# Patent

## Title
N-glycosylation in transformed Phaeodactylum tricornutum

## Abstract

Transformed Phaeodactylum tricornutum including a nucleic acid sequence operatively linked to a promoter, wherein the nucleic acid sequence encodes an N-acetylglucosaminyltransferase I and/or an α-Mannosidase II and wherein at least one β-N-acetylglucosaminidase of the transformed Phaeodactylum tricornutum has been inactivated. A method for producing a glycosylated polypeptide includes the steps of (i) culturing a transformed P. tricornutum as defined previously and (ii) purifying the polypeptide that is expressed and glycosylated in the transformed P. Tricornutum. The use of such a transformed P. tricornutum for producing a glycosylated polypeptide is also described.

## Background
FIELD OF THE INVENTION
The present invention is directed to methods for improving the N-glycosylation pattern of proteins in Phaeodactylum tricornutum in order to produce in these microalgae glycoproteins having N-glycan profiles similar to those of glycoproteins produced by animal cells, especially human cells, which can be used as human or animal therapeutic agents.
BACKGROUND OF THE INVENTION
Pharmaceutical proteins are produced as recombinant proteins by expression in eukaryotic expression systems. After the synthesis of the protein backbone, the recombinant protein is submitted to further post-translational processing including the attachment of sugar residues, a process known as glycosylation. However, eukaryotic organisms exhibit different glycosylation processing involving specific enzymes (glycosyltransferases and glycosidases), and so that the glycosylation patterns, even of the same protein, will be different depending on the eukaryotic cell in which the particular protein is being produced. Thus, the glycosylation pattern of pharmaceutical proteins expressed in eukaryotic host cells differs substantially from the glycosylation pattern of the natural proteins produced in humans and other mammals.
N-Glycosylation: a Major Post-Translational Modification of Secreted Proteins
N-glycosylation is a major post-translational modification step in the synthesis of proteins in eukaryotes. N-glycan processing in the secretory pathway is essential for proteins intended to be secreted or integrated into membranes. N-glycosylation starts when the protein is translated and translocated from the ribosome into the lumen of the endoplasmic reticulum (ER). In this processing, a dolicholphosphate oligosaccharide precursor (Glc3Man9GlcNAc2-PP-dolichol) is initially assembled at the cytoplasmic face and finished in the luminal face of the ER membrane (BURDA AND AEBI, Biochimica et Biophysica Acta, vol. 1426, p: 239-257, 1999). This precursor is used by the oligosaccharyltransferase (OST) multisubunit complex that catalyses its transfer onto the asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, of a target protein (BURDA AND AEBI, above mentioned, 1999). The precursor is then deglucosylated/reglucosylated to ensure the quality control of the neosynthesised protein through the interaction with ER-resident chaperones calreticulin and calnexin. These ER events are crucial for proper folding and oligomerization of secreted proteins (HELENIUS AND AEBI, Science, vol. 291, p: 2364-2369, 2001), highly conserved in eukaryotes investigated so far. These steps lead to the formation of a limited set of high-mannose-type N-glycans (FIG. 1). In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a large variety of organism-specific complex structures. Mannosidases located in this compartment first degrade the oligosaccharide precursor into high-mannose-type N-glycans ranging from Man9GlcNAc2 (Man-9) to Man5GlcNAc2 (Man-5). N-acetylglucosaminyltransferase I (GnT I) then transfers a first GlcNAc residue on the β(1,3)-mannose arm of Man5GlcNAc2 and opens the door to the synthesis of multiple structurally different complex-type N-glycans (FIG. 1). Then, the actions of α-mannosidase II and GnT II allow the synthesis of the core GlcNAc2Man3GlcNAc2. The complex-type oligosaccharides arise from the transfer in the Golgi apparatus of monosaccharide residues onto the core GlcNAc2Man3GlcNAc2 under the action of organism-specific glycosyltransferases. As a consequence, mature proteins leaving the secretory pathway harbour multiple organism-specific complex N-glycans allowing the protein to acquire a set of glycan-mediated biological functions. As illustration, N-glycans in mammals are maturated into poly-antennary, poly-sialylated structures harbouring an α(1,6)-linked fucose residue on the proximal N-acetylglucosamine of the core (core-α(1,6)-fucose) (FIG. 1).
Remodelling into Human-Like N-Glycans by Knock-in Strategies
Since glycosylation profiles differs between mammals and eukaryotic host cells, strategies have been developed for the in vivo remodelling of the protein N-linked glycan structures. These strategies include the knock-out of endogenous genes that are involved in the transfer of some specific monomers, and knock-in methodologies based on the expression in the host cells of mammalian enzymes. The knock-in approach results, by complementing the enzyme repertoire of the host cell, in the synthesis in the recombinant expression system of N-linked glycans similar to those found in mammalian cells. As illustration, the remodelling of plant N-glycans into mammalian-like N-glycans has been achieved by expressing a human β(1,4)-galactosyltransferase in plant cells. Targeted insertion of the human β(1,4)-galactosyltransferase in Physcomitrella patens has also been carried out leading to the addition of terminal β(1,4)-galactose to endogenous N-glycans. Human N-acetylglucosaminyltransferase III (GnT III) has also been successfully expressed in plants in order to in planta engineer endogenous N-glycans. This transferase is able to introduce β(1,4)-GlcNAc residue on the β-mannose of the core mammalian N-glycans (bisecting GlcNAc).
With the exception of IgG, human serum proteins require sialic acid on terminal positions of their N-glycans (FIG. 1). Most non-mammalian eukaryotic cells, such as plants, do not synthesize N-acetyl- and N-glycolylneuraminic acids (Neu5Ac and Neu5Gc), the two main mammalian sialic acids. As consequence, genetic manipulation has been developed for the in planta synthesis of sialylated proteins by expressing enzymes able to synthesise CMP-Neu5Ac, its Golgi transporter and the appropriate sialyltransferases (Paccalet et al., 2007, Plant Biotechnology Journal, vol. 5, p 12-25; Castilho et al., 2008, Plant Physiol., vol. 147 (11), p 331-339; Castilho et al., 2010, The journal of Biological Chemistry, vol. 285 (21), p 15923-30.



BRIEF DESCRIPTION OF DRAWINGS
The FIG. 1 shows the biosynthesis of N-linked glycans in mammals.
The FIG. 2 shows the affinodetection using concanavalin A (Con A) and immunodetection using antibodies raised against the core β(1,2)-xylose (anti-Xyl) and core α(1,3)-fucose (anti-Fuc) epitopes of proteins isolated from green onion used as a positive control (Lane 1) and from Phaeodactylum tricornutum (Lane 2).
The FIG. 3 shows the transformation vector BSJ-25 (SEQ ID No79). The expression cassette is made up of a double cauliflower mosaic virus (CaMV) 35S promoter (SEQ ID No80), a tobacco mosaic virus-Ω sequence as translation enhancer and an Arabidopsis chitinase signal peptide (SP) (SEQ ID No81) which are placed upstream of the multi-clonal site (MCS) and the sequence of FcpA terminator of Phaeodactylum tricornutum at the end. Selection marker, the bleomycin-resistant gene, ShBle is driven by the FcpB promoter. The construct also contains the ampicillin-resistant gene, Amp and the Escherichia coli replication origin.
The FIG. 4 shows the expression cassette of Phaeodactylum tricornutum GnT I. The first cassette comprised the sequence coding for the endogenous GnT I placed under the control of endogenous regulatory sequences. In the second construction, the GnT I was fused to the Green Fluorescent Protein (GFP).
The FIG. 5 shows the screening of transformed microalgae with the sequence coding for the Phaeodactylum tricornutum GnT I (Pt-GnT I). Pt-GnT I: potentially transformed microalgae; T−: water amplification negative control; T+: PCR amplification realised on the transformation vector.
The FIG. 6 shows PCR amplifications of the H4 housekeeping gene realised on the cDNA obtained from the P. tricornutum lineages which where transformed with the sequence coding for the GnT I protein (Pt-GnT I). Pt: wild strain of P. tricornutum; T−: water amplification negative control; RT+: PCR amplification realised on the cDNA; RT−: Reverse Transcription negative control.
The FIG. 7 shows PCR amplification of GnT I realised on the cDNA obtained from P. tricornutum which were transformed with the sequence coding for the endogenous GnT I (Pt-GnT I). Pt: wild strain of P. tricornutum; T−: water amplification negative control; RT+: PCR amplification realised on the cDNA; RT−: Reverse Transcription negative control.
The FIG. 8 shows Q-PCR realised on a wild type P. tricornutum lineage and on a lineage expressing the GnT I transgene. A: Fusion curve of the amplicons; B: amplification profiles of the wild-type and transformed lineages of P. tricornutum. Pt-GnT I: amplification curves obtained for 3 dilutions of cDNA from the transformed lineage with the GnT I gene. Pt-wt: amplification curves obtained for 3 dilutions of cDNA from the wild-type lineage.
The FIG. 9 shows confocal microscopy observation of P. tricornutum microalgae which were transformed with the genes coding for a cytosolic Green Fluorescent Protein (Pt-eGFP) and a GnT I-eGFP fusion protein (Pt-GnT I-eGFP).
The FIG. 10 shows the fluorescence observed in the Chloroplast and Endoplasmic Reticulum of P. tricornutum microalgae which express the eGFP protein and the fluorescence observed in the Golgi apparatus of microalgae expressing GnT I-eGFP, after a culture of said microalgae in brefeldin A (an agent capable of dismantling the structure of the Golgi apparatus by blocking the transport from the Endoplasmic Reticulum to the Golgi apparatus).
The FIG. 11 shows the detection of the GnT I-eGFP fusion protein by Western Blot. The Pt-eGFP sample corresponds to a protein extract from P. tricornutum which produce cytosolic eGFP protein. The negative sample (Pt) corresponds to protein extract from a non-transformed P. tricornutum microalga. Finally, the Pt-GnT I-eGFP correspond to protein extracts from two P. tricornutum clones which were transformed with a sequence coding for the GnT I-eGFP fusion protein and presenting fluorescence at the Golgi apparatus.
The FIG. 12 shows the constructs used for the expression of GnT I in P. tricornutum. 
The FIG. 13 shows High mannose-type N-glycans are the main oligosaccharides N-linked to P. tricornutum proteins. (A) MALDI-TOF mass spectrum of N-linked glycans released by PNGase A from glycoproteins of P. tricornutum and labelled with 2-aminobenzamide (2-AB). (B) MALDI-TOF mass spectrum of the pool of N-glycans after treatment with Jack bean α-mannosidase. (C) MALDI-TOF mass spectrum of 2-AB-labelled N-linked glycans released by PNGase F from glycoproteins of P. tricornutum. Man-3 to Man-9: paucimannose and high mannose-type N-glycans Man3GlcNAc2 to Man9GlcNAc2. *: contaminants. ▪: potassium adducts.
The FIG. 14 shows P. tricornutum GnT I complements N-glycan maturation deficiency in CHO Lec1 mutant. MALDI-TOF mass spectra of glycans N-linked to proteins extracted from CHO cells. (A) wild-type, (B) CHO Lec1 mutant and (C) transformant 4 of CHO Lec1 mutant complemented with P. tricornutum GnT I gene. Man-4 to Man-9: high mannose-type N-glycans Man4GlcNAc2 to Man9GlcNAc2. Symbols (70): black square with black outline: GlcNAc, grey circle with black outline: Man, white circle with black outline: Gal, grey triangle with black outline: Fucose.



SUMMARY OF THE INVENTION
A first aspect of the invention concerns a transformed Phaeodactylum tricornutum whose N-glycosylation pathway has been modified by the inactivation of at least one β-N-acetylglucosaminidase and/or the expression of at least one glycosylation enzyme encoded by a nucleic acid sequence operatively linked to a promoter, wherein

    
    
        (i) said nucleic acid sequence encodes an N-acetylglucosaminyltransferase I having the amino acid sequence SEQ ID No1, a fragment or a derivative thereof and said operatively linked promoter has a sequence identity of less 50% with SEQ ID No3 or a fragment thereof, and/or
        (ii) said nucleic acid sequence encodes an α-Mannosidase II having the amino acid sequence SEQ ID No5, a fragment or a derivative thereof and said operatively linked promoter has a sequence identity of less 50% with SEQ ID No7 or a fragment thereof.
    
    


In a preferred embodiment, said P. Tricornutum further comprises another nucleic acid sequence operatively linked to a promoter, said other nucleic acid sequence encoding a polypeptide that is expressed and glycosylated in the transformed P. tricornutum. 
A second aspect of the invention concerns a method for producing a glycosylated polypeptide, said method comprising the steps of

    
    
        (i) Culturing a transformed P. tricornutum as disclosed previously,
        (ii) Purifying said polypeptide that is expressed and glycosylated in said transformed P. tricornutum. 
    
    


In a preferred embodiment, said method comprises a further step (iii) of determining the glycosylation pattern of said polypeptide.
A third aspect of the invention concerns a use of a transformed P. tricornutum as disclosed previously for producing a glycosylated polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
A gene encoding a putative N-acetylglucosaminyltransferase I (GnT I) has been predicted in the Phaeodactylum tricornutum (P. tricornutum) genome, but the inventors established that this putative GnT I does not exhibit any significant activity in Phaeodactylum tricornutum under standard culture conditions, as proteins extracted from Phaeodactylum tricornutum did not exhibit any GlcNAcMan5GlcNAc2 glycosylation pattern and carry about 95-97% of high-mannose-type N-glycans ranging from Man9GlcNAc2 (Man-9) to Man5GlcNAc2 (Man-5).
However, the inventors surprisingly found in further experiments, presented in the following examples, that this putative GnT I was able to restore the maturation of N-linked glycans into complex-type N-glycans in CHO Lec1 mutants, defective in their endogenous GnT I.
Consequently, the inventors show that this putative GnT I has an enzymatic activity, which enzymatic activity can restore defective mammalian GnT I activity.
Moreover, The N-acetylglucosaminyltransferase I is not the only enzyme identified in the genome of Phaeodactylum tricornutum by the inventors. A further gene encoding an alpha-Mannosidase II (α-Man II) has been identified in the genome of Phaeodactylum tricornutum, whereas there was no detectable of α-Man II activity in P. tricornutum under standard culture conditions.
GlcNAcMan5GlcNAc2, the product of GnT I, is successively converted in the Golgi apparatus into GlcNAcMan4GlcNAc2 and then GlcNAcMan3GlcNAc2 by the action of the α-Man II, followed by the production of GlcNAc2Man3GlcNAc2 under the action of GnT II.
Nevertheless, some organisms express β-N-acetylglucosaminidases, which are enzymes responsible for the degradation of GlcNAc-terminated N-glycans after their biosynthesis in the Golgi apparatus with the action of GnT I and Man II. Elimination of terminal GlcNAc by β-N-acetylglucosaminidases in the secretory system or in compartments where proteins accumulate can then convert these oligosaccharides into Man4GlcNAc2 and Man3GlcNAc2, thus annealing the Glycosylation pathway.
The inventors identified genes encoding putative β-N-acetylglucosaminidases in the genome of Phaeodactylum tricornutum: a first β-N-acetylglucosaminidase of amino acid sequence SEQ ID No9 is encoded by the nucleic acid sequence SEQ ID No10 (Accession number 45073), whereas a second β-N-acetylglucosaminidase of amino acid sequence SEQ ID No11 is encoded by the nucleic acid sequence SEQ ID No12 (Accession number 49563).
These putative genes may explain the absence of the detectable amount of GlcNAcMan5GlcNAc2, the product of GnT I, or GlcNAcMan4GlcNAc2 and GlcNAcMan3GlcNAc2, products of GnT I and α-Man II, on P. tricornutum proteins.
Therefore, a first object of the invention is a transformed Phaeodactylum tricornutum whose N-glycosylation pathway has been modified by the inactivation of at least one β-N-acetylglucosaminidase and/or the expression of at least one glycosylation enzyme encoded by a nucleic acid sequence operatively linked to a promoter, wherein

    
    
        (i) said nucleic acid sequence encodes an N-acetylglucosaminyltransferase I having the amino acid sequence SEQ ID No1, a fragment or a derivative thereof and said operatively linked promoter has a sequence identity of less 50% with SEQ ID No3 or a fragment thereof, and/or
        (ii) said nucleic acid sequence encodes an α-Mannosidase II having the amino acid sequence SEQ ID No5, a fragment or a derivative thereof and said operatively linked promoter has a sequence identity of less 50% with SEQ ID No7 or a fragment thereof.
    
    


In a first embodiment, said transformed Phaeodactylum tricornutum comprises a nucleic acid sequence operatively linked to a promoter, wherein:

    
    
        (i) said nucleic acid sequence encodes an N-acetylglucosaminyltransferase I having the amino acid sequence SEQ ID no1, a fragment or a derivative thereof; and
        (ii) said operatively linked promoter has a sequence identity of less 50% with SEQ ID no3 or a fragment thereof.
    
    


Phaeodactylum tricornutum is a microalga which belongs to the Bacillariophyceae class, to the Naviculales order, to the Phaeodactylaceae family and to the Phaeodactylum genus.
The term “nucleic acid sequence” used herein refers to DNA sequences (e.g., cDNA or genomic or synthetic DNA), as well as analogs of DNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. Preferably, said nucleic acid sequence is a DNA sequence. This nucleic acid sequence can be in any topological conformation, like linear or circular.
The expression “Operatively linked” promoter refers to a linkage in which the promoter is contiguous with the gene of interest to control the expression of said gene.
The expression “fragment” with reference to SEQ ID No3 refers to a nucleic acid sequence of at least 100 nucleic acids of said SEQ ID No3, preferably of at least 150 nucleic acids of SEQ ID No3, most preferably of at least 200 nucleic acids of SEQ ID No3.
The term “transformed Phaeodactylum tricornutum” refers to a P. tricornutum wherein the nucleic acid sequence operatively linked to a promoter has been introduced in said microalgae by conventional methods of transformation, as described below, so as to express said nucleic acid molecule in the nucleus of said P. tricornutum. 
Transformation of P. tricornutum can be carried out by conventional methods such as microparticles bombardment, electroporation, glass beads, polyethylene glycol (PEG), silicon carbide whiskers, or use of viruses or agrobacterium. Such a protocol is disclosed in the examples. The nucleic acid sequence may be introduced into Phaeodactylum tricornutum via a plasmid, virus sequences, double or simple strand DNA, circular or linear DNA. It is generally desirable to include into each nucleic acid sequence or vector at least one selectable marker to allow selection of Phaeodactylum tricornutum that have been stably transformed. Examples of such markers are antibiotic resistant genes such as sh ble gene enabling resistance to zeocin, nat or sat-1 genes enabling resistance to nourseothricin, bar gene enabling resistance to glufosinate.
N-acetylglucosaminyltransferase I, also known as GnT I or mannoside acetylglucosaminyltransferase I (MGAT I) is an enzyme from the N-glycosylation pathway, which is capable of adding an N-acetylglucosamine (GlcNAc) residue to Man5GlcNAc2 to produce a GlcNAcMan5GlcNAc2.
The N-acetylglucosaminyltransferase I having the amino acid sequence SEQ ID No1 corresponds to the “endogenous N-acetylglucosaminyltransferase” encoded by the nucleic acid sequence SEQ ID No2 present in the genome of wild-type Phaeodactylum tricornutum. 
The expression “fragment” with reference to SEQ ID No1 refers to an amino acid sequence comprising at least SEQ ID No4 corresponding to the amino acid sequence of the catalytic site of N-acetylglucosaminyltransferase I having said SEQ ID No1.
The expression “catalytic site” refers to the amino acid sequence of an enzyme, said amino acid sequence being responsible for the enzymatic activity. Therefore, the catalytic site of the N-acetylglucosaminyltransferase I according to the invention corresponds to the amino acid sequence responsible for the addition of an N-acetylglucosamine (GlcNAc) residue to Man5GlcNAc2 to produce a GlcNAcMan5GlcNAc2.
As used herein the term “N-acetylglucosaminyltransferase I derivative” refers to an amino acid sequence capable of adding an N-acetylglucosamine (GlcNAc) residue to Man5GlcNAc2 to produce a GlcNAcMan5GlcNAc2 and having more than 85% of identity with amino acid sequence SEQ ID No1 or a fragment thereof, preferably more than 90% of identity with amino acid sequence SEQ ID No1 or a fragment thereof, and more preferably more than 95% of identity with amino acid sequence SEQ ID No1 or a fragment thereof.
As used herein, “percentage of identity” between two amino acids sequences, means the percentage of identical amino-acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences. As used herein, “best alignment” or “optimal alignment”, means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity. The best sequences alignment to perform comparison can be realized by using computer softwares using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package). To get the best local alignment, one can preferably used BLAST software, with the BLOSUM 62 matrix, or the PAM 30 matrix. The identity percentage between two sequences of amino acids is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
Many different promoters allowing the expression of a nucleic acid sequence in Phaeodactylum tricornutum are known from the skilled person. As an example of such promoters, one can cite the nuclear promoters such fcpA and fcpB from Phaeodactylum tricornutum disclosed in ZAVLASKAÏA et al. (J. Phycol., vol. 36, p: 379-386, 2000). Nevertheless, this promoter has a sequence identity of less 50% with SEQ ID no3 corresponding to the sequence of 1047 pb nucleic acid sequence upstream of the ATG of nucleic acid sequence SEQ ID No2 present in the genome of wild-type Phaeodactylum tricornutum, preferably of less than 25% and most preferably of less than 10%.
In another preferred embodiment, said transformed Phaeodactylum tricornutum further comprises a nucleic acid sequence operatively linked to a promoter, wherein:

    
    
        (i) said nucleic acid sequence encodes an alpha-Mannosidase II having the amino acid sequence SEQ ID no5, a fragment or a derivative thereof; and
        (ii) said operatively linked promoter has a sequence identity of less 50% with SEQ ID no 7 or a fragment thereof.
    
    


In a second embodiment, said transformed Phaeodactylum tricornutum comprises a nucleic acid sequence operatively linked to a promoter, wherein:

    
    
        (i) said nucleic acid sequence encodes an alpha-Mannosidase II having the amino acid sequence SEQ ID no5, a fragment or a derivative thereof; and
        (ii) said operatively linked promoter has a sequence identity of less 50% with SEQ ID no 7 or a fragment thereof.
    
    


The expression “fragment” with reference to SEQ ID No7 refers to a nucleic acid sequence of at least 100 nucleic acids of said SEQ ID No7, preferably of at least 150 nucleic acids of SEQ ID No7, most preferably of at least 200 nucleic acids of SEQ ID No7.
α-Mannosidase II, also known as α-Man II, is an enzyme which catalyzes the first committed step in the biosynthesis of complex N-glycans. α-Man II is capable of hydrolysing the terminal (1→3)- and (1→6)-linked alpha-D-mannose residues in the mannosyl-oligosaccharide GlcNAcMan5GlcNAc2. GlcNAcMan5GlcNAc2, the product of GnT I, is successively converted in the Golgi apparatus into GlcNAcMan4GlcNAc2 and then GlcNAcMan3GlcNAc2 by action of the α-Man II.
The α-Mannosidase II having the amino acid sequence SEQ ID No5 corresponds to the “endogenous α-Mannosidase II” encoded by the nucleic acid sequence SEQ ID No6 present in the genome of wild-type Phaeodactylum tricornutum. 
The expression “fragment” with reference to SEQ ID No5 refers to an amino acid sequence of at least SEQ ID No8 corresponding to the amino acid sequence of the luminal part of α-Man II, which comprises the catalytic site of α-Man II.
The expression “catalytic site” refers to the amino acid sequence of an enzyme, said amino acid sequence being responsible for the enzymatic activity. Therefore, the catalytic site of the α-Mannosidase II according to the invention corresponds to the amino acid sequence responsible for the conversion of GlcNAcMan5GlcNAc2, the product of GnT I, in the Golgi apparatus into GlcNAcMan4GlcNAc2 and then GlcNAcMan3GlcNAc2.
As used herein the term “α-Man II derivative” refers to an amino acid sequence capable of converting GlcNAcMan5GlcNAc2, the product of GnT I, in the Golgi apparatus into GlcNAcMan4GlcNAc2 and then GlcNAcMan3GlcNAc2 and having more than 85% of identity with amino acid sequence SEQ ID No5 or a fragment thereof, preferably more than 90% of identity with amino acid sequence SEQ ID No5 or a fragment thereof, and more preferably more than 95% of identity with amino acid sequence SEQ ID No5 or a fragment thereof.
Many different promoters allowing the expression of a nucleic acid sequence in Phaeodactylum tricornutum are known from the skilled person. As an example of such promoters, one can cite the nuclear promoters such fcpA and fcpB from Phaeodactylum tricornutum disclosed in ZAVLASKAÏA et al. (J Phycol., vol. 36, p: 379-386, 2000). Nevertheless, this promoter has a sequence identity of less 50% with SEQ ID no7 corresponding to the sequence of 1000 pb nucleic acid sequence upstream of the ATG of nucleic acid sequence SEQ ID No6 present in the genome of wild-type Phaeodactylum tricornutum, preferably of less than 25% and most preferably of less than 10%.
In a third embodiment, said transformed Phaeodactylum tricornutum has at least one β-N-acetylglucosaminidase which has been inactivated, preferably said at least one β-N-acetylglucosaminidase being a β-N-acetylglucosaminidase of amino acid sequence SEQ ID No9 or a β-N-acetylglucosaminidase of amino acid sequence of SEQ ID No11.
In a preferred embodiment, both β-N-acetylglucosaminidases of amino acid sequences SEQ ID No9 and SEQ ID No11 have been inactivated.
The term “inactivated” with reference to β-N-acetylglucosaminidases refers to β-N-acetylglucosaminidases which do not present any enzymatic activity.
This type of enzyme is able to hydrolyze exclusively the GlcNAc residue attached to the α-1,3/1,6-linked mannose of the core pentasaccharide of N-glycans. It is noteworthy that this enzyme could not hydrolyze Man5GlcNAc, but acted only further “downstream” on GlcNAcMan5GlcNAc2, GlcNAcMan4GlcNAc2, GlcNAcMan3GlcNAc2 or GlcNAcMan3GlcNAc2 substituted by fucose residues linked to the proximal GlcNAc.
By β-N-acetylglucosaminidase enzymatic activities, we mean the removal of the terminal GlcNAc residue linked to the mannoses of the core of complex-type glycans N-linked to proteins.
Such inactivation can be obtained by several ways known from the person skilled in the art. For example, methods of inactivation comprise gene silencing with RNA interference (miRNA, siRNA) which have been used in microalgae (Zhao T. et al 2009, The Plant Journal, vol. 58, p: 157-164; Molnar A. and al., 2009, The Plant Journal vol. 58, p: 165-174), as in P. tricornutum (De Riso V. et al., 2009, Nucleic Acids Research, p: 1-12). The inactivation of said β-N-acetylglucosaminidases can also be obtained by the knock out of the corresponding genes but also by the use of a method of inhibition of the enzymatic activity, for example by using antibody directed to the catalytic site of said β-N-acetylglucosaminidases.
The inactivation of β-N-acetylglucosaminidases can be confirmed by testing their enzymatic activity, which can be measured by techniques which are known from the skilled person in the art, for example by using one of the two different following tests of activity described in Léonard R. et al., 2006, The Journal of Biological Chemistry, Vol. 281, p: 4867-4875.
According to a first test, N-acetylglucosaminidases are incubated with the different substrates at 37° C. for 1-20 h. For experiments with p-nitrophenyl-GlcNAc, the substrate concentration is 5 mM in a total volume of 0.04 ml of 0.1 M citrate/phosphate buffer at pH 3-8. The reactions are terminated by the addition of 0.26 ml of 0.4 M glycine/NaOH buffer at pH 10.4, and absorbance at 405 nm is measured with a microtiter plate reader.
According to a second test, Pyridylaminated oligosaccharides are used at a final concentration of 0.1 mM in a total volume of 0.02 ml of 0.1 M citrate/phosphate buffer at pH 3-8. Incubation is terminated by the addition of 0.18 ml of 20 mM ice-cold sodium borate. Aliquots of 0.05 ml are analyzed by reverse-phase HPLC as described previously.
As used herein, a “transformed” Phaeodactylum tricornutum may also correspond to a P. tricornutum, wherein at least one β-N-acetylglucosaminidase has been inactivated.
In a preferred embodiment, said β-N-acetylglucosaminidases have been inactivated with the technique of RNA interference.
In a preferred embodiment, said transformed Phaeodactylum tricornutum having at least one β-N-acetylglucosaminidase which has been inactivated further comprises a nucleic acid sequence operatively linked to a promoter, wherein

    
    
        (i) said nucleic acid sequence encodes an N-acetylglucosaminyltransferase I having the amino acid sequence SEQ ID No1, a fragment or a derivative thereof, and
        (ii) said operatively linked promoter has a sequence identity of less 50% with SEQ ID No3 or a fragment thereof.
    
    


In another preferred embodiment, said transformed Phaeodactylum tricornutum having at least one β-N-acetylglucosaminidase which has been inactivated as disclosed previously further comprises a nucleic acid sequence operatively linked to a promoter, wherein

    
    
        (iii) said nucleic acid sequence encodes an α-Mannosidase II having the amino acid sequence SEQ ID No5, a fragment or a derivative thereof, and
        (iv) said operatively linked promoter has a sequence identity of less 50% with SEQ ID No7 or a fragment thereof.
    
    


Preferably, all the transformed P. tricornutum as described previously further comprises a further nucleic acid sequence operatively linked to a promoter, said further nucleic acid sequence encoding a polypeptide that is expressed and glycosylated in the transformed P. tricornutum. 
The term “polypeptide” as used herein refers to an amino acid sequence comprising more than 50 amino acids which are linked by peptide bonds.
After transformation of P. tricornutum, transformants producing the desired polypeptide are selected. Selection can be carried out by one or more conventional methods comprising: enzyme-linked immunosorbent assay (ELISA), mass spectroscopy such as MALDI-TOF-MS, ESI-MS chromatography, characterization of cells using fluorescence activated cell sorter, spectrophotometer, fluorimeter, immunocytochemistry by exposing cells to an antibody having a specific affinity for the desired protein. Such methods are detailed in examples below.
The glycosylated polypeptides have at least one GlcNAcMan5GlcNAc2 structure. Preferably, said glycosylated polypeptides have at least one GlcNAcMan4GlcNAc2, GlcNAcMan3GlcNAc2 or GlcNAc2Man3GlcNAc2.
Advantageously, the polypeptide expressed and glycosylated by the transformed Phaeodactylum tricornutum is a polypeptide of animal origin, preferably of mammalian origin, and most preferably of human origin.
In a still preferred embodiment, the polypeptide expressed and glycosylated by the transformed P. tricornutum of the invention is a polypeptide having a therapeutic interest. Preferably, said polypeptide is selected in the group comprising erythropoietin, cytokines such as interferons, antibodies and their fragments, coagulation factors, hormones, beta-glucocerebrosidase, pentraxin-3, anti-TNFs, acid α-glucosidase, α-L-iduronidase and derivatives thereof.
An antibody is an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, two identical heavy (H) chains (about 50-70 kDa when full length) and two identical light (L) chains (about 25 kDa when full length) inter-connected by disulfide bonds. Light chains are classified as kappa and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Each heavy chain is comprised of a N-term heavy chain variable region (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains (CH1, CH2, and CH3) for IgG, IgD, and IgA; and 4 domains (CH1, CH2, CH3, and CH4) for IgM and IgE. Each light chain is comprised of a N-term light chain variable region (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The HCVR and LCVR regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with well-known conventions. The functional ability of the antibody to bind a particular antigen depends on the variable regions of each light/heavy chain pair, and is largely determined by the CDRs.
The term “antibody”, as used herein, refers to a monoclonal antibody per se. A monoclonal antibody can be a human antibody, chimeric antibody and/or humanized antibody.
The term “antibody fragments” as used herein refers to antibody fragments that bind to the particular antigens of said antibody. For example, antibody fragments capable of binding to particular antigens include Fab (e.g., by papain digestion), Fab′ (e.g., by pepsin digestion and partial reduction) and F(ab′)2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or ScFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention.
Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab′)2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and/or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
The term “Cytokines” refers to signalling proteins which are released by specific cells of the immune system to carry a signal to other cells in order to alter their function. Cytokines are immunomodulating agents and are extensively used in cellular communication. The term cytokines encompasses a wide range of polypeptide regulators, such as interferons, interleukins, chemokins or Tumor Necrosis Factor.
The term “Coagulation factors” refers to the plasma proteins which interact with platelets in a complex cascade of enzyme-catalyzed reactions, leading to the formation of fibrin for the initiation of a blood clot in the blood coagulation process. Coagulation factors, at the number of 13, are generally serine proteases, but also comprise glycoproteins (Factors VIII and V) or others types of enzyme, such as transglutaminase (Factor XIII).
The term “Hormones” refers to chemical messengers secreted by specific cells in the plasma or the lymph to produce their effects on other cells of the organism at a distance from their production sites. Most hormones initiate a cellular response by initially combining with either a specific intracellular or cell membrane associated receptor protein. Common known hormones are, for example, insulin for the regulation of energy and glucose in the organism, or the Growth Hormone which stimulates growth and cell reproduction and regeneration.
In another preferred embodiment, the invention relates to a transformed P. tricornutum as described above, further comprising another nucleic acid sequence operatively linked to a promoter, wherein said nucleic acid sequence encodes an N-acetylglucosaminyltransferase II, a fragment or a derivative thereof.
N-acetylglucosaminyltransferase II, also known as GnT II or mannoside acetylglucosaminyltransferase II (MGAT II) is an enzyme from the N-glycosylation pathway, which is capable of adding an N-acetylglucosamine (GlcNAc) residue to GlcNAcMan3GlcNAc2, product of α-Man II, to produce a GlcNAc2Man3GlcNAc2 
Examples of GnT II, also known as mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT 2), include GnT II from Mus musculus (SEQ ID No13. Accession number NP_666147), from Homo sapiens (SEQ ID No14, Accession number NP_002399) or from Phaeodactylum tricornutum (SEQ ID No5).
Preferably, N-acetylglucosaminyltransferase I 1 has an amino acid sequence of SEQ ID No5, a fragment or a derivative thereof and said operatively linked promoter has a sequence identity of less 50% with SEQ ID No7 or a fragment thereof.
In still another preferred embodiment, said N-acetylglucosaminyltransferase II comprises the amino acid sequence SEQ ID No15 and said operatively linked promoter has a sequence identity of less 50% with SEQ ID no 7 or a fragment thereof.
Inventors have noticed that α-Mannosidase II have an N-acetylglucosaminyltransferase II domain of amino acid sequence SEQ ID No15.
The expression “fragment” with reference to SEQ ID No5 and GnT II refers to an amino acid sequence of at least SEQ ID No15 corresponding to the amino acid sequence of the GnT II domain of α-Mannosidase II.
The expression “catalytic site” refers to the amino acid sequence of an enzyme, said amino acid sequence being responsible for the enzymatic activity. Therefore, the catalytic site of the GnT II according to the invention corresponds to the amino acid sequence responsible for the addition of an N-acetylglucosamine (GlcNAc) residue to GlcNAcMan3GlcNAc2 to produce a GlcNAc2Man3GlcNAc2.
As used herein the term “GnT II derivative” refers to an amino acid sequence capable of adding an N-acetylglucosamine (GlcNAc) residue to GlcNAcMan3GlcNAc2, product of α-Man II, to produce a GlcNAc2Man3GlcNAc2 and having more than 85% of identity with the amino acid sequence SEQ ID No5 or a fragment thereof, preferably more than 90% of identity with amino acid sequence SEQ ID No5 or a fragment thereof, and more preferably more than 95% of identity with amino acid sequence SEQ ID No5 or a fragment thereof.
In another preferred embodiment, the invention relates to a transformed P. tricornutum as described above, further comprising another nucleic acid sequence operatively linked to a promoter, said nucleic acid sequence encoding at least an enzyme of the human N-glycosylation pathway such as N-acetylglucosaminyltransferases III, IV, V, VI, and glycosyltransferases such as galactosyltransferases, fucosyltransferases or sialyltransferases. Said enzymes are expressed in said transformed P. tricornutum and enable the N-glycosylation of a polypeptide.
In another preferred embodiment, said transformed P. tricornutum as described above, further comprising another nucleic acid sequence operatively linked to a promoter comprises a nucleic acid sequence encoding N-acetylglucosaminyltransferases III, IV, V and VI.
In still another preferred embodiment said transformed P. tricornutum as described above, further comprising another nucleic acid sequence operatively linked to a promoter comprises a nucleic acid sequence encoding glycosyltransferases comprising galactosyltransferases, fucosyltransferases and sialyltransferases.
GnT III, GnT IV, GnT V, GnT VI, fucosyltransferase, galactosyltransferase (GalT) and sialyltransferases (ST) are well known from one of skilled in the art.
Examples of GnT III, also known as mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase (MGAT 3), include GnT III from Mus musculus (SEQ ID No17, Accession number NP_034925) or from Homo sapiens (SEQ ID No18, Accession number NP_002400). Preferably, said N-acetylglucosaminyltransferase III (GnT III) corresponds to SEQ ID No18 (Accession number NP_02400).
Examples of GnT IV, also known as mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase (MGAT4), include GnT IV isozyme A from Mus musculus (SEQ ID No19, Accession number NP_776295), isozyme B from Mus musculus (SEQ ID No20, Accession number NP_666038), isozyme C from Mus musculus (SEQ ID No21, Accession number NP_080519), GnT IV isozyme A from Homo sapiens (SEQ ID No22, Accession number NP_036346), GnT IV isozyme B from Homo sapiens (isoform 1, SEQ ID 23, Accession number NP_055090 or isoform 2, SEQ ID No24, Accession number NP_463459) or GnT IV isozyme C from Homo sapiens (SEQ ID No25, Accession number NP_037376).
Examples of GnT V, include GnT V from Mus musculus (SEQ ID No26, Accession number NP_660110), GnT V isozyme B from Mus musculus (SEQ ID No27, Accession number NP_766536), GnT V from Homo sapiens (SEQ ID No28, Accession number NP 002401), GnT V isozyme B from Homo sapiens (isoform 1, SEQ ID No29, Accession number NP_653278 or isoform 2, SEQ ID No30, Accession number NP_945193).
Example of GnT VI includes GnT VI from Gallus gallus (SEQ ID No31, Accession number NP_990012).
Fucosyltransferases are well known from the skilled person and include, as an example alpha (1.6) fucosyltransferase (fucosyltransferase 8 (FUT8)), like FUT8 from Mus musculus (SEQ ID No32, Accession number NP_058589) or FUT8 from Homo sapiens (SEQ ID No33, Accession number Q9BYC5). Preferably, said fucosyltransferase corresponds to SEQ ID No33 (Accession number Q9BYC5).
Galactosyltransferase are well known from the skilled person and include, as an example, one beta-(1,4)-galactosyltransferase (B4GALT1), like B4GALT1 from Homo sapiens (SEQ ID No34, Accession number. NP_001488), or B4GALT1 from Mus musculus (SEQ ID No35, Accession number CAM14782). Preferably, said galactosyltransferase corresponds to SEQ ID No34 (Accession number NP_001488).
Sialyltransferase are well known from the skilled person and include, as an example Alpha 2,6 Sialyltransferase (ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1) or beta galactoside alpha 2,6 sialyltransferase 2 (ST6GAL2)), like ST6GAL2 from Mus musculus (SEQ ID No36, Accession number NP_766417) or ST6GAL1 from Homo sapiens (isoform a, SEQ ID 37, Accession number NP_775323 or isoform b, SEQ ID No38, Accession number NP_775324), or Alpha 2,3 Sialyltransferase (ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1), ST3 beta-galactoside alpha-2,3-sialyltransferase 2 (ST3GAL2), ST3 beta-galactoside alpha-2,3-sialyltransferase 3 (ST3GAL3), like ST3GAL1 from Mus musculus (SEQ ID No39, Accession number NP_033203) or from Homo sapiens (SEQ ID No40, Accession number NP_003024), ST3GAL2 from Homo sapiens (SEQ ID No41 Accession number NP_008858), ST3GAL3 from Homo sapiens (isoform a, SEQ ID No42, Accession number NP_777623, isoform b, SEQ ID No43, Accession number NP_777624, isoform c, SEQ ID No44, Accession number NP_777625, isoform f, SEQ ID No45, Accession number NP_777628, isoform j, SEQ ID No46, Accession number NP_006270, isoform d, SEQ ID No47, Accession number NP_777626, isoform e, SEQ ID No48, Accession number NP 777627, isoform i, SEQ ID No49, Accession number NP_777631, isoform g, SEQ ID No50, Accession number NP_777629, isoform h, SEQ ID No51, Accession number NP_777630), or ST3GAL6 from Homo sapiens (SEQ ID No52, Accession number NP_006091).
For a glycosyltransferase to function satisfactorily in the Golgi apparatus, it is necessary for the enzyme to be provided with sufficient concentrations of an appropriate nucleotide sugar, which is the high-energy donor of the sugar moiety added to a nascent glycoprotein. In humans, the full range of nucleotide sugar precursors are generally synthesized in the cytosol and transported into the Golgi apparatus, where they are attached to the core oligosaccharide by glycosyltransferases. The Applicant observed in microalgae a sufficient concentration of GlcNAc, mannose, fucose and galactose but not of sialic acid.
Therefore, for a sialyltransferase to function satisfactorily in the Golgi apparatus, it is necessary to express in the microalgae one or more enzymes needed for sialic acid synthesis, its activation and its transport within the Golgi apparatus among UDP-GlcNAc 2-epimerase, GlcNAc 2-epimerase, GlcNAc-6P 2-epimerase, NeuAc synthase. NeuAc-9P synthase, CMP-NeuAc synthase and CMP-sialic acid transporter (see for example works done in plants: Misaki R et al. Biochem Biophys Res Commun. 2006 Jan. 27; 339 (4): 1184-9; Paccalet T et al. Plant Biotechnol J. 2007 January; 5(1): 16-25). Castihlo et al., 2008 and Castilho et al., 2010 as described above).
UDP-GlcNAc 2-epimerase, which is also known as glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE), is well known from the skilled person and include, as an example GNE from Mus musculus (SEQ ID No53, Accession number NP_056643) or GNE from Homo sapiens (SEQ ID No54, Accession number NP_005467). Preferably, said GNE corresponds to SEQ ID No54 (Accession number NP_005467).
GlcNAc 2-epimerase is well known from the skilled person and includes, as an example, the renin binding protein (RENBP) from Homo sapiens (SEQ ID No55, Accession number NP_002901).
NeuAc-9-P synthase, also called N-acetylneuraminic acid synthase (NANS), is well known from the skilled person and include, as an example, NANS from Homo sapiens (SEQ ID No56, Accession number NP_061819).
CMP-NeuAc synthase, which is also known as cytidine monophospho-N-acetylneuraminic acid synthetase (CMAS), is well known from the skilled person and include, as an example CMAS from Mus musculus (SEQ ID No57, Accession number NP_034038) or from Homo sapiens (SEQ ID No58, Accession number NP_061156). Preferably, said CMAS corresponds to SEQ ID No58 (Accession number NP_061156).
CMP-sialic acid transporters are also well known from the skilled person and include, as an example, solute carrier family 35 (CMP-sialic acid transporter), member A1 (SLC35A1) from Mus musculus (SEQ ID No59, Accession number NP_036025) or from Homo sapiens (SEQ ID No60, Accession number NP_006407). Preferably, said CMP-sialic acid transporter corresponds to SLC35A1 from Homo sapiens (SEQ ID No60, Accession number NP_006407).
The added transporter protein conveys a nucleotide sugar from the cytosol into the Golgi apparatus, where the nucleotide sugar may be reacted by the glycosyltransferase, e.g. to elongate an N-glycan. The reaction liberates a nucleoside diphosphate or monophosphate, UDP, GDP, or CMP. As accumulation of a nucleoside diphosphate inhibits the further activity of a glycosyltransferase, it is frequently also desirable to provide an expressed copy of a gene encoding a nucleotide diphosphatase. The diphosphatase (specific for UDP or GDP as appropriate) hydrolyzes the diphosphonucleoside to yield a nucleoside monosphosphate and inorganic phosphate. The nucleoside monophosphate does not inhibit the glycosyltransferase and in any case is exported from the Golgi by an endogenous cellular system.
Another object of the invention is a method for producing a glycosylated polypeptide, said method comprising the steps of:

    
    
        (i) Culturing a transformed P. tricornutum as described here above;
        (ii) Purifying said polypeptide expressed and glycosylated in said transformed P. tricornutum. 
    
    


In a preferred embodiment, said method for producing a glycosylated polypeptide comprises a former step of transforming a Phaeodactylum tricornutum so as to obtain a P. tricornutum as defined previously.
Methods which can be employed for the transformation of P. tricornutum are described here above. Such transformation, culture of P. tricornutum and purification of glycosylated polypeptides are also exemplified below.
Advantageously, the method of the invention further comprises a step (iii) of determining the glycosylation pattern of said polypeptide.
This glycosylation pattern can be determined by method well known from the skilled person. As an example, preliminary informations about N-glycosylation of the recombinant glycoprotein can be obtained by affino- and immunoblotting analysis using specific probes such as lectins (CON A; ECA; SNA; MAA . . . ) and specific N-glycans antibodies (anti-β1,2-xylose; anti-α-1,3-fucose; anti-Neu5Gc, anti-Lewis . . . ). This is made according to FITCHETTE et al., (Methods Mol. Biol., vol. 355, p: 317-342, 2007) and could be completed by deglycosylation assays.
To investigate the detailed N-glycan profile of recombinant protein, N-linked oligosaccharides is then released from the protein in a non specific manner using enzymatic digestion or chemical treatment (FITCHETTE et al., above mentioned, 2007; SEVENO et al., Anal. Biochem., vol. 379(1), p: 66-72, 2008). The resulting mixture of reducing oligosaccharides can be profiled by HPLC and/or mass spectrometry approaches (ESI-MS-MS and MALDI-TOF essentially) (BARDOR et al., Curr Opin Struct Biol., vol. 16 (5), p: 576-583, 2006; SEVENO et al., above mentioned, 2008). These strategies, coupled to exoglycosidase digestion, enable N-glycan identification and quantification (SEVENO et al., above mentioned, 2008).
Another alternative to study N-glycosylation profile of recombinant protein is to work directly on its glycopeptides after protease digestion of the protein, purification and mass spectrometry analysis of the glycopeptides as disclosed in BARDOR et al. (Plant Biotechnol. J., vol. 1 (6), p: 451-462, 2003).
Another object of the invention relates to the use of a transformed P. tricornutum as defined previously for producing a glycosylated polypeptide.
In the following, the invention is described in more detail with reference to methods. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.
EXAMPLES
Example 1
No Significant GnT-I Activity was Detected in Phaeodactylum tricornutum 
A gene encoding a putative GnT-I was predicted in the P. tricornutum genome (Pt54844; http://genome.jgi-psf.org/Phatr2/Phatr2.home.html) (SEQ ID No2). In eukaryotes, this enzyme is involved in the N-glycan maturation into complex-type N-glycan by transfer of a terminal GlcNAc onto Man-5 (FIG. 1). This putative GnT I sequence SEQ ID No2 is predicted to be a type II protein with a luminal part sharing high structural homologies with the catalytic domain of the rabbit GnT I.
In order to determinate if this gene encodes for an active GnT I, the glycosylation pattern of P. tricornutum proteins was analyzed.
Phaeodactylum tricornutum was cultivated using a standard batch culture method using a scale-up from 2 to 10 L glass carboys in sterilized Conway media (WALNE, L. Fish Invest Serie II, vol. 25(4), p: 1-53, 1966) with seawater (salinity=3.3-3.4%), 1 μm-filtered and aerated with a 2% CO2/air mixture to maintain the pH in a range of 7.5-8.1. Sodium metasilicate was added to the media to the 40 mg/L final concentration.
Phaeodactylum tricornutum were grown at 22-23° C. under continuous illumination (280-350 μmol photons m2s−1). The concentrated culture (about 20·106 cells/mL) is first centrifuged at 5,000 g for 20 min at 4° C. and the pellet was then lyophilised.
Two grams of lyophilised microalgae were grind in presence of sand in a mortar using a 750 mM Tris-HCl pH 8 buffer containing 15% (w/v) of sucrose, 2% (v/v) of β-mercaptoethanol and 1 mM phenylmethylsulfonylfluoride and then centrifuged at 4° C. for 30 min at 11,500 g. Proteins from the supernatant were then precipitated with 90% ammonium sulfate during 2 hours at room temperature. The pellet was solubilized in water and then, dialysed against water overnight at 4° C. Finally, the total protein extract was ultra-centrifuged at 100 000 g for 1 hour at 4° C. and resuspended in the smallest volume of water, prior to protein quantification and further analyses. Protein quantification was performed on the total protein extracts from Phaeodactylum tricornutum using the BCA protein assay kit from PIERCE according to the manufacturer's instructions.
Structural analysis of glycans N-linked to P. tricornutum proteins was then investigated by western-blot analysis on a total protein extract using probes specific for glycan epitopes. For this analysis, 50 μg of total proteins were separated by SDS-PAGE. Onion proteins were used as a control. The separated proteins were transferred onto nitrocellulose membrane and stained with Ponceau Red in order to control transfer efficiency. Affinodetection using concanavalin A was performed by incubation with the lectin at 25 μg·mL−1 during 2 h at RT in TBS-T, complemented with 1 mM CaCl2 and 1 mM MgCl2. After washing with TBS-T complemented with CaCl2 and MgCl2 (6 times, 5 minutes), binding of this lectin was detected using horseradish peroxidase diluted at 50 μg·mL−1, 1 h at RT in TBS-T complemented with 1 mM CaCl2 and 1 mM MgCl2. After washing with the same TBS-T and then TBS, final development of the blots was performed by using 4-chloro-1-naphtol as previously described (FITCHETTE et al., Methods in Molecular Biology published by Humana Press, USA (Totowa, N.J.), p: 317-342, 2006).
Immunodetection using home-made specific core-β(1,2)-xylose and core-α(1,3)-fucose antibodies (1:1,000 in TBS containing 1% of gelatin, 2 h, RT) was also performed. After washing with TBS-T (6 times, 5 minutes), binding of antibodies was detected using a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted at 1:3,000 in TBS containing 1% gelatin for 90 min at RT (Bio-Rad). Final development of the blots was performed by using 4-chloro 1-naphtol as previously described (FITCHETTE et al., above mentioned, 2006).
The results are presented in FIG. 2 showing affinodetection using concanavalin A (Con A) and immunodetection using antibodies raised against the core β(1,2)-xylose (anti-Xyl) and core α(1,3)-fucose (anti-Fuc) epitopes of proteins isolated from green onion as a positive control (Lane 1) and from Phaeodactylum tricornutum grown in standard conditions (Lane 2)
The results show that the proteins expressed in Phaeodactylum tricornutum do not exhibit any β(1,2)-xylose (anti-Xyl) and core α(1,3)-fucose (anti-Fuc) epitopes. Nevertheless, Phaeodactylum tricornutum proteins exhibit high-mannose sequences as revealed by Con A binding, a lectin specific for high-mannose sequences. The presence of high-mannose sequences in Phaeodactylum tricornutum proteins was also confirmed by deglycosylation assays using peptide N-glycosidase F (PNGase F) and Endoglycosidase H (Endo H) (data not shown).
In order to determine the specific glycosylation pattern of Phaeodactylum tricornutum proteins, said proteins were digested prior to mass spectrometry analysis.
Total proteins were digested by successive treatments with pepsin and PNGase A as previously described in FITCHETTE et al. (above mentioned, 2006). 4 mg of proteins were digested with 6 mg of pepsin in 2 mL of 10 mM HCl, pH 2.2, at 37° C. for 48 h. After neutralization with 1 M ammonium hydroxide, the solution was heated for 5 min at 100° C. and lyophilized. Glycopeptides were then deglycosylated overnight at 37° C. with PNGase A (10 mU, BOEHRINGER MANNHEIM) in a 100 mM sodium acetate buffer, pH 5.0. N-Glycans were purified by successive elution through an AG 50W-X2 column (BIO-RAD) and a C18 cartridge (VARIAN) according to FITCHETTE et al. (above mentioned, 2006). The purified N-glycans were then labelled by 2-aminobenzamide (2-AB) using the optimized protocol described in BIGGE et al. (Anal Biochem, vol. 230, p: 229-238, 1995). Then, labelled N-glycans were detected with an UV light and eluted using water. The eluted labelled N-glycans were finally lyophilised prior to exoglycosidase digestion and MALDI-TOF mass spectrometry analysis. For exoglycosidase digestion, 200 milliunits of Jack bean α-mannosidase (SIGMA-ALDRICH) were desalted by ultrafiltration using a Centricon and incubated overnight at 37° C. with approximately 50 pmoles of 2-AB labelled N-glycan mixture. Then, the 2-AB labelled N-glycans was directly analysed by matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry on a Voyager DE-Pro MALDI-TOF instrument (APPLIED BIOSYSTEMS) 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 in a 70:30 acetonitrile/0.1% TFA, was mixed with the water 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 externally calibrated using des-Arg1-bradykinin (904.4681 Da), angiotensin I (1296.6853), Glu1-fibrinopeptide B (1570.6774 Da), ACTH18-39 (2465.1989 Da) and bovine insulin (5730.6087 Da). 1000 Laser shots were accumulated for each spectrum and several spectra were accumulated (between 5 to 10 spectra) in order to obtain a good signal to noise ratio.
The results in FIG. 13 have shown that the major ions (95-97%) for the resulting pool of labeled N-glycans correspond to (M+Na)+ ions of 2-AB derivatives of Hexose5-9GlcNAc2. Nevertheless, no derivatives GlcNAc-Hexose5GlcNAc2 was detected.
Consequently, we show that P. tricornutum does not exhibit a significant GnT I activity.
Example 2
A Putative GnT I Detected in P. Tricornutum GnT I Complement CHOΔGnT-I Cells
Even if no detectable GnT I activity was identify in Phaeodactylum tricornutum, we try to express the nucleic acid sequence corresponding to the P. tricornutum putative GnT I full-length sequence (Pt54844) (SEQ ID No2) in CHO Lec1 mutant, which is mutated on its endogenous GnT I. Said P. tricornutum sequence was expressed in the CHO mutant in fusion with a V5 epitope to monitor the expression of the fusion protein in transformants.
On the basis of the detection of this epitope with anti-V5 antibodies, we show that most of the CHO transformants were found to express the V5 fusion protein (data not shown). Two cell lines expressing the V5 fusion protein were selected for N-linked glycan analysis.
Proteins from these lines as well as from wild-type and Lec1 CHO cells were isolated and their N-linked glycans were released by treatment with PNGase F followed by MALDI-TOF mass spectrometry analysis.
The results shown that the CHO Lec1 mutant accumulates high-mannose-type N-glycans, in contrast to wild-type CHO cells which exhibited both high-mannose-type and complex-type N-glycans. Surprisingly, the proteins from the CHO Lec1 mutant expressing V5 fusion protein carry both high-mannose-type and a complete set of complex N-glycans identical to the one observed in wild-type CHO cells (FIG. 14).
Finally, these results show that even if no GnT I activity was significantly detectable in P. tricornutum, the expression of the Pt54844 gene was able to restore the biosynthesis of complex N-glycans in mammalian cells.
In conclusion, Pt54844 gene encodes for a functional transferase designed as Pt GnT I.
Example 3
Expression of GnT I in Phaeodactylum tricornutum and Validation by RT-PCR
The Pt54844 gene disclosed previously (SEQ ID No2) was cloned under of an enhanced promoter of Cauliflower Mosaic Virus in a pPha-T1 based vector called BSJ-25 vector. As a control, vectors comprising GnT I from Arabidopsis thaliana (AtGnT I, At4g38240) and GnT I from human cell (hGnT I, MGAT1) were also constructed. BSJ-25 (SEQ ID No79) was derived from pPha-T1 (Zaslayskaia and Lippmeier, 2000, J. Phycol. vol. 36(2), p: 379-386.) vector by replacing the FcpA promoter of the expression cassette by the double enhanced Cauliflower Mosaic Virus (CaMV35S) (SEQ ID No80) fused to the plant signal peptide. For this, pPha-T1 containing bleomycin-resistance cassette driven by FcpB promoter was digested with Nde1 and EcoR1 to remove the FcpA promoter. This construct was designated as “pPha-T1-PfcpA deleted”. The double enhanced Cauliflower Mosaic Virus 35S promoter of the expression cassette was amplified by PCR with forward primer, CaMV35Sfwd (5′-GAACATATGGTGGATTGATGTGATCTACTCC-3′) (SEQ ID No61) and reverse primer, CaMV35Srev (5′-AATTCTCGAGGAATTCGGCCGAGG-3′) (SEQ ID No62) on the PS1 construct (Kotzer et al., 2004, J Cell Sci, vol. 117(Pt 26), p: 6377-89). PS1 construct contains a double Cauliflower Mosaic Virus 35S promoter (SEQ ID No80), a tobacco Mosaic Virus—Ω sequence as translation enhancer fused to the tobacco chitinase signal peptide of SEQ ID No81 (Haseloff et al., 1997, Proc Natl Acad Sci USA, vol. 94(6), p: 2122-7.; Batoko et al., 2000, Plant Cell, vol. 12(11), p: 2201-18). The PCR product was digested by Nde1 and EcoR1 and then was cloned into “pPha-T1-PfcpA deleted” to generate BSJ-25 vector.
To resume, the expression cassette of BSJ-25 vector contain a double CaMV promoter, a translational enhancer, a signal peptide, a multi-cloning site and the FcpA terminator (FIG. 3). The GnT I gene from various origin has been introduced in this vector using enzymatic digestion from the multi-cloning site and the inserts had been cloned between the signal peptide and the terminator sequence.
Specific vectors expressing the abovementioned genes in fusion to Green Fluorescent Protein (GFP) were also constructed to investigate the cellular localisation in the Golgi apparatus of the corresponding fusion proteins.
The constructs can be seen in FIG. 12.
Said vectors were used to transform Phaeodactylum tricornutum. 
Transformation of Phaeodactylum tricornutum was carried out as described by Zaslayskaia et al. (2000, J. Phycol. Vol. 36(2), p: 379-386). P. tricornutum 1.8.6 are cultivated (flask or agar plate 10%) in sterile sea water (0.22 μm filtered) enriched with 0.1% (v/v) of the nutritive medium (Conway) and 0.1% (v/v) of a silica solution (0.4 g/ml). All the cultures are maintained at 20° C. under continuous lighting in sterile conditions. For genetic transformation, cultures of microalgae in exponential phase of growth are counted and concentrated by centrifugation, diluted in sterile sea water and approximately 108 cells were inoculated as a plaque of 2.5 cm of diameter on the surface of the medium agar plate. Five hundred micrograms or gold microcarrier (0.6 μm of particle size) was coated with 1 μg of vector DNA in the presence of 1.25M of CaCl2 and 20 μM of spermidin. The transformation was carried out with the BIORAD PDS-1000/He biolistic particle delivery system for particles bombardment. Experiments are performed under a hood. The bombardment was performed at 900 and 1100 psi under a negative pressure of 27 Hg with different target distance (6-8 cm). After bombardment, cells transformed were suspended in 600 μL of nutritive sea water and were cultivated 1 day under illumination at 20° C. Transformed cells were spread on agar plate medium containing 100 μg·mL−1 of Zeocin and incubated to grown 3 or 4 weeks under continuous light.
The presence of transcripts for the recombinant GnT I was then monitored by reverse transcription PCR(RT-PCR). Microalgae pellet were resuspended in 1.2 mL of Trizol (INVITROGEN) and homogenized in 2 mL Lysing Matrix E by 10 Fastprep-24 run (6.5 m/s, 60 sec, MP BIOMEDICALS). 300 μL of chloroform were added to the supernatant, vigorously homogenized and incubated 3 min at room temperature. After a 15 min centrifugation at 4° C., the aqueous was mixed with 1 volume of absolute ethanol and the RNA were purified by RNEASY MINI kit (QIAGEN). The purified RNA were eluted in 50 μL of RNase-free water, dosed with NanoDrop (THERMO SCIENTIFIC) and were digested by RQ1 DNase (PROMEGA). After second purification by RNEASY MINI kit and Nanodrop quantification, RNA were conserved at −80° C.
Reverse Transcription (RT) was performed on 1 μg of purified RNA. The first cDNA strand was synthesised by 200 units of M-MLV RT RNase H minus (PROMEGA) with oligodT primers. Two μL of cDNA were used for PCR with GoTaq polymerase (PROMEGA) in 50 μL. The annealing was performed with specific primers of GnT I and actin sequence.
The results confirmed the expression of transcripts for the recombinant GnT in the selected recombinants (data not shown).
The glycosylation pattern of proteins expressed in said transformants is then performed as disclosed previously. Simultaneously, the cellular localisation of the fusion proteins is investigated by observing the GFP fluorescence of GnT I-GFP fusion proteins by confocal microscopy.
Another set of tests were run in order to demonstrate the expression and localization of endogenous GnT I in transformed Phaeodactylum tricornutum. 
Example 4
Expression of Endogenous GnT I and Validation by RT-PCR and Q-PCR
1) Constructions Comprising the Sequence of GnT I
Different constructs comprising the sequence coding for the endogenous GnT I from Phaeodactylum tricornutum were realised with the cloning vector pPHA-T1 built by Zavlaskaïa et al. (2000) for the genetic transformation of Phaeodactylum tricornutum, said vector including sequences of P. tricornutum promoters fcpA and fcpB (fucoxanthin-chlorophyll a/c-binding proteins A and B) and the terminator of fcpA. It contains a selection cassette with the gene she ble and a MCS flanking the fcpA promoter.
Said constructs are schematised in FIG. 4.
The first cassette comprised the sequence coding for the endogenous GnT I placed under the control of endogenous regulatory sequences. In the second construction, the GnT I was fused to the Green Fluorescent Protein (GFP).
The endogenous GnT I sequence of Phaeodactylum tricornutum was cloned in an expression vector with regulatory sequences from said microalgae. The GnT I was cloned alone or fused to the Green Fluorescent Protein (eGFP). The expression of said fusion protein enabled to visualise the expression and localization of the GnT I in the microalgae.
The vectors used for the transformation of Phaeodactylum tricornutum also comprised a selection cassette comprising a zeocin resistance gene. They enabled the genetic transformation of the Pt186 Phaeodactylum tricornutum strain.
The obtained clones were isolated and cultured in order to be analysed.
The transformation of Phaeodactylum tricornutum was realized as described previously.
2) Screening of Transformed Microalgae
The clones obtained after the transformation of the Pt186 Phaeodactylum tricornutum strain were isolated on fresh culture medium. The insertion of the construction comprising a gene coding for GnT I in the genome of the microalga was verified by PCR amplification with a set of specific primers for the transgene (SEQ ID No69 for the vector and SEQ ID No70 for the GnT I).
The PCR reaction was carried out in a final volume of 50 μl consisting of 1× PCR buffer, 0.2 mM of each dNTP, 5 μM of each primer, 20 ng of template DNA and 1.25 U of Taq DNA polymerase (Taq DNA polymerase, ROCHE). Thirty cycles were performed for the amplification of template DNA. Initial denaturation was performed at 94° C. for 3 min. Each subsequent cycle consisted of a 94° C. (1 min) melting step, a 55° C. (1 min) annealing step, and a 72° C. (1 min) extension step. Samples obtained after the PCR reaction were run on agarose gel (1%) stained with ethidium bromide.
The results are shown on FIG. 5 and present the screening obtained for 5 different lineages of potentially transformed Phaeodactylum tricornutum. The amplification of a specific sequence of 120 bp from the transgene obtained for the positive control (plasmidic DNA) was also found in the 5 analysed clones. The clones of the microalga therefore did integrate the genetic construction comprising the gene coding for the endogenous GnT I.
3) Screening of Microalgae Expressing the GnT I
a) Analysis of the Expression of GnT I by RT-PCR
A transcriptomic analysis was realised on transformed clones with the sequence coding for the P. tricornutum GnT I. The total RNAs from the different clones were purified. The corresponding cDNA were synthesized then analysed by Reverse Transcription PCR amplification according to the instructions as disclosed in Example 3 with specific primers for the transgene (SEQ ID No71 and SEQ ID No72) and with the H4 housekeeping gene (SEQ ID No73 and SEQ ID No74).
The results of the amplifications realised with specific primers from the H4 housekeeping gene are presented in FIG. 6. The absence of amplification for the RT-samples enables to validate that the samples did not contain genomic DNA.
The amplifications realised on the RT+ samples enabled to obtain amplicons at 150 bp which were homogenous between all the Pt-GnT I clones. This study therefore enabled to validate the experimental conditions.
The Pt-GnT I clones were then analysed with primers enabling the highlight of the expression of GnT I (see FIG. 7).
The FIG. 7 represents the RT-PCR results obtained for the expression of GnT I in the P. tricornutum transformed clones. The absence of amplification in the RT-samples validates the quality of the samples. Furthermore, the amplification fragment of 212 bp with a higher intensity than the one of the wild type strain is obtained for the clones 1 and 3. This difference of signal corresponds to a surexpression of GnT I. Therefore, the inventors were able to identify microalgae clones which surexpressed the GnT I. A thinner analysis of the expression of GnT I was proceeded by quantitative PCR (Q-PCR).
b) Analysis of the Expression of GnT I by Q-PCR
A lineage from transformed P. tricornutum with a construction comprising the GnT I gene was selected by RT-PCR. The expression of GnT I was then analysed by Q-PCR as disclosed in Siaut et al., 2007, Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum, Gene 406 (1-2): 23-35, by using primers directed to the transgene (SEQ ID No75 and SEQ ID No76) and the H4 housekeeping gene (SEQ ID No 77 and SEQ ID No78).
The FIG. 8A corresponds to a fusion curve. It enables to verify that only one PCR product has been amplified. The presence of one peak shows that only one amplification fragment was obtained. The dissociation temperature of 81.2° C. shows a strong specificity of the amplicon-primers association.
The FIG. 8B presents the detection profile of GnT I in the transformed cells (Pt-GnT I) and the wild-type cells (Pt-wt). For each strain, 3 dilutions of cDNA were analysed. The results were expressed in number of cycles compared to the relative values of detection of the GnT I amplicon.
The 3 sigmoids corresponding to negative controls (cDNA- and water) show a late detection, which is indicative of a non-significative amplification of GnT I.
The analysis of the transformed lineage with GnT I showed a detection which was significantively earlier than the wild-type cells. The data obtained were normalized with a housekeeping gene H4. The normalisation compared to the housekeeping gene by using the comparative Ct method also called as 2−[delta][delta]Ct (ΔΔCT method) revealed a clear difference of expression profile between the strain expressing the GnT I transgene and the wild-type cells (ΔΔCT=7).
Example 5
Display of the Expression and Localisation of the GnT I-eGFP Protein
1) By Confocal Microscopy
The clones of the P. tricornutum microalgae which were transformed with a sequence coding for a GnT I-eGFP fusion protein were analyzed by confocal microscopy with standard parameters. The FIG. 9 presents the results of said analysis.
The observations realized clearly show a difference of fluorescence localization between the microalgae expressing a cytosolic eGFP and the GnT I-eGFP fusion protein. In that case, the fluorescence is much more localised and is presented under the form of a bowl or a bean. This marking is typical of the Golgi apparatus in P. tricornutum microalgae. The inventors therefore demonstrated that the endogenous GnT I was expressed and localized in the suitable cell compartment.
2) By Analysis after Treatment of the Culture with Brefeldin A and Epifluorescence Microscopy
In order to validate those results, the inventors analyzed the clones expressing a GnT I-eGFP fusion protein after a treatment of the culture of the transformed microalgae with brefeldin A compared to microalgal strain of P. tricornutum expressing eGFP in the chloroplast, in the endoplasmic reticulum or in the Golgi apparatus after a culture with brefeldin A (FIG. 10).
The brefeldin A is a molecule presenting a particularity of dismantling the structure of the Golgi apparatus in the cells. Therefore, it is possible to confirm the localization of the fusion protein which is produced in P. tricornutum microalgae. The cells were incubated with 50 μM of brefeldin A and the observations were conducted with an epifluorescence microscopy on a period of 48 hours.
The FIG. 10 presents the results which were obtained. No fluorescence variation was observed in the P. tricornutum microalgae expressing eGFP in the chloroplast or in the Endoplasmic Reticulum after a culture with brefeldin A. In the microalgae expressing the GnT I-eGFP fusion protein, small fluorescent vesicles rapidly appeared (after 5 hours on the picture), which is indicative of the effect of brefeldin A on the Golgi apparatus. Said effect became more and more visible after 24 and 48 hours of culture with a marking spreading to the whole cell. The Golgi marking which was very targeted almost disappeared.
This analysis enabled to validate that the GnT I-eGFP fusion protein is expressed and localized in the Golgi apparatus of transformed P. tricornutum microalgae.
3) Immunoblotting Analysis by Western Blot
Aliquotes of wild-type and transformed cells of P. tricornutum culture at exponential phase of growth are collected and cells are separated from the culture medium by centrifugation (10 minutes, 2150 g, 20° C.). Cell pellets are resuspended in Tris-HCl 0.15 M pH 8, saccharose 15%, SDS 0.5%, PMSF 1 mM, protease inhibitor cocktail 1% (SIGMA) and sonicated for 30 min. Cell suspensions obtained are centrifuged (60 minutes, 15000 g, 4° C.) to remove cell debris and supernatants correspond to the intracellular fraction.
Ten μL of intracellular fractions from Pt-eGFP, Pt-GntI-eGFP transformed and wild-type cells are separated by SDS-PAGE using a 12% polyacrylamide gel. The separated proteins are transferred onto nitrocellulose membrane and stained with Ponceau Red in order to control transfer efficiency. The nitrocellulose membrane is blocked overnight in milk 5% dissolved in TBS for immunodetection. Immunodetection is then performed using horseradish peroxidase-conjugated anti-GFP (Santa Cruz, sc-9996-HRP) (1:2000 in TBS-T containing milk 1% for 2 h at room temperature). Membranes are then washed with TBS-T (6 times, 5 minutes, room temperature) followed by a final wash with TBS (5 minutes, room temperature). Final development of the blots is performed by chemiluminescence method.
The total protein extracts from wild-type P. tricornutum microalgae, from P. tricornutum microalgae expressing the eGFP protein (Pt-eGFP) and from P. tricornutum microalgae expressing the GnT I-eGFP fusion protein (Pt-GnT I-eGFP) were analyzed by Western Blot with anti-GFP antibodies which were coupled to peroxidase.
The FIG. 11 presents the results obtained for the detection of the GnT I-eGFP fusion protein by Western Blot. The figure shows a detection at around 28 kDa for the cytosolic eGFP and around 75 kDa for the clones which were transformed with a sequence coding for the GnT I-eGFP fusion protein, which theoretical size corresponds to 77 kDa.
The presence of the fusion protein in the protein samples from transformed microalgae is therefore demonstrated. The absence of degradation is a proof that the fusion protein is stable.
This analysis therefore demonstrates that it is possible to express the P. tricornutum endogenous GnT I sequence and that said protein is addressed to the suitable cell compartment, i.e. the Golgi apparatus.
Example 6
Glycosylation Pattern of Proteins Expressed in Transformed P. Tricornutum with Endogenous GnT I
In order to investigate the presence in P. tricornutum proteins of complex-type N-glycans carrying terminal GlcNAc, we treat the protein extract with a β1,4-galactosyltransferase, an enzyme able to transfer a galactose residue onto terminal GlcNAc residues, and then we analyze by affinoblotting the resulting protein preparation either with RCA 120 or ECA, which are lectins that bind specifically to Galβ1-4GlcNAc sequences according to a strategy previously reported (Bardor et al., 2003, Plant Biotech J, vol. 1, 451-462). Alternatively, the glycosylation pattern of proteins expressed in transformed P. tricornutum with endogenous GnT I is study by approaches which has been mentioned above.
Example 7
Expression of α-Man II in Phaeodactylum tricornutum 
A gene encoding a putative α-Man II was predicted in the P. tricornutum genome (Pt52108).
The Pt52108 gene (SEQ ID No6) is cloned under of an enhanced promoter of Cauliflower Mosaic Virus in a pPha-T1 based vector called BSJ-25 vector. As a control, vectors comprising α-Man II from Arabidopsis thaliana (NP_196999) and from human cell (Q16706 or AAC50302) are also constructed. BSJ-25 is derived from pPha-T1 vector (Zaslayskaia and Lippmeier, 2000, J. Phycol. vol. 36(2), p: 379-386) by replacing the FcpA promoter of the expression cassette by the double enhanced Cauliflower Mosaic Virus (CaMV35S) fused to the plant signal peptide. For this, pPha-T1 containing bleomycin-resistance cassette driven by FcpB promoter is digested with Nde1 and EcoR1 to remove the FcpA promoter. This construct was designated as “pPha-T1-PfcpA deleted”. The double enhanced Cauliflower Mosaic Virus 35S promoter of the expression cassette is amplified by PCR with forward primer, CaMV35Sfwd (5′-GAACATATGGTGGATTGATGTGATCTACTCC-3′) (SEQ ID No61) and reverse primer, CaMV35Srev (5′-AATTCTCGAGGAATTCGGCCGAGG-3′) (SEQ ID No62) on the PS1 construct (Kotzer et al., 2004, J Cell Sci, vol. 117(Pt 26), p: 6377-89). PS1 construct contains a double Cauliflower Mosaic Virus 35S promoter, a tobacco Mosaic Virus—Ω sequence as translation enhancer fused to the tobacco chitinase signal peptide (Haseloff et al., 1997, Proc Natl Acad Sci USA, vol. 94(6), p: 2122-7.; Batoko et al., 2000, Plant Cell, vol. 12(11), p: 2201-18). The PCR product is digested by Nde1 and EcoR1 and then is cloned into “pPha-T1-PfcpA deleted” to generate BSJ-25 vector.
To resume, the expression cassette of BSJ-25 vector contain a double CaMV promoter, a translational enhance, a signal peptide, a multi-cloning site and the FcpA terminator (FIG. 3).
Specific vectors expressing the abovementioned genes in fusion to Green Fluorescent Protein (GFP) are also constructed to investigate the cellular localisation in the Golgi apparatus of the corresponding fusion proteins.
Said vectors are used to transform Phaeodactylum tricornutum. 
Transformation of Phaeodactylum tricornutum is carried out as disclosed previously in the example of expression of GnT I in Phaeodactylum tricornutum. 
The presence of transcripts for the recombinant α-Man II is then monitored by reverse transcription PCR(RT-PCR). Microalgae pellet are resuspended in 1.2 mL of Trizol (INVITROGEN) and homogenized in 2 mL Lysing Matrix E by 10 Fastprep-24 run (6.5 m/s, 60 sec, MP BIOMEDICALS). 300 μL of chloroform are added to the supernatant, vigorously homogenized and incubated 3 min at room temperature. After a 15 min centrifugation at 4° C., the aqueous is mixed with 1 volume of absolute ethanol and the RNAs were purified by RNEASY MINI kit (QIAGEN). The purified RNAs are eluted in 50 μL of RNase-free water, dosed with NanoDrop (Thermo SCIENTIFIC) and are digested by RQ1 DNase (PROMEGA). After second purification by RNEASY MINI kit and Nanodrop quantification, RNAs are conserved at −80° C.
Reverse Transcription (RT) is performed on 1 μg of purified RNA. The first cDNA strand is synthesised by 200 units of M-MLV RT RNase H minus (PROMEGA) with oligodT primers. Two μL of cDNA are used for PCR with GoTaq polymerase (PROMEGA) in 50 μL. The annealing is performed with specific primers of GnT I and actin sequence.
The results confirm the expression of transcripts for the recombinant α-Man II in the selected recombinants (data not shown).
The glycosylation pattern of proteins expressed in said transformants is then performed as disclosed previously. Simultaneously, the cellular localisation of the fusion proteins is investigated by observing the GFP fluorescence of GnT I-GFP fusion proteins by confocal microscopy.
Example 8
Glycosylation Pattern of Proteins Expressed in Transformed P. Tricornutum with Endogenous Alpha Man II
The glycosylation pattern of proteins expressed in transformed P. tricornutum with endogenous alpha Man II is studying by HPLC and Mass spectrometry approaches as mentioned above.
Example 9
Inactivation of N-acetylglucosaminidases in Phaeodactylum tricornutum with RNA Interference
a) By the Use of Vectors
This example is based on the use of constructs containing antisense RNA or inverted-repeat RNA for the inactivation of β-N-acetylglucosaminidase expression as described in De Riso V. et al., 2009, Nucleic Acids Research, p: 1-12.
For the generation of a β-N-acetylglucosaminidase vector, two fragments from the β-N-acetylglucosaminidase cDNA are amplified with specific primers, wherein one of the fragments is longer than the other fragment and also contains the total sequence of the shorter fragment. The two obtained fragments are then digested with restriction enzymes which can also be used for the linearization of the vector.
For the antisense construct, one of the two fragments obtained is inserted into the vector in the antisense orientation.
For the inverted-repeat construct, fragments are ligated in sense and antisense orientations in the integration site of the vector.
Said vectors are then used together or separately by choosing one vector to transform Phaeodactylum tricornutum. Transformation of Phaeodactylum tricornutum is carried out as described in example 3.
b) By the Use of siRNAs
The gene silencing of β-N-acetylglucosaminidases can also be obtained with the transformation of Phaeodactylum tricornutum with the use of siRNAs specific of at least one gene encoding a β-N-acetylglucosaminidase in said microalga (1 μg for each siRNA). Transformation of Phaeodactylum tricornutum is carried out as described in example 3.
siRNAs used for the transformation of Phaeodactylum tricornutum have the following sequences:

    
    
        siRNAs specific of a first β-N-acetylglucosaminidase of amino acid sequence SEQ ID No9 which is encoded by the nucleic acid sequence SEQ ID No10
    
    









siRNA1(β-N-acetylglucosaminidase SEQ ID No 10)


SEQ ID No 63


GGCCAUUCGUUACUAGCAA


 


siRNA2(β-N-acetylglucosaminidase SEQ ID No 10)


SEQ ID No 64


GUGGUUCGUUGGGAAAUGA


 


siRNA3(β-N-acetylglucosaminidase SEQ ID No 10)


SEQ ID No 65


CCGUCUGUGUGAAAUUGGU






    
    
        siRNAs specific of a second β-N-acetylglucosaminidase of amino acid sequence SEQ ID No11 which is encoded by the nucleic acid sequence SEQ ID No12
    
    









siRNA1(β-N-acetylglucosaminidase SEQ ID No 12)


SEQ ID No 66


CGGUAGUAGUGCUUGUUGU


 


siRNA2(β-N-acetylglucosaminidase SEQ ID No 12)


SEQ ID No 67


CUGCCAUGGAUAUUGUCAA


 


siRNA3(β-N-acetylglucosaminidase SEQ ID No 12)


SEQ ID No 68


GCCUUUGGUCCUGAAGAAA






Example 10
Expression and Glycosylation of a Polypeptide in Transformed P. tricornutum 
As explained previously, polypeptides of interest to be expressed and glycosylated in transformed P. tricornutum according to the invention are proteins of therapeutic interest. We choose the erythropoietin to exemplify the expression and glycosylation of such proteins in transformed P. tricornutum according to the invention. However, the invention is not limited to said erythropoietin and could be applied to any protein of therapeutic interest which needs to be glycosylated to present the adequate glycosylation pattern.
a) Expression Construct for Erythropoietin
The vector used for the expression of Erythropoietin comprises a nucleic acid sequence operably linked to a promoter, said nucleic acid sequence encoding Erythropoietin. Said vector preferably contains a selectable marker distinct from the selectable markers present on the BSJ-25 vector according to the example 3.
b) Transformation of P. tricornutum 
Transformed P. tricornutum of example 3 are also transformed with a vector according to step a).
c) Glycosylation Pattern Analysis of Expressed and Glycosylated Erythropoietin
The glycosylation pattern of Erythropoietin that is expressed and glycosylated in the transformed P. tricornutum according to the invention is described in the example 1.

## Claims

1. A transformed Phaeodactylum tricornutum, whose N-glycosylation pathway has been modified by inactivation of terminal N-acetylglucosamine residue removal activity of at least one β-N-acetylglucosaminidase.

2. The transformed P. tricornutum of claim 1, further comprising a nucleic acid sequence operatively linked to a promoter, wherein said nucleic acid sequence encodes an N-acetylglucosaminyltransferase II corresponding to an amino acid sequence capable of adding an N-acetylglucosamine residue to GlcNAcMan3GlcNAc2 to produce GlcNAcMan3GlcNAc2, and having more than 85% of identity with the amino acid sequence SEQ ID NO: 5.

3. The transformed P. tricornutum of claim 2, wherein
(i) said N-acetylglucosaminyltransferase II comprises the amino acid sequence SEQ ID NO: 15, and
(ii) said operatively linked promoter has a sequence identity of less 50% with SEQ ID NO: 7 or a fragment thereof.

4. The transformed P. tricornutum of claim 1, wherein said P. Tricornutum further comprises another nucleic acid sequence operatively linked to a promoter, said another nucleic acid sequence encoding a polypeptide that is expressed and glycosylated in the transformed P. tricornutum.

5. The transformed P. tricornutum of claim 4, wherein said glycosylated polypeptide has at least one GlcNAcMan5GlcNAc2 structure.

6. The transformed P. tricornutum of claim 4, wherein said polypeptide is selected from the group consisting of erythropoietin, cytokines, antibodies and their fragments, coagulation factors, hormones, beta-glucocerebrosidase, pentraxin-3, anti-TNFs, acid α-glucosidase, α-L-iduronidase and derivatives thereof.

7. The transformed P. tricornutum of claim 1, wherein said P. tricornutum further comprises another nucleic acid sequence operatively linked to a promoter, said another nucleic acid sequence encoding at least one enzyme selected among N-acetylglucosaminyltransferase III, IV, V and VI.

8. The transformed P. tricornutum of claim 7, wherein said P. tricornutum further comprises another nucleic acid sequence operatively linked to a promoter, said nucleic acid sequence encoding at least one glycosyltransferase enzyme selected from the group consisting of galactosyltransferases, fucosyltransferases and sialyltransferases.