Kevin Bellande

Alexandre Lalo

Laetitia Ligat

David Roujol

Elisabeth Jamet

Hervé Canut
email: canut@lrsv.ups-tlse.fr

Recombinant N-glycosylation isoforms of Legume lectins: production and purification from Nicotiana benthamiana leaves following RuBisCO depletion

Keywords: Arabidopsis thaliana, N-glycosylation, Nicotiana benthamiana, Recombinant lectin, RuBisCO depletion

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Legume-type lectins have been identified as carbohydrate binding proteins for a long time [START_REF] Van Damme | Plant lectins[END_REF]. They are ubiquitous proteins in plants and play important roles in cell-to-cell communication, development and defence strategies. The sequencing of plant genomes has revealed that the Legume lectin domain is mainly present in chimeric proteins and associated with intracellular kinase domains to constitute receptor-like kinases (LecRKs) [START_REF] Bellande | Plant lectins and lectin receptorlike kinases: how do they sense the outside?[END_REF]. For example, in Arabidopsis thaliana, Legume-type LecRKs form a family of 43 genes while only seven genes encode cell wall proteins (Lecs) and four genes receptor-like proteins: the Legume lectin domain is therefore mainly found at the cell surfaces. Many studies suggest specific functions for Legume-type LecRKs in plant innate immunity [START_REF] Singh | Lectin receptor kinases in plant innate immunity[END_REF][START_REF] Balagué | The Arabidopsis thaliana lectin receptor kinase LecRK-I.9 is required for full resistance to Pseudomonas syringae and affects jasmonate signalling[END_REF]. The functions of Legume-type Lecs have been rarely addressed and their involvement in cell wall organisation has been proposed [START_REF] Hijazi | Arabinogalactan protein 31 (AGP31), a putative network-forming protein in Arabidopsis thaliana cell walls?[END_REF]. The potential ligands for Legume-type LecRKs, i.e. eATP, eNAD + , eNADP + or/and RGD-containing peptides [START_REF] Gouget | Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis[END_REF][START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF][START_REF] Wang | A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana[END_REF][START_REF] Wang | Extracellular pyridine nucleotides trigger plant systemic immunity through a lectin receptor kinase/BAK1 complex[END_REF], are far from what could be expected for a carbohydratebinding domain. Modelling of LecRK lectin domain has confirmed the β-sandwich fold of Legume lectins, the conserved residues involved in Ca 2+ and Mn 2+ binding, and the potential carbohydrate binding site [START_REF] Gouget | Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis[END_REF][START_REF] Nguyen | Computational analysis of the ligand binding site of the extracellular ATP receptor, DORN1[END_REF]. In most cases, the carbohydrate-binding activity of Legume-type LecRKs is not demonstrated, except in poplar, where such an activity is detected with α-L-rhamnose as a ligand [START_REF] André | Evidence for lectin activity of a plant receptor-like protein kinase by application of neoglycoproteins and bioinformatic algorithms[END_REF]). The A.

thaliana Legume-type LecRKs are suspected to be devoid of any significant monosaccharidebinding activity. Indeed, the invariant Asp81 key residue responsible for the sugar recognition by canonical Legume lectins is replaced by a His residue [START_REF] Adar | Mutational studies of the amino acid residues in the combining site of Erythrina corallodendron lectin[END_REF][START_REF] Gouget | Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis[END_REF]). However, one cannot exclude that the lectin domain of Legume-type LecRKs accommodates complex carbohydrates or recruit another lectin domain to restore a functional protein. Finally, A. thaliana Legume-type lectin domains are predicted to be posttranslationally modified by glycosylation and carry one to several N-glycans. In this context, the development of efficient methods to produce and purify recombinant Legume lectin domains in sufficient amount is necessary to decipher their recognition capability.

Nicotianeae is one of the most efficient platforms for producing heterologous recombinant proteins [START_REF] Yusibov | Antibody production in plants and green algae[END_REF]. In particular, agroinfiltration of Nicotiana benthamiana leaves together with viral vectors and coexpression of a suppressor of gene silencing (Garabagi et al. 2012) allows accumulating high levels of different heterologous proteins, ranging from reporter fluorescent proteins to complex molecules such as immunoglobulins [START_REF] Yusibov | Antibody production in plants and green algae[END_REF]. Agroinfiltration is an effective and inexpensive technique that can be easily set up in a laboratory. It is also beneficial because plant cells can synthesise complex proteins with post-translational modifications such as glycosylation and disulfide bonds. However, protein recovery and purification processes from plant material, especially from green leaves, remain challenging. Indeed, plants contain numerous molecules including phenolics, proteases and highly abundant proteins that must be neutralised or removed from crude samples. Also, to simplify the downstream processes of purification, recombinant proteins are often translationally fused to small affinity tags.

Protein recovery and purification procedures from leaf extracts are complicated by the high abundance of ribulose biphosphate carboxylase/oxygenase (RuBisCO) which comprises about 30-60% of soluble proteins. Because it is a simple non-chromatographic step, inexpensive and easy to scale up, the selective precipitation of RuBisCO is often used in the early stages of purification processes [START_REF] Zhang | Lysozyme purification from tobacco extract by polyelectrolyte precipitation[END_REF]. The objective is to increase the ratio of recombinant to host proteins that is critical to ensure the overall purification efficiency.

Accordingly, many attempts have been performed including isoelectric and polyelectrolyte precipitations [START_REF] Zhang | Lysozyme purification from tobacco extract by polyelectrolyte precipitation[END_REF][START_REF] Holler | Purification of an acidic recombinant protein from transgenic tobacco[END_REF][START_REF] Kim | Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method[END_REF][START_REF] Stephan | Simple purification of Nicotiana benthamiana-produced recombinant colicins: high-yield recovery of purified proteins with minimum alkaloid content supports the suitability of the host for manufacturing food additives[END_REF], polyethyleneglycol (PEG) fractionation [START_REF] Xi | Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome[END_REF][START_REF] Widjaja | Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense[END_REF]Arfi et al. 2015), or RuBisCO IgY affinity [START_REF] Cellar | Cross species availability of abundant protein depletion columns for ribulose-1,5-biphosphate carboxylase/oxygenase[END_REF][START_REF] Widjaja | Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense[END_REF]). Most of these procedures have been applied to enrich protein samples in low-abundant proteins for proteomic studies and did not take into account the conservation of protein structures and activities. Isoelectric precipitation requires acidic conditions that may result in protein denaturation. Polyelectrolyte precipitations using polyethyleneimine (PEI), protamine sulphate (PS) or polyacrylic acid (PAA) are quite effective and selective in depleting RuBisCO from the extracts of various plants. However, it is still difficult to remove them from protein samples [START_REF] Holler | Purification of an acidic recombinant protein from transgenic tobacco[END_REF]. The same difficulty occurs with PEG. Finally, RuBisCO IgY affinity efficiently removes RuBisCO from protein samples but with limited amounts of material and frequent contamination with IgY fragments have been observed [START_REF] Widjaja | Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense[END_REF]).

Here we used agroinfiltration in N. benthamiana leaves to produce four recombinant Legume lectin domains being part of two LecRKs and two Lecs of A. thaliana. These recombinant lectin domains consist of around 250 amino acid residues with high sequence identity to which have been added the V5 epitope and a 6xHis tag at their C-terminus. We have developed a procedure to selectively deplete RuBisCO from total protein extracts. Based on citrate or succinate buffers at mild acidic pH, it was designed to facilitate the downstream purification process which could be achieved for three recombinant lectin domains. Then, their N-glycosylation status was addressed.

Materials and Methods

Plant expression constructs

The nucleotide sequences of LecRK-I.5 (At3g45430), LecRK-I.9 (At5g60300), Lec-I.3 (At1g53070) and Lec-I.6 (At3g16530) were retrieved from TAIR (www.arabidopsis.org). The amino acid residues 1-258 (LecRK-I.5), 1-287 (LecRK-I.9), 1-272 (Lec-I.3) and 1-276 (Lec-I.6) covered the signal peptide together with the Lectin_legB domain sequences (PF00139www.pfam.xfam.orgversion 32.0 September 2018) (Supplementary Fig. S1 and Fig. S2).

The four coding sequences (single exon coding sequences) were amplified by PCR from genomic DNA of A. thaliana ecotype Col-0 using the Pfu DNA polymerase (Promega) and specific primers. The resulting products were cloned into the vector pBAD-TOPO® (Invitrogen) allowing the fusion in-frame to the V5 epitope and the 6xHis tag sequences: these two tags were located at the C-terminus of the lectin domains (Supplementary Fig. S1 and Fig. S2).

Each of the four DNA fragments encoding the signal peptide::Lectin_legB domain::V5 epitope::6xHis tag fusion protein was subcloned by recombination into the entry vector pDONR207 (Invitrogen, CA) and the pCaMV35S overexpression vector pK7WG2D.1.

A particular feature of the vector pK7WG2D.1 was the presence of the rolD promoter::enhanced green-fluorescent protein (eGFP) gene carried by its T-DNA.

Transient expression in N. benthamiana leaves

N. benthamiana transient expression was performed by agroinfiltration of leaves using a needle-less syringe. The four binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404 using a freeze/thaw transformation method and the cultures were maintained in LB medium supplemented with 100 µg.mL -1 spectinomycin and 25 µg.mL -1 rifampicin. For infiltration, bacteria were grown to stable phase at 28°C to an OD 600 of 1.0 and collected by centrifugation. The bacterial pellets were resuspended in 10 mM MgCl 2 (pH 5.8) containing 100 µM acetosyringone by keeping a final OD 600 value of 0.5. The transformed A. tumefaciens suspensions were infiltrated into the leaves of 6 week-old N. benthamiana plants (from the third to the fifth leaf from the apical meristem) after mixing with an equal volume of an A. tumefaciens suspension (final OD 600 value of 0.5) carrying a p19 suppressor of post-transcriptional gene silencing. The overall surface of the leaves was infiltrated. The plants were maintained in a growth chamber under 16 h light (24°C)/8 h dark (20°C) photoperiod, at 120 µmol.m -2 .s -1 and 75% relative humidity. Infiltrated tissues were harvested three days post-infection and the material was frozen in liquid nitrogen, processed into fine powder and stored at -80°C until use.

Floral dip transformation of A. thaliana

A. thaliana Col 0 ecotype transformation was performed by floral dipping. The previous binary vector containing the DNA fragment encoding the lectin domain of LecRK-I.9 described above was introduced into the A. tumefaciens strain C58 containing the pMP90 (GV3101) plasmid using a freeze/thaw transformation method. The culture was maintained in LB medium supplemented with 100 µg.mL -1 gentamycin and 25 µg.mL -1 rifampicin. One colony of the A. tumefaciens strain was grown overnight in LB medium. Cells were harvested by centrifugation and resuspended into the infiltration medium (see below) to a final OD 600 of approximately 1.0. Inoculations were performed by dipping aerial parts of the plants for a few seconds in 500 mL of a solution containing 5% (w/v) sucrose, 0.03% (v/v) Silwett L-77, 10 mM MgCl 2 and the A. tumefaciens cells. The plants were kept into the dark with high humidity for 24 h and then transferred to the growth chamber. Seeds were sown on medium supplemented with 100 µg.mL -1 kanamycin to select transformants.

Extraction and fractionation of total soluble proteins from N. benthamiana leaves

Ground leaf material (1 g) was homogenised with 5 mL ice-cold protein extraction buffer consisting of 20 mM NaH 2 PO 4 /Na 2 HPO 4 buffer, pH 7.4, 150 mM NaCl, 10 mM βmercaptoethanol, 100 mg polyvinylpyrrolidone. The buffer also contained 100 µL of a protease inhibitor cocktail (P8849, Sigma-Aldrich), along with 100 µM L-benzylsuccinic acid as a carboxypeptidase inhibitor. After 15 min vortexing vigorously at 4°C, the slurry was clarified by centrifugation at 40,000 x g for 10 min at 4°C. The resulting pellet was resuspended with 5 mL ice-cold protein extraction buffer for a second extraction following the same procedure. The two supernatants were combined and filtered through glass wool.

The final filtrate was referred to as total soluble proteins (TSP). The protein fractionation started with protein precipitation using either ammonium sulphate, protamine sulphate (PS), polyethyleneimine (PEI) or polyethylene glycol (PEG). The four different procedures were compared to evaluate their efficiency with regard to the purification process of the lectin domains.

Solid (NH 4 ) 2 SO 4 was added to the TSP to a final concentration of 80 % and allowed to fully dissolve for 30 min at 4°C under continuous vortexing. Following centrifugation at 40,000 x g for 10 min, the resulting 80 % pellet was resuspended in an ice-cold 10 mM citrate solution containing 150 mM NaCl buffered to pH 5.0 (NaOH), unless otherwise indicated.

The solubilisation step was carried out at a ratio of 1.5 mL citrate solution to a pellet corresponding to 1 g of ground leaf material. The mixture was kept on ice for 10 min to ensure protein solubilisation and centrifuged at 12,000 x g for 5 min at 4°C. The supernatant was collected and the precipitate was solubilised in 20 mM NaH 2 PO 4 /Na 2 HPO 4 buffer, pH 7.4, 150 mM NaCl. Alternatively, the precipitate was solubilised in 10 mM citrate or succinate buffer at different pH ranging from 3.0 to 6.0, 150 mM NaCl.

For the three other procedures, TSP was divided into equal portions and mixed with (i) the desired PS concentration (1% stock solution in distilled water), from 0.02% to 0.20% (PS salt from salmon, P4020, Sigma-Aldrich), (ii) the desired PEI concentration (50% aqueous stock solution), from 0.1% to 0.5% (PEI long chain average molecular mass 750 kDa, 181978, Sigma-Aldrich), or (iii) 5, 10, 15, and 20% w/v polyethylene glycol (PEG) (PEG approx. molecular mass 3350, P3640, Sigma-Aldrich). The mixtures were kept on ice for 10 min to ensure protein precipitation and centrifuged at 12,000 x g for 5 min at 4°C. The supernatants were collected and the precipitates were solubilised in 20 mM NaH 2 PO 4 /Na 2 HPO 4 buffer, pH 7.4, 150 mM NaCl.

All the protein fractions were stored at -20°C before protein amount determination, SDS-PAGE and immune-or lectin-blot analysis. Protein concentration was determined by the Bradford dye binding assay (Pierce™).

Extraction and fractionation of total soluble proteins from A. thaliana and Brachypodium distachyon leaves

To know whether the procedures described above could be applied to other plants, TSP from A. thaliana and B. distachyon leaves were prepared as for N. benthamiana. Young leaves from four week-old rosettes of A. thaliana and young leaves (3 to 6 cm-long) from two-month-old plants of B. distachyon were collected.

Purification of recombinant proteins

Recombinant lectin domains were isolated according to the same purification scheme involving three consecutive chromatographic steps. The first two steps (anion-exchange and hydroxyapatite chromatographies) were used in the negative mode in order to submit a protein fraction enriched as much as possible in lectin domains to the third chromatographic step (immobilized metal-ion chromatography) that relies on the presence of a 6xHis tag at the Cterminus of the proteins. All the purification processes started after protein depletion of TSP using the citrate procedure (see above).

The depleted protein fractions were desalted using Econo-Pac® 10DG columns (Bio-Rad) equilibrated with 20 mM Tris pH 7.5 (HCl), 50 mM NaCl. In the case of the protein fractions containing the lectin domain of LecRK-I.5, the equilibration buffer was 20 mM Tris pH 7.5 (HCl), 100 mM NaCl. DEAE Sepharose FF (GE Healthcare) equilibrated in their respective buffers was added to the desalted fractions in the following ratio: 1 mL of chromatographic support to a protein fraction obtained from 1 g of ground leaf material. The mixture was then introduced into a column and the flow-through fraction was desalted as before with columns equilibrated with 10 mM NaH 2 PO 4 /Na 2 HPO 4 buffer, pH 6.5, 500 mM NaCl. Hydroxyapatite (Bio-Gel HTP, Bio-Rad) equilibrated in the same buffer was added to the new desalted fractions (500 µL of chromatographic support /1 g of ground leaf material).

The mixture was loaded onto a column and the flow-through fraction was adjusted to pH 8.5

(NaOH 1 M). Ni-NTA Superflow (Qiagen) equilibrated with 50 mM NaH 2 PO 4 /Na 2 HPO 4 buffer, pH 8.5, 500 mM NaCl was added to the hydroxyapatite flow-through fraction (500 µL of chromatographic support /1 g of ground leaf material) and a protein fraction was eluted in the same buffer plus 20 mM imidazole. All steps were carried out at 4°C. All protein fractions were stored at -20°C before protein amount determination, SDS-PAGE, immuno-or lectinblot analysis, and enzymatic deglycosylation. During the purification steps, protein concentration was determined by the Bradford dye binding assay.

SDS-PAGE, immunoblotting and lectin blot analysis

Electrophoretic separation of the proteins was performed by 11% (w/v) SDS-PAGE in reducing conditions. The resolved proteins were stained with Coomassie colloidal blue to visualise protein patterns, or electrotransferred onto nitrocellulose membranes for immunodetection with mouse anti-V5 epitope antibody (Invitrogen) and alkaline phosphataseconjugated goat anti-mouse IgG as secondary antibody (Promega) followed by a staining reaction using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP).

Presence of N-glycans on purified lectin fractions was analysed by reducing SDS-PAGE and lectin-blotting, followed by detection with DIG-labelled lectins according to the instructions supplied by the manufacturer (DIG Glycan differentiation Kit, Roche Diagnostics). Two DIG conjugated lectins were used in this analysis: Datura stramonium agglutinin (DSA) recognises unsubstitued galactose-(1-4)-N-acetylglucosamine; Galanthus nivalis agglutinin (GNA) recognises terminal mannose, (1-3), (1-6) or (1-2) linked to mannose. Lectin binding was detected by alkaline phosphatase-conjugated anti-digoxigenin followed by a staining reaction using NBT/BCIP.

Enzymatic deglycosylation

N-glycans were released from proteins with Endoglycosidase H (EndoH) and Peptide N-Glycosidase F (PNGaseF) according to the instructions supplied by the manufacturer (EndoH, V4871; PNGaseF, V483A; Promega). The purified protein samples (0.5 -2 µg) were first denaturated for 5 min at 95°C by adding 100 mM DTT and 0.5% (w/v) SDS final concentrations. EndoH digestions were carried out at 37°C for 24 h in 50 mM sodium citrate buffer (pH 5.5) containing 500 units EndoH and 1 µL of a protease inhibitor cocktail (P9599, Sigma-Aldrich). PNGaseF digestions were carried out at 37°C for 3 h in 50 mM sodium phosphate buffer (pH 7.5) containing 20 units PNGaseF, 2% (v/v) TritonX-100 and 1 µL of a protease inhibitor cocktail (P9599, Sigma-Aldrich). The final volume of the reaction mixtures was 25 µL. The enzymatic reactions were stopped by adding Laemmli buffer and immediately stored at -20°C until SDS-PAGE and lectin-blot analysis.

Results

Multiple isoforms of the recombinant lectin domains are produced in N. benthamiana leaves

Recombinant lectin domains originating from either lectin receptor kinases (LecRK-I.5 and LecRK-I.9) or cell wall lectins (Lec-I.3 and Lec-I.6) were produced in transiently transformed N. benthamiana leaves. The four constructs contained the coding sequences of lectin domains including their original signal peptides targeting the recombinant proteins to the secretory pathway as well as sequences encoding V5 epitope and 6xHis tags. Total soluble proteins (TSP) were analysed by SDS-PAGE and immunoblotting (Fig. 1). Recombinant lectin domains exhibited significant molecular mass heterogeneity with multiple closely spaced bands separated from each other by about 2 kDa. The apparent molecular masses were between 40 and 30 kDa for LecRK-I.5 (4 bands), 55 and 35 kDa for LecRK-I.9 (6 bands), 40 and 35 kDa for Lec-I.3 (1 major plus 2 minor bands) and around 35 kDa for Lec-I.6 (1 thick plus 2 minor bands). The expected molecular masses were lower than those observed: 28.7, 32.4, 30.9 and 31.5 kDa for LecRK-I.5, LecRK-I.9, Lec-I.3 and Lec-I.6 respectively (Supplementary Fig. S1 and Fig. S2). This result suggests that the observed bands correspond to N-glycosylated forms of the lectin domains. Indeed, bioinformatics predictions indicate the presence of 4, 7, 3 and 3 N-glycosylation sites for LecRK-I.5, LecRK-I.9, Lec-I.3 and Lec-I.6 respectively (Supplementary Fig. S1 and Fig. S2). The Legume lectin of LecRKs thus appeared to be highly glycosylated with most of the N-glycosylation sites occupied by carbohydrates while cell wall lectins showed one prevailing glycoform.

Citrate buffers deplete RuBisCO from leaf protein extracts during a salting-in step

In order to clarify protein extracts by removing residual particulates while maintaining a high extraction efficiency of the recombinant lectins, we have developed a method using citrate buffers during a salting-in step, the flow chart of which is presented in Fig. 2. Once total soluble proteins (TSP) were precipitated by ammonium sulphate to 80% saturation (salting-out step), citrate buffers at different pH ranging from 3.0 to 6.0 and containing 0.15 M NaCl were added in order to solubilise proteins (salting-in step) from pellets. After centrifugation, proteins from supernatants and pellets were analysed by SDS-PAGE and immunoblotting (Fig. 3). Protein patterns revealed that RuBisCO was fully depleted from the supernatants obtained with citrate buffer at pH 5.0 or below (Fig. 3A) while the recombinant lectin domains of LecRK-I.9 and Lec-I.6 remained present (Fig. 3B andC). Similar patterns were obtained for the lectin domains of LecRK-I.5 and Lec-I.3 (not shown). Notably, all the isoforms of each lectin domain present in TSP were also detected in depleted protein extracts. many proteins other than RuBisCO were present in the pellets. Besides, two other proteins, namely a recombinant GFP (Green Fluorescent Protein) and native actin, were assessed and showed a distinct behaviour during the salting-in step (Fig. 3D andE): while soluble in citrate buffers at pH 4.5 to 6.0, GFP partly precipitated while actin completely precipitated at more acidic pH (3.0 and 4.0). From these results, it appears that the citrate buffer at pH 5.0 is the most effective in depleting RuBisCO and enriching the abundance of other proteins within the supernatants with little or no effect on their depletion. In fact, any citrate buffer below pH 5.0 might be selected if we consider only the selective extraction of lectin domains. However, a citrate buffer at pH 5.0 was chosen because the potential deleterious effects of a more acidic pH on protein structures and activities. In addition, this step significantly contributed in clarifying the protein samples as judged by the chlorophyll pigments present in the pellets.

RuBisCO depletion by citrate buffers is beyond a simple acidic precipitation

The selective extraction observed during the salting-in step apparently relied on the differential behaviour of proteins towards the pH of the added solutions. Citrate buffers depleted RuBisCO in the acidic range starting at pH 5.0 while lectin domains remained soluble even under highly acidic conditions (pH 3.0 -Fig. 3B andC). Citrate buffers were ineffective in precipitating RuBisCO at pH 6.0. To further examine this step, succinate and MES buffers at different pH ranging from 3.0 to 6.0 and containing 0.15 M NaCl were tested in a procedure identical to that used for the citrate buffers (Fig. 2). The resulting protein supernatants and pellets were analysed by SDS-PAGE and immunoblotting (Fig. 4). The succinate buffers precipitated RuBisCO at pH 5.0 or below (Fig. 4A) while the recombinant lectin domains of LecRK-I.9 remained present (Fig. 4B): the overall protein profiles were identical to those obtained with citrate buffers. In contrast, MES buffers failed to precipitate RuBisCO at pH 5.0 (Fig. 4C): RuBisCO was only depleted from supernatant of pH 3.0 and recovered as a precipitate in the corresponding pellet. MES buffers let lectin domains mainly soluble whatever the pH (Fig. 4D). These results suggest an acidic precipitation of RuBisCO under highly acidic conditions (pH 3.0) whatever the buffer used, and a distinctive effect of citrate and succinate in precipitating RuBisCO in mild acidic conditions.

RuBisCO was equally depleted from supernatants and precipitated in the pellets obtained from A. thaliana and B. distachyon leaves using citrate buffers during the salting-in step (Fig. 5). The most effective citrate buffer appeared to be at pH 4.5: the protein depletion was still a non-specific process with other proteins being precipitated together with RuBisCO.

The procedure thus seems reliable for other plants with a condition of pH adjustment.

Comparison of the procedure using citrate with those using polyelectrolyte precipitation and PEG fractionation

Several methods to enrich plant extracts in recombinant proteins by depleting RuBisCO have been previously established. The most common methods are based on either polyethyleneglycol (PEG) fractionation or polyelectrolyte precipitation using polyethyleneimine (PEI) and protamine sulphate (PS). We therefore applied them for comparison with the citrate procedure following the flow chart presented in Fig. 2.

PEI precipitation was conducted on TSP from N. benthamiana leaves obtained in phosphate buffer with a protein concentration of 1 mg.mL -1 . A nearly complete precipitation of RuBisCO and a maximum recovery in the pellet fractions were observed at concentrations of 0.1% and 0.2% (Fig. 6A). Increasing the PEI concentration actually decreased the amount of precipitated RuBisCO. Similarly to the citrate procedure, the process was non-specific since many other proteins were recovered in the pellets. All the isoforms of the lectin domain of LecRK-I.9 were found in equal amounts in the different supernatants (Fig. 6B). However, they were also present in the pellets thus leading to the loss of the protein of interest.

Using PS, a very efficient RuBisCO precipitation started at the lowest concentration of 0.02% (Fig. 6C). Although still efficient, higher concentrations of PS left some RuBisCO in the supernatants in the same way as PEI: a protein-polyelectrolyte complex with a stoichiometry below 1 is usually insoluble, while overdosed system might be soluble [START_REF] Romanini | Applications of calorimetric techniques in the formation of protein-polyelectrolytes complexes[END_REF]. Once again, protein removal using PS was non-specific. All the isoforms of the lectin domain of LecRK-I.9 were recovered at high yield in all supernatants in a way similar to the citrate procedure (Fig. 6D). PEG precipitated RuBisCO in a dose-dependent manner (Supplementary Fig. S3A).

However, as PEG concentration increased, numerous proteins pulled down together with RuBisCO in the pellets, and particularly all the isoforms of the lectin domain of LecRK-I.9

(Supplementary Fig. S3B).

Protein determination (Fig. 7) confirmed the protein profiles and indicated that 60 to 70% of TSP were removed using succinate and citrate buffers. Such a protein depletion was comparable to that obtained with the most efficient concentrations of PEI and PS.

Purified recombinant lectin domains from N. benthamiana are N-glycosylated

Purification of the recombinant lectin domains started with 2 g of agro-infiltrated N.

benthamiana leaves. After the selective extraction of the lectin domains, using citrate buffer pH 5.0 during a salting-in step, three consecutive chromatographic steps were performed for their purification (see Materials and Methods). In the course of establishing the purification process, cation exchange and hydrophobic interaction chromatographies were excluded as well as Concanavalin A (ConA) affinity chromatography. In the former cases, the recombinant lectin domains did not bind efficiently to the columns. In the latter case, leaking of ConA, a member of the Legume lectin family, heavily contaminated the eluted fractions.

This was a major drawback for the biochemical characterisation of the recombinant Legume lectin domains. Of the four lectin domains to be purified, three of them bound to the Ni-NTA support consistently with the presence of an efficient 6xHis tag at the C-terminus of the proteins (LecRK-I.9, LecRK-I.5, Lec-I.3). However, the recombinant lectin domain of Lec-I.6

did not bind to the Ni-NTA column. The eluted fractions were analysed by SDS-PAGE.

After immunoblotting using an antibody against the V5 epitope, the purified recombinant lectin domains appeared as groups of bands (Fig. 8A) with patterns very similar to those obtained from TSP (Fig. 1) indicating an analogous behaviour of the different isoforms during the purification process. After Coomassie colloidal blue staining, the purified lectin domains appeared as the major bands of the eluted fractions with no visible or only a few protein contaminants. Only the fainter bands detected with the anti-V5 antibody were not stained by Coomassie colloidal blue (Fig. 8A). The recombinant lectin content in the eluted fraction related to LecRK-I.5 amounted to 14 µg.g -1 fresh mass, corresponding to 0.26% of TSP. For the recombinant lectin domains of LecRK-I.9 and Lec-I.3 similar purification yields were achieved (17 and 21 µg.g -1 fresh mass, corresponding to 0.28 and 0.37% of TSP, respectively).

The glycosylation status of the recombinant lectin domains was first examined using 8B, the enzymatic digestions of the recombinant lectin domains caused a decrease in the molecular mass of all them. The molecular masses of the deglycosylated lectin domains were around 30 kDa as expected for their peptide chains (Supplementary Fig. S1 and Fig. S2). The elimination of the carbohydrates from the lectin domains was very similar regardless the type of enzyme, suggesting that the complex-class of N-glycans was not present.

The glycosylation status of the lectin domains was further examined using lectin-blots revealed with the digoxigenin (DIG)-labelled lectins GNA (Galanthus nivalis agglutinin) and DSA (Datura stramonium agglutinin) suitable to identify different types of N-glycans (see Material and Methods). For LecRK-I.5::V5::6xHis, the two major bands were detected by both GNA and DSA (Fig. 8). A third band, detected by Coomassie colloidal blue staining and the anti-V5 antibody, was not revealed in the lectin blot experiment, probably due to a lower amount of protein or to a N-glycan unrecognised by GNA and DSA. For LecRK-I.9::V5::6xHis, more complex patterns were observed: the three upper bands were detected by both GNA and DSA, the fifth band was detected only by GNA while the two others were not detected. Finally, for Lec-I.3::V5::6xHis, the two bands were detected by both GNA and DSA. Taken together, the results indicate that the recombinant lectin domains produced in N.

benthamiana leaves were N-glycosylated.

Finally, the recombinant lectin domain of LecRK-I.9 was obtained after stable genetic transformation of A. thaliana using the same construct as for transient transformation of N.

benthamiana. After RuBisCO depletion of TSP, SDS-PAGE and immunoblotting, recombinant lectin domains produced in A. thaliana exhibited similar features to that produced in N. benthamiana, i.e. molecular mass heterogeneity with multiple closely spaced bands ranging from 55 to 40 kDa (Supplementary Fig. S4). After enzymatic deglycosylation with PNGaseF, the molecular mass of the lectin domains was around 30 kDa, thus showing the presence of N-glycans.

Discussion

In the present study, we present a simple and robust procedure to purify recombinant Legume lectin domains from transiently transformed N. benthamiana leaves. We found that the depletion of the high-abundant protein RuBisCO from protein extracts can be obtained by differential precipitation using citrate buffers. This allowed improving the efficiency of further chromatographic steps and the recovery of multiple N-glycosylation isoforms of lectin domains in the purified fractions.

Protein precipitation relies on the disruption of protein hydration shell that forms around proteins and which modifies the repulsive electrostatic forces between proteins to favour their aggregation. The most common contributing parameters include pH, ionic strength, identity of buffer species and additives, most of them being linked to each other. In particular, citrate buffers were considered in studies aiming at deciphering aggregation mechanisms and protein-protein interactions for immunoglobulins [START_REF] Barnett | Specific-ion effects on the aggregation mechanisms and protein-protein interactions for anti-streptavidin immunoglobulin gamma-1[END_REF][START_REF] Roberts | Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody[END_REF]. At low citrate concentrations (below 50 mM) and mild acidic pH, the electrostatic repulsions between proteins were shown to be reduced because of the accumulation of citrate ions at the protein surface. Interestingly, the effectiveness of the buffer at reducing the repulsion forces in solutions at pH 5.0 is the highest for citrate buffers, followed by succinate buffers [START_REF] Roberts | Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody[END_REF]. In addition, in the process of precipitating protein to form crystals, di and tri-carboxylic acids play a dual role as buffer components and protein cross-linking agents [START_REF] Mcpherson | The role of small molecule additives and chemical modification in protein crystallization[END_REF]). In our conditions, i.e. a salting-in step after ammonium sulphate precipitation, RuBisCO could preferentially bind citrate or succinate, thus leading to protein aggregates which were removed by centrifugation. Finally, the overall procedure including the chromatographic steps provided purification yields in the range of those frequently described in the literature, i.e. between 0.1% and 1% of TSP [START_REF] Lombardi | Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation[END_REF]Nishimoto et al. 2014). High-level transient protein expression in N.

benthamiana leaves was obtained by constructing hydrophobin fusion proteins with the exceptional yield of 51% of TSP [START_REF] Joensuu | Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana[END_REF]. Recombinant proteins were targeted to the ER in which they accumulated into protein bodies. However, glycoproteins to be localised at the plasma membrane or in the cell wall require going through the secretory pathway in order to have fully processed N-glycans. Consequently, a proper N-glycosylation is not compatible with hydrophobin fusion constructs.

Glycosylation is a very common post-translational modification for plant receptors and cell wall proteins [START_REF] Song | N-Glycan occupancy of Arabidopsis N-glycoproteins[END_REF][START_REF] Canut | Post-translational modifications of plant cell wall proteins and peptides: a survey from a proteomics point of view[END_REF]. Glycans contribute to protein folding and function, and take part in many physiological processes [START_REF] Strasser | Biological significance of complex N-glycans in plants and their impact on plant physiology[END_REF]): thus, the activity of receptors and cell wall proteins may be dependent on their glycosylation status [START_REF] Häweker | Pattern recognition receptors require N-glycosylation to mediate plant immunity[END_REF][START_REF] Yamamoto | Site-specific N-glycosylation of the S-locus receptor kinase and its role in the self-incompatibility response of the brassicaceae[END_REF]Méndez-Yaňez et al. 2017). In particular, proteins harbouring a N-terminal signal peptide will enter the sequential biosynthesis of N-glycans within the endomembrane system. The lectin domains of the two receptors LecRK-I.5 and LecRK-I.9 but also that of the cell wall protein Lec-I.3 display several predicted Nglycosylation sites. Our results indicate that most of the predicted N-glycosylation sites were occupied by carbohydrates as revealed by (i) the presence of multiple glycoforms, (ii) the decrease in molecular mass of the lectin domains after EndoH and PNGaseF digestions and

(iii) the recognition by DSA and GNA which are specific for different N-glycan structures.

The enzymatic deglycosylation indicated that the complex-class of N-glycans is not present within the recombinant lectin domains. DSA preferentially binds to tri-or higher-branched Nglycans containing the disaccharide GalGlcNAc with an unsubstituted Gal [START_REF] Nishimoto | Datura stramonium agglutinin: cloning, molecular characterization and recombinant production in Arabidopsis thaliana[END_REF]: for instance, the GalGlcNAcMan 7 GlcNAc 2 N-glycan structure belonging to the hybridclass is a possible target. GNA strongly interacts with the high Man-class of N-glycans and preferentially recognition of terminal Man residues, (1-3), (1-6) or (1-2) linked to Man [START_REF] Fouquaert | Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity[END_REF]) including the Man 5-9 GlcNAc 2 structures. For LecRK-I.9, six isoforms of the recombinant lectin domain were detected in a purified fraction. Starting from the lowest molecular mass isoform, an incremental increase of about 2 kDa separated the five other bands suggesting that the lectin domain could be decorated by either only one or up to six Nglycan moieties. However, we cannot rule out the possibility that some N-glycosylation sites are occupied by low molecular mass N-glycans such as the GlcNAc-only class [START_REF] Zeng | N-linked glycan micro-heterogeneity in glycoproteins of Arabidopsis[END_REF]. A similar conclusion can be drawn for the recombinant lectin domains of LecRK-I.5

and Lec-I.3.

Heterogeneity within glycoforms of the recombinant lectin domains could also result from differences in the structure of the N-glycan moieties. Indeed, a number of bands were revealed both by DSA and GNA meaning that the N-glycan hybrid-class with terminal unsubstituted Gal and high Man-class could be present on the same lectin domain. This applied particularly for the two major isoforms of LecRK-I.5 and Lec-I.3. The example of LecRK-I.9 was even more intricate. The three first isoforms were revealed by both DSA and GNA. The fifth isoform was only detected by GNA meaning that (i) it did not contain the complex-class of N-glycans harbouring Gal such as the Lewis epitope, and (ii) the high Manclass was present, without excluding other structures. Finally, the fourth isoform was labelled neither by DSA nor by GNA: the presence of a structure belonging to the hybrid-class with a substituted Gal could be assumed. Recently, a comprehensive analysis of N-glycopeptides using advanced mass spectrometry has revealed the N-glycan micro-heterogeneity present in A. thaliana N-glycoproteins [START_REF] Zeng | N-linked glycan micro-heterogeneity in glycoproteins of Arabidopsis[END_REF] 3)Fuc( 1)Pent(1), HexNAc(2)Hex( 4)Pent(1) and HexNAc(3)Hex(3)Pent(1).

Our study also revealed that the recombinant lectin domains of LecRK-I.9 produced in A.

thaliana were highly N-glycosylated. There is still the question of whether this high glycosylation status for the recombinant lectin domains is of biological relevance. It will also be necessary to demonstrate the presence of N-glycans in the native proteins.

Our work also noted a marked difference between receptors and cell wall proteins in the number of their lectin domains glycoforms (Fig. 1). Furthermore, using bioinformatic prediction, more N-glycosites were detected in the Legume lectin domains of A. thaliana and Medicago truncatula receptors (Supplementary Fig. S5). That pointed out, respectively, an average of 5.5 and 5.6 predicted N-glycosites for receptors, and an average of 2.5 and 2.1 for cell wall proteins. Plant receptor-like kinases were already noted as heavily N-glycosylated proteins [START_REF] Schoberer | Plant glyco-biotechnology[END_REF] but the biological function of the complex N-glycans is largely unexplored. The example of the S-locus receptor kinase involved in the selfincompatibility response of the Brassicaceae indicated that N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking to the plasma membrane through the ER quality control machinery [START_REF] Yamamoto | Site-specific N-glycosylation of the S-locus receptor kinase and its role in the self-incompatibility response of the brassicaceae[END_REF]. The two closely related pattern-recognition receptors EFR and FLS2 were distinguished by a differential requirement for ER quality control components that could be linked to N-glycosylation (e.g. position, number of glycosylated sites, glycan structures) [START_REF] Nekrasov | Control of the pattern-recognition receptor EFR by an ER protein complex in plant immunity[END_REF]. In particular, a single N-glycosite plays a critical role for EFR abundance and ligand recognition [START_REF] Häweker | Pattern recognition receptors require N-glycosylation to mediate plant immunity[END_REF]. Noteworthy, in mammals, the number of N-glycans per protein and the branching of their structures provided mechanisms to regulate developmental processes [START_REF] Lau | Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation[END_REF][START_REF] Hang | N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4[END_REF][START_REF] Medina-Cano | High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect[END_REF][START_REF] Chandler | N-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2)[END_REF]. It is then possible that the lectin domains being part of receptor-like kinases have evolved to a high number of Nglycosites in order to ensure regulatory mechanisms through the ER quality control machinery.

In summary, our present results provide a purification scheme for the multiple Nglycoforms of recombinant Legume lectin domains produced by N. benthamiana leaves.

Through depletion of the high-abundant protein RuBisCO from protein samples, the citrate buffer served as an effective non-chromatographic step for the initial fractionation and clarification of protein extracts. The next two chromatographic steps performed in the negative mode were dedicated to the enrichment in lectin domains before a Ni-NTA final affinity chromatography using the 6xHis tag: the multiple N-glycosylation lectins have the same chromatographic behaviour and were recovered as proteins purified near to homogeneity. The procedure can be readily adapted to other plant species. Despite being designed for the purification of recombinant proteins, the procedure is fully compatible with proteomic studies. It reveals the high degree of heterogeneity in the N-glycosylation patterns of the Legume lectin domains, and particularly of those of LecRKs. We believe that our procedure holds significant promise to reveal the functional characterization of A. thaliana

Legume lectins in cell signalling and cell wall organisation.

Fig. 1 .

 1 Fig. 1. Immunoblotting analysis of recombinant lectin domains from total protein extracts of N. benthamiana leaves after transient expression. (A) SDS-PAGE analysis of a total soluble protein extract (TSP) from leaves transformed with the LecRK-I.9::V5::6xHis construct as an example, followed by staining with Coomassie colloidal blue: the abundant RuBisCO large (LSU) and small (SSU) subunits are marked by black arrowheads; (B) Immunoblots to reveal the recombinant lectin domains (red arrowheads) of LecRK-I.5, LecRK-I.9, Lec-I.3 and Lec-I.6 using anti-V5 antibody.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Flow chart describing the procedures for the depletion of RuBisCO from plant leaf protein extracts. TSP: total soluble proteins. PEI: polyethyleneimine. PS: protamine sulphate. PEG: polyethylene glycol.

Fig. 4 .

 4 Fig. 4. Effect of succinate and MES buffers on RuBisCO precipitation and recombinant lectin domain recovery. Total soluble proteins of N. benthamiana leaves after transient transformation for the production of recombinant lectin domains of LecRK-I.9: after ammonium sulphate precipitation, protein precipitates were re-suspended in succinate (A) and MES buffers (C) of different pH ranging from 3.0 to 6.0, or in phosphate buffer pH 7.4 as a reference (*). After centrifugation, proteins from supernatants and pellets were analysed by SDS-PAGE and stained with Coomassie colloidal blue. Abundant RuBisCO large (LSU) and small (SSU) subunits are marked by black arrowheads. Immunoblots (B) and (D) corresponding to (A) and (C) were revealed with anti-V5 antibody to detect recombinant lectin domains of LecRK-I.9. Details are given in the experimental section.

Fig. 5 .

 5 Fig. 5. Effect of citrate buffers on RuBisCO precipitation in leaf extracts of A. thaliana and B.distachyon. After ammonium sulphate precipitation, protein precipitates were re-suspended in citrate buffers of different pH ranging from 4.0 to 6.0, or in phosphate buffer pH 7.4 as reference (*). After centrifugation, proteins from supernatants and pellets were analysed by SDS-PAGE and stained with Coomassie colloidal blue. TSP, total soluble proteins. LSU, RuBisCO large subunit.

Fig. 6 .

 6 Fig. 6. Effect of polyethyleneimine (PEI) and protamine sulphate (PS) on RuBisCO precipitation and recombinant lectin domain recovery. (A) Total soluble extracts of N. benthamiana leaves after transient transformation for the production of recombinant lectin domains of lecRK-I.9 were subjected to different concentrations of PEI ranging from 0.1% to 0.5%. After centrifugation, proteins from supernatants and pellets were analysed by SDS-PAGE and stained by Coomassie colloidal blue. Abundant RuBisCO large (LSU) and small (SSU) subunits are marked by black arrowheads. (B) Immunoblots corresponding to (A) were revealed to detect recombinant lectin domains of LecRK-I.9 with anti-V5 antibody. (C) Total soluble extracts were subjected to different concentrations of PS ranging from 0.02% to 0.2%. (D) Immunoblots corresponding to (C) were revealed with anti-V5 antibody to detect recombinant lectin domains of LecRK-I.9.

the

  EndoH and PNGaseF glycosidases. EndoH cleaves the chitobiose core of N-linked oligosaccharides belonging to the high-mannose-class or the hybrid-class of N-glycans. It does not cleave the complex-class of N-glycans. PNGaseF cleaves all N-linked oligosaccharides, except when an α1-3 Fuc is present on the core GlcNAc. As shown in Fig.

Fig. 8 .

 8 Fig. 8. N-glycosylation analysis of the purified recombinant lectin domains. After RuBisCO depletion of protein extracts of transformed N. benthamiana leaves, the recombinant lectin domains of LecRK-I.9, LecRK-I.5 and Lec-I.3 were purified by immobilised metal affinity chromatography. (A) The three purified recombinant lectin domains were detected by (i) immunoblotting using anti-V5 antibody (lanes V5), (ii) SDS-PAGE followed by Coomassie colloidal blue staining (lanes C), (iii) lectin-blotting using Galanthus nivalis agglutinin (lanes GNA), (iv) lectin-blotting using Datura stramonium agglutinin (lanes DSA). Stars indicate isoforms of the different recombinant lectin domains, open circles isoforms that were not detected by Coomassie colloidal blue staining, dots contaminating proteins and dashes isoforms revealed by agglutinins. (B) Enzymatic deglycosylation of the three recombinant lectin domains by Peptide-N-Glycosidase F (lane PNGaseF) or Endoglycosidase H (lane EndoH). Stars indicate isoforms of the different lectin domains in the control lanes (without enzyme). Dashes indicate the remaining high molecular mass bands for Lec-I.3 after enzymatic digestions.

Fig. S4 .

 S4 Fig. S4. Deglycosylation of the partially purified recombinant lectin domains of LecRK-I.9 produced in A. thaliana. After RuBisCO depletion of protein extracts from transformed A. thaliana plants, the recombinant lectin domains of LecRK-I.9 were deglycosylated using Peptide-N-Glycosidase F (lane PNGaseF) or Endoglycosidase H (lane EndoH). Stars indicate isoforms of the LecRK-I.9 lectin domains in the control lane (without enzyme). Dashes indicate remaining high molecular mass bands after enzymatic digestions.

. Protein depletion from N. benthamiana extracts. Several

	of leaves)
	Protein recovery (mg/g
	1	0.2	0.3	0.4	0.5	7.4	0.02	0.05	0.10	0.15	0.20	7.4	5	10	15	20	7.4
	Fig. 7methods were tested for protein
	removal: 10 mM citrate, succinate or MES solutions at different pHs; polyethyleneimine (PEI),
	protamine sulphate (PS) or polyethyleneglycol (PEG) solutions in phosphate buffer (pH 7.4) ranging
	from concentrations of 0.1% to 0.5%, 0.02% to 0.2% and 5% to 20% respectively. Protein contents
	were determined on supernatants obtained after treatments. Extracts obtained in phosphate buffer pH
	7.4 without additives (orange columns) were used as reference (100) to indicate yields in selected
	columns. Errors bars indicate standard deviation (n=4).

72 55

Acknowledgements

The authors are thankful to the Paul Sabatier-Toulouse III University (France) and CNRS for supporting their research work. KB has been granted by the Paul Sabatier-Toulouse III University for his PhD work. AL and LL have been supported by the ANR grant WALLARRAY (ANR-2010-Genom-BTV-010).This work was also supported by the ANR French Laboratory of Excellence project entitled "TULIP" (ANR-10-LABX-41; ANR-11-IDEX-0002-02).

Abbreviations

DSA, Datura stramonium agglutinin ; DIG, digoxigenin ; eGFP, enhanced green-fluorescent protein; GNA, Galanthus nivalis agglutinin; LB, Luria-Bertani medium; TSP, total soluble proteins; PEI, polyethyleneimine; PEG, polyethyleneglycol; PS, protamine sulphate; RuBisCO, ribulose biphosphate carboxylase/oxygenase.

Legends for supplementary material:

Fig. S1. The lectin domains of LecRK-I.9 and LecRK-I.5 are potentially highly glycosylated. ---------------------------------------------- -----------------------------------------------A -LecRK-I.9::V5::6xHis ------------------------------------------------ -------------------------------------------------Fig. S1. The lectin domains of LecRK-I.9 and LecRK-I.5 are potentially highly glycosylated. Amino acid sequences of the recombinant lectin domain of LecRK-I.9 (A) and LecRK-I.5 (B) showing: predicted signal peptides (1-21 and 1-22), predicted N-glycosylation sites (Asn-Xaa-Ser/Thr sequons in the sequence are highlighted in blue; Asn residues predicted to be N-glycosylated are highlighted in red), the V5 epitope and the 6xHis tag (red italics). The results for the prediction of Nglycosylation sites in the recombinant lectin domains were performed using the NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/). The sequons that are predicted to be N-glycosylated are highlighted in yellow boxes (threshold 0.5).

B -

Position Potential

Jury N-Glyc agreement result ------------------------------------------------- ------------------------------------------------ -------------------------------------------------- ------------------------------------------------- and1-19), predicted N-glycosylation sites (Asn-Xaa-Ser/Thr sequons in the sequence are highlighted in blue; Asn residues predicted to be N-glycosylated are highlighted in red), the V5 epitope and the 6xHis tag (red italics). The results for the prediction of N-glycosylation sites in the recombinant lectin domains were performed using the NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/). All the sequons are predicted to be N-glycosylated and highlighted in yellow boxes (threshold 0.5).


D Wei

Antoine Bruneau-Voisine

M Dubois

Stéphanie Bastin

Jean-Baptiste Sortais

Manganese-Catalyzed Transfer Hydrogenation of Aldimines

Keywords: manganese, imines, reduction, transfer hydrogenation, amines

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Manganese-catalyzed transfer hydrogenation of aldimines

Duo Wei, [a,b]# Antoine Bruneau-Voisine, [a,b]# Maxime Dubois, [b] Stéphanie Bastin [b] and Jean-Baptiste Sortais* [b,c] Abstract: The reduction of imines to amines via transfer hydrogenation was achieved promoted by phosphine-free manganese(I) catalyst. Using isopropanol as reductant, in the presence of tBuOK (4 mol%) and manganese complex [Mn(CO)3Br( 2 N,N-PyCH2NH2)] (2 mol%), a large variety of aldimines (30 examples) were typically reduced in 3 hours at 80 °C with good to excellent yield.

The development of catalysts based on earth abundant 3d transition metals, as sustainable and economical alternative to precious transition metals, is one of the current challenge in the field of homogeneous catalysis. [1] In the case of reduction reactions, [2] despite its natural abundance, the potential of manganese [3] was disregarded until recently compared to iron, [4] cobalt, [5] and nickel. [6] Indeed, the first efficient manganese based catalyst for hydrogenation of polar bonds was reported latterly, in 2016, by the group of Beller. [7] Since then, hydrogenation catalysts were developed for the reduction of carbonyl derivatives, [8] nitriles, [9] esters, [10] amides, [11] carbonates, [12] and CO2. [13] To our knowledge, only two examples of hydrogenation of imines are reported, one with a manganese nano-sheet [14] and one with an aminophosphinepyridine manganese catalyst developed by our group. [15] As an alternative to molecular dihydrogen (or hydrosilanes [16] ) as reductants, secondary alcohols, typically isopropanol, are convenient source of hydrogen. [17] Advantages of transfer hydrogenation over hydrogenation are that reduction reactions can be performed in conventional equipments, i.e. without requiring the use of high-pressure autoclaves, and that alcohols are usually benign chemicals, safer to handle than dihydrogen. Compared to hydrogenation, only a few catalytic systems based on manganese were developed for transfer hydrogenation reactions, including the asymmetric version (Scheme 1). [8f, 18] The first system was developed by the group of Beller, with a tridendate ligand, namely dipicolamine, for the reduction of ketones. [18a] Zirakzadeh and Kirchner reported the application of a chiral catalyst supported by a tridendate PNP ligand bearing a ferrocenyl moiety for the enantioselective production of alcohols. [18b] Similar catalytic system, developed by Clarke, performed the reduction of ketones in the presence of alcohols, but required hydrogen pressure to exhibit enantioselectivity. [8f] More recently, the groups of Leitner [18c] and Morris, [18d] reported respectively an aminotriazole and a chiral tridentate manganese catalyst efficient in transfer hydrogenation reactions. Our group demonstrated that simple manganese complex based on a bidendate aminomethylpyridine ligand could perform with high efficiency the reduction of ketones and aldehydes, even at room temperature. [18e] We also showed that the combination of [Mn(CO)5Br], as commercial manganese precursor, and a diamine ligand, for example ethylenediamine or a chiral diamine, namely (1R,2R)-N,N-dimethyl-1,2-diphenylethane-1,2-diamine, could promote the (asymmetric-) transfer hydrogen of carbonyl compound. [18f] So, to date, manganese-catalyzed transfer hydrogenation reactions were limited to the reduction of ketones and aldehydes. Furthermore, examples of reduction of imines via this methodology involving non-noble metals such as iron, [19] nickel, [20] and cobalt [21] , are quite scarce. Starting from this statement, and taking into account the importance of amines in organic synthesis, [START_REF] Lawrence | Amines: synthesis, properties and applications[END_REF] we found relevant to explore the manganesecatalyzed reduction of imines by hydrogen transfer [23] as a complementary method in the chemist tool box to hydrogenation, [START_REF] De Vries | The Handbook of Homogeneous Hydrogenation[END_REF] hydrosilylation [START_REF] Li | [END_REF] or hydrogen borrowing, [2b] for example.

Scheme 1. Manganese catalysts used in transfer hydrogenation. In line with our research program to develop manganese complexes for reduction reactions, [8d, 26] we have selected the well-defined Mn(I) complex 1 (Scheme 1) featuring an 2-(aminomethyl)-pyridine ligand for this study as it was the most efficient catalyst for the reduction of carbonyl derivatives. [18e] Besides, the synthesis of this complex is very straightforward, as 1 can be obtained in high yield in only one step from commercially available reactants. [18e] Table 1. Optimization of the parameters of the reduction of imine with catalyst 1. [a] Entry 16 [b] 2 tBuOK (4) 0.1 80 3 94

[a] Reaction conditions: Under inert atmosphere, a 20 mL Schlenk tube was filled sequentially with the imine a1 (0.5 mmol), anhydrous 2-propanol, complex 1, and the base. The reaction was heated in an oil bath. Conversion was determined by 1 H NMR spectroscopy [b] In the presence of Hg (300 equiv.).

The conditions of the reaction were optimized for the reduction of N-benzylidene-4-methylaniline a1 in 2-propanol as solvent (Table 1). At 80 °C, in the presence of tBuOK (2 mol%) and manganese complex 1 (1 mol%), the corresponding amine b1, namely Nbenzyl-4-methylaniline, was formed in 77% yield. Various bases were first screened (entries 1-6), showing that tBuOK and KHMDS gave the best results (77%, entries 1 and 3). Interestingly, common base KOH could also be used, albeit affording a slightly lower conversion than the former bases (65%, entry 4) The concentration of the reaction mixture is often crucial for the reduction of carbonyl derivatives, as transfer hydrogenation is a reversible reaction. [START_REF]The dehydrogenation of amine b1 to imine a1 was tested in acetone, at 80 °C[END_REF] Therefore, the influence of the concentration of a1 was examined ranging from 0.5 to 0.05 mol.L - 1 . An optimal concentration of 0.1 mol.L -1 (entries 1, 7-9) was identified. With 1 mol% catalyst, lengthen the reaction time (3 h) allowed to improve the conversion to 88% (entry 10). However, lowering the temperature had a detrimental effect on the -activity as the conversion dropped to 39% at 50 °C and to 7% at 30 °C (entries 11 and 12). A satisfying yield (93%) was obtained using 2 mol% of catalyst and 4 mol% of tBuOK at 80 °C after 3 h (entry 13). Finally, lowering the catalyst loading to 0.5 mol% resulted in a moderate TON of 120 (1, 3 h, 60% conversion, entry 14). It is worth noting that under the optimized conditions, but in the absence of manganese complex 1, no reaction occurred (entry 15, Table S2), demonstrating a metal-catalyzed reduction. [28] Additionally, the reaction proceeded in the presence of Hg (300 equiv.) without observing any significant changes in reactivity (Table 1, entry 16), suggesting a homogeneous catalytic system. Given the promising activity of catalyst 1 for the reduction of aldimine 1a, we then investigated its potential for the more challenging reduction of ketimines, with N-[1-phenylethylidene]aniline as a model subtrate (See Table S1). Even under harsch conditions (1 (5 mol%), tBuOK (10 mol%), 130 °C, 18 h), the corresponding amine was obtained in low yield (20%), demonstrating that this catalytic system is not suitable for the reduction of ketimines.

Having optimized the conditions for the reduction of aldimines (complex 1 (2 mol%), tBuOK (4 mol%), iPrOH (C = 0.1 mol.L-1), 80 °C, 3 h), we then explored the scope of this transformation (Table 2). In general, a large variety of aldimines, prepared by the condensation of benzaldehyde with aniline derivatives, was reduced in high yield. Steric hindrance has an influence as increasing the number of ortho substituents on both the benzaldehyde (a3, a4) or the aniline (a5, a6) moieties, from one to two, led to the sterically hindered amines b4 and b6 in lower yield than b3 and b5. The steric hindrance on the aniline moieties has a more drastic influence than on the benzaldehyde part (b4 vs b7). Halogen substituents (F, Cl, Br, I) were well tolerated (b8-b11, isolated yield > 90%), and no product resulting from dehalogenation was detected. Interestingly, heteroaromatic imines (a12-a15) affording N-substitutedaminomethylheterocycles (b12-b15) were also surprisingly reduced without significant loss of activity. It should be noticed that the structure of the reaction products are very similar to the one of the ligand used to support the manganese center, namely 2-(aminomethyl)-pyridine. Under the conditions of the catalysis, a dynamic exchange of ligands can not be ruled out. Since diverse diamines proved to be active ligands in transfer hydrogenation of ketones, such an exchange may not alter the reduction. [18f] Likewise, organometallic 4-methyl-N-(ferrocenylmethylidene)aniline a16 was totally reduced and led to the corresponding amine b16 in excellent isolated yield (88%). The tolerance toward various functional groups such as amino (a17), methoxy (a18), cyano (a19), nitro (a20), ester (a21), amido (a22), acetal (a23), vinyl (a24) and alkynyl (a25) groups was tested. Table 1. Generality of the reduction of aldimines catalyzed by complex 1. [a] [a] ] Reaction conditions: Under inert atmosphere, a 20 mL Schlenk tube was filled sequentially with the imine (0.5 mmol), anhydrous 2-propanol (5 mL), complex 1 (3.3 mg, 2 mol%), and tBuOK (2.2 mg, 4 mol%). The reaction was heated in an oil bath at 80 °C for 3 h. Conversion was determined by 1 H NMR spectroscopy. Isolated yield in parenthesis. [b] Isolated as mixture containing 20% of saturated amine. [c] complex 1 (6.6 mg, 4 mol%), tBuOK (4.4 mg, 8 mol%) Except for the nitro-substituted imine (a20), [START_REF]Competitve experiments for the reduction of model imine 1a in the presence of p-nitrotoluene, N,N-dimethylaniline and Nmethylbenzylamine[END_REF] all the functional groups did not affect the catalytic system and remained intact after the reduction. In the case of the conjugated imine (a26), the unsaturated amine b26 was obtained as major product (80%) along with 20% of the corresponding saturated amine. The unability of our catalytic system to reduce ketimines allowed the selective reduction of substrates displaying both ketimine and aldimine moieties. Gratifyingly, for the diimine a27 derived from 4formylacetophenone, only the aldimine moiety was reduced affording the corresponding amino-ketimine b27. Finally, imines bearing aliphatic substituents were difficult to convert to the desired amines (b28-b31, yield 17%-38%).

In conclusion, we reported the first application of Mn-based catalyst in transfer hydrogenation of imines. We have shown that manganese (I) pre-catalyst bearing a 2-aminomethylpyridine ligand catalyzes efficiently the transfer hydrogenation of aldimines in the presence of iPrOH as reductant under mild conditions (2 mol% catalyst, 4 mol% base, 80 °C, 3 to 18 h). The catalytic process displayied a high tolerance towards a large variety of functional groups. This protocol enlarges the scope of reactions catalyzed by manganese, highlighting the rising potential of this transition metal in homogeneous catalysis.

Experimental Section

Typical catalytic reduction. A 20 mL Schlenk tube was charged with imine (0.5 mmol), anhydrous isopropanol (5.0 mL), manganese complex 1 (3.3 mg, 2 mol%), and tBuOK (2.2 mg, 4 mol%) under argon, in that order. Then the mixture was stirred at 80 o C in an oil bath for 3 h. After cooling to room temperature, the solution was diluted with ethyl acetate (2.0 mL) and filtered through a small pad of celite (2 cm in a Pasteur pipette). The celite was washed with ethyl acetate (2×2.0 mL). The filtrate was evaporated and the crude residue was purified by column chromatography (SiO2, mixture of petroleum ether/ethyl acetate as eluent) to afford the desired product.

  , Dr. A. Bruneau-Voisine, M. Dubois Dr. S. Bastin and Prof. Dr. J.-B. Sortais LCC-CNRS, Universite´ de Toulouse, CNRS, UPS, Toulouse, France E-mail: jean-baptiste.sortais@lcc-toulouse.fr [c] Prof. Dr. J.-B. Sortais Institut Universitaire de France 1 rue Descartes, F-75231 Paris Cedex 05, France # Equal contributionSupporting information for this article is given via a link at the end of the document.

  reductant, in the presence of tBuOK and manganese complex [Mn(CO)3Br( 2 N,N-PyCH2NH2)], a large variety of aldimines (30 examples) were reduced to the corresponding amines with good to excellent yield.

Acknowledgements

We thank the Centre National de la Recherche Scientifique (CNRS), the Université de Rennes 1, the Université Toulouse III, Paul Sabatier, the Institut Universitaire de France (IUF) and the Agence National de la Recherche (ANR agency, program JCJC ANR-15-CE07-0001 "Ferracycles").