein localized it to the ER. However, there is no evidence that these signals are the main retention signals involved in the ER localization of the full length GCSI.
Based on the observation that replacement of Arg-6, Arg-7, Arg-10 and Arg-12 by leucine or alanine resulted in disruption of ER-directing information for the GCS90 construct, the N-terminal 13 aa were deleted from the full-length sequence of the GCSI to evaluate the significance of the motifs (RXR and RR) in ER targeting of wild type enzyme (D13GCSI,
In order to validate this hypothesis, the 81 amino acid residues 70-150 or the 61 aa 90-150 were fused to the luminal domain of the medial Golgi marker XYLT35 (
It has been shown in mammalian cells that ER residency can be accomplished by direct retention involving association of protein subunits to give large oligomeric complexes via their transmembrane and/or luminal domains, as previously described in the kin-recognition model for Golgi-located membrane proteins. (Nilsson et al., 1994 (ref. 46); Opat et al., 2000 (ref. 48)). These large protein oligomers were assumed to escape packaging into transport vesicles, thus preventing their export from the organelle. This type of mechanism may be functional in the ER retention of subunit components of the hetero-oligomeric oligosaccharyltransferase complex but were not described in plants yet.
In conclusion, both the cytosolic tail and the luminal domain of AtGCSI contain ER targeting determinants Consistent with its specificity and in complete agreement with observations made in other eukaryotic systems, the results demonstrate that AtGCSI is localized in the plant ER and exclusively in this compartment. Other glycosylation enzymes acting early in the plant N-glycan maturation such as N-acetylglucosaminyltransferase (GNT1) and a1,2 mannosidase (ManI) have been shown to be located both in the ER and in the cis-Golgi. The GNTI and ManI, ER and cis-Golgi have been shown to contain targeting information resides in the cytosolic tail whereas the first 13 N-terminal amino acids of GCSI cytosolic tail contain ER targeting information. The present example shows which were the key amino-acids in the cytosolic tail involved in ER targeting but also demonstrates that an arginine-rich cytosolic tail is not the only ER targeting determinant in the whole protein sequence.
Indeed, the deletion of the arginine-rich sequence from GCS150, does not permit the exit of the ER to a later compartment such as the Golgi and D13GCS90 is still exclusively located in the ER.
Together these results indicate that at least two domains, sufficient to confer ER retention to a Golgi type II membrane protein, coexist in the GCSI sequence. Deletions in the luminal domain of GCSI have indicated that information for ER retention is contained in a 60 amino acid peptide located between the amino acids 90 and 150 in the GCSI sequence.
Comparable results were obtained for human glucosidase I concerning the presence of a luminal ER targeting signal but to date the luminal sequence involved is not yet characterized (Hardt et al., 2003 (ref. 28)). As mentioned above, when associated to the Golgi reporter protein XYLT35 the plant luminal peptide is sufficient to relocate XYLT35 to the ER. The luminal domain of GCSI could facilitate the formation of complexes between AtGCSI and soluble and/or membrane bound ER resident proteins. However, in contradiction with the kin recognition model, it has been shown that some large protein complexes are located in the plasma membrane and have to be completely assembled to be transported out of the ER.
In fact, it does not seem necessary to consider the size of these complexes to explain retention. Indeed, due to the very high concentration of proteins in the ER lumen, it is probably more difficult to leave the ER than to stay inside for a protein having the capacity to form protein-protein complexes. As previously described for another ER chaperone system involving BiP (we are currently investigating whether the nascent glycoprotein folding machinery could form a large multi protein complex made of GCSI, glucosidases II, calnexin, calreticulin and ERp57 in the plant ER
It has been shown that the arginine-rich sequence (MTAGASRRSARGRI (SEQ ID No 1) is sufficient to target the Golgi marker XYL35 in the ER. Based on previous studies on membrane protein targeting in the secretory pathway of mammalian cells, the present example was made to identify if the four arginines residues were containing ER retention information. Mutation of the four arginines into alanines or leucines residues completely abolished ER retention capacity of this sequence as L/GGCS90 was found in the Golgi, thus validating the key role of arginine residues in ER retention.
Further analysis based on directed mutagenesis in this arginine-rich sequence has shown that in fact two arginine residues (RR, RXR or RXXR SEQ ID No 70) are sufficient to confer ER retention of GCS90 and that consequently the 13 aa peptide contains three distinct di-arginine motifs sufficient for ER retention that co-exist in the cytosolic tail of GCSI. Interestingly, when only one out these three di-arginine motifs is present in the cytosolic tail of a GCS90, fluorescence is detected not only in the ER but also in small spots associated to- and moving with the Golgi stacks along ER tracks.
In addition, a soluble ER marker protein GFP-HDEL is excluded from these small spots. The hypothesis was that these small spots where R/L6-7GCS90, R/L6-12GCS90 and R/L10-12GCS90 are detected, correspond to secretion units (ERES) located at the ER surface and mediating material exchange between the ER and the Golgi apparatus (Runion et al., 2006 (ref. 55). Further investigations validating this hypothesis would be in favor of an ER/Golgi transport model based on a single secretion unit connected to and moving with a dictyosome at the ER surface. However, mini-spots do not colocalize with Sar1p. Di-arginine motifs have been extensively studied in mammalian membrane proteins but they were never characterized before the present study in their plant homologues. Interestingly a di-arginine motif previously identified in human GCSI has been shown to mediate ER retention in plant cell (Hardt et al., 2003 (ref. 28)).
However di-arginine motifs identified in AtGCSI look more flexible than their human homologue. Indeed the di-arginine motif of human a glucosidase I is made of two arginine residues in position +7 and +8 and of a basic amino acid in position +9. In AtGCSI the distance between two arginine residues looks more flexible but cannot exceed two amino acids for a good efficiency. Furthermore this motif should be in a close proximity of the N-terminal end of the protein. Indeed, in GCSI a di-arginine motif RR in position +23 and +24 is still present in the fusion protein D13GCSS90-GFP but is not sufficient to confer ER retention.
Finally a comparison of GCSI sequences available has shown that di-arginine motifs at the terminal end of these ER resident proteins are highly conserved (Table 2, Boisson et al., 2001(ref. 6); Hong et al., 2004 (ref. 30)).
As shown in the previous examples, each signal listed in Table 3, is sufficient to target a reporter protein such as the green fluorescent protein to the ER and/or the GA (see annexed
thaliana glucosidase I
Nicotiana tabacum
Medicago sativa alpha
As disclosed in the previously, SEQ ID No 1 to 3 represents a set of anchoring sequences for membrane protein targeting to the ER. These sequences are located in the cytosolic tail of the membrane protein located at the C- or N-terminal end.
It has been shown that the following di-Arg motif(s) motifs sequences present in these cytosolic tails (SEQ ID No 1 to 3) have been sufficient to retain a reporter membrane protein in the ER:
Motif Sequences:
As illustrated in
The other following sequences have been tested accordingly:
SEQ ID No 4 to 7 are responsible of a strict retention of the recombinant membrane polypeptide in the ER. These sequences are located in the ER lumen.
It is estimated that every sequence having at least 70% of homology with the SEQ ID no 4 of the present invention have the same effect that the targeting signal of the present invention.
In
A strict retention of the recombinant polypeptide in the ER (see
A transmembrane domain (GS2) of from 16 to 23 amino acids has been shown to be sufficient to address a protein to the Golgi. The use of this domain is sufficient to anchor a recombinant protein or an enzyme in the Golgi membranes. Examples of tested transmembrane domains (GS2) that are included in the peptidic signal of the present invention are as following:
Arrangement between the cytosolic tail (GS1), the transmembrane domain (GS2) and the stem (GS3) from Golgi enzymes is responsible of membrane protein retention in the cis, medial or trans Golgi (see annexed
Examples of peptide signal issued from the GS3 domain are the followings:
These signals have been used to target the expression of several membrane proteins in tobacco, soybean, tomato or radish cells after transient or stable transformation. These signals can be added either to the N-terminal (type II membrane protein) or to the C-terminal end (type I membrane protein) of membrane proteins with the same targeting efficiency and specificity (it seems that the signal could be also added to a type III or IV membrane protein).
The structural analysis of plant ER-resident proteins has shown that they bear exclusively high-mannose-type N-glycans (Navazio et al., 1997 (ref. 41), 1998 (ref. 42); Pagny et al., 2000 (ref. 49)). These oligosaccharide structures are common to plants and mammals, and therefore are not immunogenic. This observation has suggested a strategy to prevent the association of immunogenic residues such beta1,2 xylose or alpha1,3 fucose to plant-made pharmaceuticals (PMPs) N-glycans. This strategy consists in the storage of recombinant proteins within the ER, i.e., upstream of Golgi cisternae, where immunogenic glyco-epitopes are added to maturing plant N-glycans. It was first shown that the addition of H/KDEL amino acid sequences at the C-terminal end of a recombinant soluble protein is sufficient for its retention in the plant ER (Gomord at al., 1997 (ref. 24), 1999 (ref. 22)).
In the present example, using the same strategy, KDEL-ER signal sequence was fused to both heavy and light chains of the antibody of two different antibodies.
The sequence SEQ ID No 1 to 8 have been used to target heavy and light chain of the antibodies to the ER.
The sequence SEQ ID No 32 to 36 have been used to target heavy and light chain of antibodies to the ER and GA.
These antibodies present exclusively non immunogenic high-mannose-type N-glycans (Sriraman et al., 2004 (ref. 59); Petrucelli et al., 2006 (ref. 51)), indicating a very efficient recycling based on glycan maturation limited to enzymes located in the ER and cis-Golgi, such as a-mannosidase I (Nebenfuhr et al., 1999 (ref. 43)). Therefore, preventing the association of immunogenic N-glycans to PMPs through the fusion to ER retention signals is possible.
The expression of an antibody or an antibody fragment in the ER and/or in the GA offer different strategies to improve recombinant protein production. For example it can lead to target non modified (mutated) therapeutical proteins to the ER and/or to the GA.
Two major research directions have been established since the original demonstration of a functional expression antibody in a plant. The first was the use of plants as bio-reactors for large-scale production of therapeutic antibodies or antibody fragments. In the second, antibody or an antibody fragments are expressed in a host cell to affect a physiological process by a mechanism termed immunomodulation. The potential immunomodulation was recently illustrated when the expression of an antibody specific for a herbicide was shown to confer resistance in planta (Almquist K C et al. 2004, (ref. 1) and see Annexed
In plants, as in other eukaryotic cells, N-glycosylation starts in the ER, with a cotranslational addition of an oligosaccharide precursor (Glc3Man9GlcNAc2) to specific asparagine residue on the nascent polypeptide. Once transferred on to the protein, and while the secreted glycoprotein is transported along the secretory pathway, the oligosaccharide undergoes several maturations resulting in complex N-glycan. Many pharmaceuticals, including antibodies used for their effector functions, such as the triggering of the immune response (Wright and Morrison, 1994 (ref. 64)), require glycosylation for their in vivo activity and stability. This is why use the potential plants can offer for the production of recombinant antibodies, it becomes necessary to inhibit these plant-specific maturations in order to obtain ‘humanized’ non-immunogenic N-glycans.
An attractive strategy to humanize plant N-glycans is to express mammalian glycosyltransferases in the plant, which would complete (or compete with) the endogenous machinery of N-glycan maturation in the plant Golgi apparatus. Based on these complementation strategies, the expression of human beta(1,4)-galactosyltransferase, in the Golgi of plant cells, lead to a partial humanization of plant N-glycans and, possibly, compete with the addition of beta(1,2)-xylose and alpha(1,3)-fucose.
The expression of human beta(1,4)-galactosyltransferase in alfalfa or tobacco plants, transfers galactose residues on to the terminal N-acetylglucosamine residues of plant N-glycans. However only, 30 to 40% of N-glycans carried by glycoprotein produced in tobacco or alfalfa plants expressing this human galactosyltransferase, bear terminal N-acetyllactosamine sequences of the mammalian type (Bakker et al., 2001 (ref. 2);
The human beta(1,4)-galactosyltransferase was fused with the golgi targeting signals.
Plasmids for hGalT and GNTIhGalT expression were assembled from pBLTI121 (Pagny et al., 2003 ref 50). The CaMV 35S promoter was replaced by the alfalfa plastocyanin promoter at HindIII-XbaI sites. The human β(1,4)-galactosyltransferase (hGalT) gene (UDP galactose: β-N-acetylglucosaminide: ε(1,4)-galactosyltransferase; EC 2.4.1.22) was isolated from pUC19-hGalT with EcoRI digestion. After klenow treatment, the 1.2 kb hGalT fragment was cloned into pBLTI221 at SmaI sites. A flag tag was then fused to the C-terminal end of the coding region by PCR using the FGalT forward (5′-GACTCTAGAGCGGGAAGATGAGGCTTCGGGAGCCGCTC-3′ SEQ ID No 92) and the reverse RGalTFlagStu (5′-AAGGCCTACGCTACTTGTCATCGTCATCTTTGTAGTCGCACGGTGTCCCGAAGTCCAC-3′ SEQ ID No 93) primers. R622 was then produced by cloning this XbaI-StuI fragment into the binary vector pBLTI121 under control of Plasto promotor and Nos terminator.
The first N-terminal 38 a.a. from N. tabacum N-acetylglucosaminyltransferase I (GNTI) corresponding to the transmembrane domain were amplified by PCR using the forward FGNT (5′-ATCGAAATCGCACGATGAGAGGGTACAAGTTTTGC-3′ SEQ ID No 94) and reverse RGNTspe (5′-CCCATGATGCGATCTGCATATTCTGACTGTGTCGC-3′ SEQ ID No 95) primers and successively cloned into pGEM-T vector, and into pBLTI221 by ApaI/BamHI. For the fusion between GNTI and hGalT, PCR amplification was done from pUC19-hGalT to eliminate its own TMD and create SpeI and StuI sites. The forward FGalTspe (5′-GGACTAGTGCACTGTCGCTGCCCGCCTGC-3′ SEQ ID No 96) and reverse RgalTFlagStu (5′-AAGGCCTACGCTACTTGTCATCGTCATCTTTGTAGTCGCACGGTGTCCCGAAGTCCAC-3′ SEQ ID No 97) were used to amplify the SpeI/StuI hGalT fragment.
This fragment was then cloned into pBLTI221-GNTI. Finally, digestion by the surrounding sites XbaI/StuI allowed to isolate a 1030 bp fragment and R622 was then produced by cloning this 1030 stretch into the binary vector pBLTI121-Plasto.
Transformation of alfalfa plants was done as follows.
Alfalfa (Medicago sativa L.), ecotype R2336, was transformed using an Agrobacterium tumefaciens AGL1 and modified as follow: petiole, stem and leaf explants were co-cultured with Agrobacterium for 5 to 7 days. The co-culture step was performed with an undiluted culture of Agrobacterium at 0.8 to 1 OD and 3% sucrose (instead of 1.5% sucrose) in the SH2K medium Well-established plants resulting of embryo development of responsive explants were transferred into soil in the greenhouse and leaves were analyzed.
The analysis of N-linked glycans isolated from wild-type and GalT or GNTI/GalT-transformed alfalfa plants was done as follow.
Proteins were extracted for 30 minutes at 4° C. from 500 mg of fresh alfalfa leaves in 5 mL of extraction buffer (0.7 M Saccharose, 0.5 M Tris, 30 mM HCl, 0.1 M KCl, 2% beta-mercaptoethanol). Insoluble material was eliminated by centrifugation during 10 minutes, 5,000 g, at 4° C. The resulting supernatant is treated by adding 1 volume of water saturated phenol, during 30 minutes at 4° C. Then, proteins and glycoproteins contained in the phenolic fraction were precipitated, overnight, at −20° C., by volumes of PB (0.1 M Ammonium acetate dissolved in Methanol). After washing the pellet with 5 mL of PB, the proteins and glycoproteins were digested by successive treatments with pepsin and PNGase A as previously described in Bakker et al., 2001. Then, the N-glycans were fluorescent labelled by 2-Amino Benzamide (2-AB).
MALDI-TOF mass spectra of these derivatized N-glycans were acquired on a Voyager DE-Pro MALDI-TOF instrument (Applied Biosystems, USA) equipped with a 337-nm nitrogen laser. Mass spectra were performed in the reflector, delayed extraction mode using 2,5-dihydroxybenzoic acid (Sigma-Aldrich) as matrix.
The matrix, freshly dissolved at 5 mg·mL−1 in a 70:30% acetonitrile/0.1% TFA, was mixed with the solubilized oligosaccharides in a ratio 1:1 (V/V). These spectra were recorded in a positive mode, using an acceleration voltage of 20,000 V with a delay time of 100 ns. They were smoothed once and externally calibrated using commercially available mixtures of peptides and proteins (Applied Biosystems). In this study, the spectra have been calibrated using des-Arg1-Bradykinin (904.4681 Da), Angiotensin I (1296.6853), Glu1-Fibrinopeptide B (1570.6774 Da), ACTH clip 18-39 (2465.1989) and bovine insulin (5730.6087). Laser shots were accumulated for each spectrum in order to obtain an acceptable signal to noise ratio.
As disclosed in the previously, the present invention provides a large panel of signals, which are used to target only glycosyltransferase activity in subcellular compartment and subdomains. This offer a large panel to improve the efficiency of N- and O-glycosylation in host expression system and to optimize post transcriptional modification of heterologous proteins by co-expression with enzymes presented table 5
As disclose in the previous examples, the present invention provide a method for producing protein, for modifying expressing protein in subcompartment of plant cells, for expressing heterologous proteins in the RE and/or GA of plant cells. The invention provides also post transcriptional modified proteins.
In the present example, a fused protein comprising ZERA® and sequence signal (SEQ ID No 8) and mannosidase I was made in order to accumulate the glycosidase as a membrane protein in the protein bodies. The targeting signal (SEQ ID no 8) was used to accumulate the enzyme in the membrane ER and allow the production of protein bodies by forming aggregates via the ZERA peptide.
The aim was to accumulate, in protein bodies, glycoprotein harbouring N-glycan (Man5GlcNAc2)
Plasmid Construct: The ADNc encoding the ZERA fused to the human mannosidase I (Access No Q9UKM7) was amplified by PCR and sub-cloned in the pBLTI121 containing the targeting signal (SEQ ID no 8) at the SPeI and SacI endonucleases sites.
Agrobacterium-Mediated Tobacco BY-2 Cell Transformation
pVKH18En6-mRFP, PBLTI121-GFP and pBIN20-GFP-fusions were transferred into Agrobacterium tumefaciens (strain GV3101 pMP90, Koncz and Schell, 1986) by heat shock. Transgenic Agrobacterium were selected onto YEB medium (per liter, beef extract 5 g, yeast extract 1 g, sucrose 5 g, MgSO4-7H2O 0.5 g) containing kanamycin (100 mg·mL−1) and gentamycin (10 mg·mL−1) and were used to transform Nicotiana tabacum (c.v. Bright Yellow-2) BY-2 cells, as described in Gomord et al., 1998. Transformed tobacco cells were selected in the presence of kanamycin (100 mg·mL−1) for PBLTI121-GFP and pBIN20-GFP-fusions or hygromycin (40 mg·mL−1) for pVKH18En6-mRFP and cefotaxime (250 mg·mL−1). For the double transformants coexpressing GFP and mRFP fusions, microcalli were first selected onto kanamycin plates, and were then transferred onto hygromycin plates. After screening, calli expressing the GFP and or mRFP-fusions were used to initiate suspension cultures of transgenic cells. 3-4 days old BY-2 suspension-cultured cells were used for experiments.
The fused proteins made in the present examples was express in the transformed cells and accumulate the enzyme in the membrane ER and allow the production of protein bodies by forming aggregates via the ZERA peptide.
As disclosed in the previous example, the present invention permit to target proteins to specific domain of cells, to increase the yield of production of recombinant polypeptides, to prevent immunogenicity of recombinant polypeptides and to obtain therapeutically active recombinant polypeptides that are the exact copy of their natural counterpart. It also permits to produce post transcriptional modified proteins
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2007/004234 | 11/8/2007 | WO | 00 | 10/29/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60857524 | Nov 2006 | US |
