Plant N-glycans Research Articles

Nutrient supplementation-induced metabolic profile changes and early appearance of free N-glycans in nutrient deficient tomato plants revealed by mass spectrometry.

Inorganic fertilizers are routinely used in large scale crop production for the supplementation of nitrogen, phosphorus, and potassium in nutrient poor soil. To explore metabolic changes in tomato plants grown on humic sand under different nutritional conditions, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was utilized for the analysis of xylem sap. Variations in the abundances of metabolites and oligosaccharides, including free N-glycans (FNGs), were determined. Statistical analysis of the sample-related peaks revealed significant differences in the abundance ratios of multiple metabolites, including oligosaccharides, between the control plants, grown with no fertilizers, and plants raised under "ideal" and "nitrogen deficient" nutritional conditions, i.e., under the three treatment types. Among the 36 spectral features tentatively identified as oligosaccharides, the potential molecular structures for 18 species were predicted based on their accurate masses and isotope distribution patterns. To find the spectral features that account for most of the differences between the spectra corresponding to the three different treatments, multivariate statistical analysis was carried out by orthogonal partial least squares-discriminant analysis (OPLS-DA). They included both FNGs and non-FNG compounds that can be considered as early indicators of nutrient deficiency. Our results reveal that the potential nutrient deficiency indicators can be expanded to other metabolites beyond FNGs. The m/z values for 20 spectral features with the highest variable influence on projection (VIP) scores were ranked in the order of their influence on the statistical model.

α-Cyano-3-aminocinnamic acid: A novel reactive matrix for qualitative and quantitative analysis of plant N-glycans by MALDI-MS

N-glycans have a diversity of crucial biological roles in organisms. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an indispensable analytical instrument for biomolecules. However, due to the inherent low abundance, high structural heterogeneity, and poor ionization efficiency of N-glycans, as well as the extremely inhomogeneous co-crystal property using traditional matrices, the qualitation and quantitation of N-glycans by MALDI-MS remains challenging. In the present study, α-cyano-3-aminocinnamic acid (3-CACA) was reasonably designed and synthesized as a novel reactive matrix for N-glycan analysis. Combining with traditional matrix α-cyano-4-hydroxycinnamic acid (CHCA) as an acidic catalyst, a combinational matrix 3-CACA/CHCA was obtained with homogeneous co-crystallization and high derivatization efficiency, achieving the sensitive qualitation with the limits of detection low to femtomole and reproducible quantitation with good linearity (R2 > 0.998). As a result, the established method was successfully applied to the on-target derivatization and high-throughput quantification of N-glycans in eight varieties of the peach complex system, indicating that N-glycan has the potential to become a new biomarker for food allergy, and elucidating the prospective correlation between N-glycan epitopes and allergic reactions.

Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana.

N-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N-glycan. Therefore, N-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine β1,4-galactosyltransferase (β1,4-GALT) to modify plant N-glycan with β1,4-linked Gal residue(s) and Arabidopsis thaliana β1,3-galactosylatransferase (β1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine β1,4-GALT in soil-grown N. benthamiana had 42.5% of N-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N-glycan profile analysis of IgG co-expressed with β1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary β1,4-galactosylated N-glycan structures.

Comparative analysis of plant transient expression vectors for targeted N-glycosylation.

While plant-based transient expression systems have demonstrated their potency to rapidly express economically feasible quantities of complex human proteins, less is known about their compatibility with posttranslational modification control. Here we investigated three commonly used transient expression vectors, pEAQ, magnICON and pTra for their capability to express a multi-component protein with controlled and modified N-glycosylation. Cetuximab (Cx), a therapeutic IgG1 monoclonal antibody, which carries next to the conserved Fc an additional N-glycosylation site (GS) in the Fab-domain, was used as model. While pEAQ and pTra produce fully assembled Cx at similar levels in N. benthamiana, the yield of magnICON-Cx was twice as high. When expressed in wild type plants, both Cx-GSs exhibited typical plant N-glycans decorated with plant-specific xylose and fucose. Likewise, Cx generated in the glycoengineered ΔXTFT line carried mainly complex N-glycans lacking plant specific residues. Exposure to different engineering settings (encompassing stable lines and transient approaches) towards human galactosylation and sialylation resulted in Cx carrying targeted N-glycans at similar quantities using all three expression vectors. Collectively, our results exhibit the universal application of plant-based glycoengineering, thereby increasing the attractivity of the ambitious expression platform.

Plant N-glycan breakdown by human gut Bacteroides

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

O-methylated N-glycans Distinguish Mosses from Vascular Plants.

In the animal kingdom, a stunning variety of N-glycan structures have emerged with phylogenetic specificities of various kinds. In the plant kingdom, however, N-glycosylation appears to be strictly conservative and uniform. From mosses to all kinds of gymno- and angiosperms, land plants mainly express structures with the common pentasaccharide core substituted with xylose, core α1,3-fucose, maybe terminal GlcNAc residues and Lewis A determinants. In contrast, green algae biosynthesise unique and unusual N-glycan structures with uncommon monosaccharides, a plethora of different structures and various kinds of O-methylation. Mosses, a group of plants that are separated by at least 400 million years of evolution from vascular plants, have hitherto been seen as harbouring an N-glycosylation machinery identical to that of vascular plants. To challenge this view, we analysed the N-glycomes of several moss species using MALDI-TOF/TOF, PGC-MS/MS and GC-MS. While all species contained the plant-typical heptasaccharide with no, one or two terminal GlcNAc residues (MMXF, MGnXF and GnGnXF, respectively), many species exhibited MS signals with 14.02 Da increments as characteristic for O-methylation. Throughout all analysed moss N-glycans, the level of methylation differed strongly even within the same family. In some species, methylated glycans dominated, while others had no methylation at all. GC-MS revealed the main glycan from Funaria hygrometrica to contain 2,6-O-methylated terminal mannose. Some mosses additionally presented very large, likewise methylated complex-type N-glycans. This first finding of the methylation of N-glycans in land plants mirrors the presumable phylogenetic relation of mosses to green algae, where the O-methylation of mannose and many other monosaccharides is a common trait.

Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins.

Plants are excellent production hosts for the in vivo synthesis of complex glycosylated proteins such as antibodies. The plant N-glycosylation machinery is largely similar to that found in humans and other mammalian organisms, which is an advantage in comparison to microbial production systems in particular. However, there are some differences in the identity and chemical linkage of the sugars that plants and mammals use to build their N-glycans. These differences can affect important properties of glycosylated proteins produced recombinantly in plants. Here we describe the complete procedure of multiplex targeted gene knockout with CRISPR/Cas9 in Nicotiana benthamiana in order to eliminate the undesirable sugars α-1,3-fucose and β-1,2-xylose from the plant N-glycans. The workflow includes target gene identification, guide RNA design and testing, plant transformation, and the analysis of the regenerated transgenic plants by Sanger sequencing, immunoblot, and mass-spectrometric analysis of recombinant and endogenous proteins.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

β-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins.

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

Lewis A Glycans Are Present on Proteins Involved in Cell Wall Biosynthesis and Appear Evolutionarily Conserved Among Natural Arabidopsis thaliana Accessions

N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species: Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.

Transcriptomic Analysis of Dark-Induced Senescence in Bermudagrass (Cynodon dactylon).

Leaf senescence induced by prolonged light deficiency is inevitable whenever turfgrass is cultivated in forests, and this negatively influences the survival and aesthetic quality of the turfgrass. However, the mechanism underlying dark-induced senescence in turfgrass remained obscure. In this study, RNA sequencing was performed to analyze how genes were regulated in response to dark-induced leaf senescence in bermudagrass. A total of 159,207 unigenes were obtained with a mean length of 948 bp. The differential expression analysis showed that a total of 59,062 genes, including 52,382 up-regulated genes and 6680 down-regulated genes were found to be differentially expressed between control leaves and senescent leaves induced by darkness. Subsequent bioinformatics analysis showed that these differentially expressed genes (DEGs) were mainly related to plant hormone (ethylene, abscisic acid, jasmonic acid, auxin, cytokinin, gibberellin, and brassinosteroid) signal transduction, N-glycan biosynthesis, and protein processing in the endoplasmic reticulum. In addition, transcription factors, such as WRKY, NAC, HSF, and bHLH families were also responsive to dark-induced leaf senescence in bermudagrass. Finally, qRT-PCR analysis of six randomly selected DEGs validated the accuracy of sequencing results. Taken together, our results provide basic information of how genes respond to darkness, and contribute to the understanding of comprehensive mechanisms of dark-induced leaf senescence in turfgrass.

N-glycans of the microalga Chlorella vulgaris are of the oligomannosidic type but highly methylated

Microalgae of the genus Chlorella vulgaris are candidates for the production of lipids for biofuel production. Besides that, Chlorella vulgaris is marketed as protein and vitamin rich food additive. Its potential as a novel expression system for recombinant proteins inspired us to study its asparagine-linked oligosaccharides (N-glycans) by mass spectrometry, chromatography and gas chromatography. Oligomannosidic N-glycans with up to nine mannoses were the structures found in culture collection strains as well as several commercial products. These glycans co-eluted with plant N-glycans in the highly shape selective porous graphitic carbon chromatography. Thus, Chlorella vulgaris generates oligomannosidic N-glycans of the structural type known from land plants and animals. In fact, Man5 (Man5GlcNAc2) served as substrate for GlcNAc-transferase I and a trace of an endogenous structure with terminal GlcNAc was seen. The unusual more linear Man5 structure recently found on glycoproteins of Chlamydomonas reinhardtii occurred - if at all - in traces only. Notably, a majority of the oligomannosidic glycans was multiply O-methylated with 3-O-methyl and 3,6-di-O-methyl mannoses at the non-reducing termini. This modification has so far been neither found on plant nor vertebrate N-glycans. It’s possible immunogenicity raises concerns as to the use of C. vulgaris for production of pharmaceutical glycoproteins.

Molecular characterization of second tomato α1,3/4-fucosidase (α-Fuc'ase Sl-2), a member of glycosyl hydrolase family 29 active toward the core α1,3-fucosyl residue in plant N-glycans.

In a previous study, we molecular-characterized a tomato (Solanum lycopersicum) α1, 3/4-fucosidase (α-Fuc'ase Sl-1) encoded in a tomato gene (Solyc03g006980), indicating that α-Fuc'ase Sl-1 is involved in the turnover of Lea epitope-containing N-glycans. In this study, we have characterized another tomato gene (Solyc11g069010) encoding α1, 3/4-fucosidase (α-Fuc'ase Sl-2), which is also active toward the complex type N-glycans containing Lea epitope(s). The baculovirus-insect cell expression system was used to express that α-Fuc'ase Sl-2 with anti-FLAG tag, and the expression product (rFuc'ase Sl-2), was found as a 65 kDa protein using SDS-PAGE and has an optimum pH of around 5.0. Similarly to rFuc'ase Sl-1, rFuc'ase Sl-2 hydrolyzed the non-reducing terminal α1, 3-fucose residue on LNFP III and α1, 4-fucose residues of Lea epitopes on plant complex type N-glycans, but not the core α1, 3-fucose residue on Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc or Fucα1-3GlcNAc. However, we found that both α-Fuc'ases Sl-1 and Sl-2 were specifically active toward α1, 3-fucose residue on GlcNAcβ1-4(Fucα1-3)GlcNAc, indicating that the non-substituted β-GlcNAc linked to the proximal GlcNAc residue of the core tri-saccharide moiety of plant specific N-glycans must be a pre-requisite for α-Fuc'ase activity. A 3 D modelled structure of the catalytic sites of α-Fuc'ase Sl-2 suggested that Asp192 and Glu236 may be important for binding to the α1, 3/4 fucose residue.

Trimming of N-Glycans by the Golgi-Localized α-1,2-Mannosidases, MNS1 and MNS2, Is Crucial for Maintaining RSW2 Protein Abundance during Salt Stress in Arabidopsis

Asparagine (Asn/N)-linked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mns1 mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mns1 mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-β-1,4-endoglucanase (RSW2/KOR) that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mns1 mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.

Molecular cloning and functional expression of Lewis type α1,3/α1,4-fucosyltransferase cDNAs from Mangifera indica L.

In higher plants, complex type N-glycans contain characteristic carbohydrate moieties that are not found in mammals. In particular, the attachment of the Lewis a (Lea) epitope is currently the only known outer chain elongation that is present in plant N-glycans. Such a modification is of great interest in terms of the biological function of complex type N-glycans in plant species. However, little is known regarding the exact molecular basis underlying their Lea expression. In the present study, we cloned two novel Lewis type fucosyltransferases (MiFUT13) from mango fruit, Mangifera indica L., heterologously expressed the proteins and structurally and functionally characterized them. Using an HPLC-based assay, we demonstrated that the recombinant MiFUT13 proteins mediate the α1,4-fucosylation of acceptor tetrasaccharides with a strict preference for type I-based structure to type II. The results and other findings suggest that MiFUT13s are involved in the biosynthesis of Lea containing glycoconjugates in mango fruits.

Structural features of N-glycans linked to glycoproteins expressed in three kinds of water plants: Predominant occurrence of the plant complex type N-glycans bearing Lewis a epitope

The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galβ1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts.

Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.

Glycan arrays have become a technique of choice to screen glycan-protein interactions in a high-throughput manner with high sensitivity and low sample consumption. Here, the synthesis of a new multifunctional fluorescent linker for glycan labeling via aminoxy ligation and immobilization is described; the linker features a fluorescent naphthalene group suitable for highly sensitive high-performance liquid chromatography-based purification and an azido- or amino-modified pentanoyl moiety for the immobilization onto solid supports. Several glycoconjugates displaying small sugar epitopes via chemical or chemoenzymatic synthesis were covalently attached onto a microarray support and tested with lectins of known carbohydrate binding specificity. The glycan library was extended using glycosyltransferases (e.g. galactosyl-, sialyl- and fucosyltransferases); the resulting neoglycoconjugates, which are easily detected by mass spectrometry, mimic antennal elements of N- and O-glycans, including ABH blood group epitopes and sialylated structures. Furthermore, an example natural plant N-glycan containing core α1,3-fucose and β1,2-xylose was also successfully conjugated to the fluorescent linker, immobilized and probed with lectins as well as antihorseradish peroxidase. These experiments validate our linker as being a potentially valuable tool to study glycozyme and lectin specificities, sensitive enough to allow purification of natural glycans.

The tonoplast--where sweetness is dispensable.

The proteolytic machinery of certain cellular organelles is potentially harmful to resident proteins. In mammals, the large luminal domains of integral lysosomal proteins are heavily glycosylated to protect them from degradation. In this issue of Journal of Experimental Botany (pages 1769–1781) Pedrazzini and co-workers reveal that plants are very different. Glycosylation is one of the most common protein modifications in all eukaryotes. Resident proteins of different intracellular organelles including the endoplasmic reticulum (ER), Golgi apparatus and hydrolysing compartments are often glycosylated at multiple sites with oligosaccharide chains of varying length and composition. The attached carbohydrates modulate protein stability, activity and trafficking and mediate protein–protein interactions. N-glycosylation, the most prevalent form of protein glycosylation in eukaryotes, is defined by the covalent linkage of an oligosaccharide (N-glycan) to selected asparagine residues of newly synthesized secretory proteins as they are translocated into the ER. Once attached to the polypeptide chain, the N-glycans play crucial roles in protein folding and quality control processes (Helenius and Aebi, 2001) – for example, they regulate a specific cell death event in Arabidopsis (de Oliveira et al., 2016). Immediately after the transfer to the protein, N-glycans are subjected to step-wise processing reactions that alter the composition of the N-glycan and by doing so control the fate of glycoproteins. As a consequence, the exposure of a defined glyco-code can promote protein folding or alternatively serve as a signal for the degradation of terminally misfolded or orphan glycoproteins. In principle, these essential N-glycan-dependent processes look highly conserved between animals and plants (Hong et al., 2012; Huttner and Strasser, 2012). By contrast, in the Golgi apparatus where the N-glycans acquire their final composition (referred to as complex N-glycans) N-glycan processing steps differ substantially in eukaryotes. A large variety of structurally and functionally diverse complex oligosaccharides are generated in the Golgi in mammals. In different plant species, on the other hand, only a limited number of complex N-glycan structures have been reported so far (Wilson et al., 2001). An outstanding question is therefore whether the reduced structural diversity of Golgi-processed N-glycans in plants also leads to fundamental differences in the biological role of these protein-linked carbohydrates. While most of the machinery and pathways involved in biosynthesis and processing of N-glycan structures have been identified, the study of the biological role associated with distinct plant N-glycan structures on individual proteins has proved challenging so far (Strasser, 2014). More detailed and comprehensive (glyco)proteomic approaches of different organelles are needed for understanding the structure and function of glycoproteins in a specific cellular environment. Now Pedrazzini et al. (2016) reveal that the Arabidopsis tonoplast is almost completely lacking glycoproteins with complex N-glycans and even proteins with ER-derived N-glycans are highly underrepresented.

Rice α-fucosidase active against plant complex type N-glycans containing Lewis a epitope: purification and characterization.

Rice α-fucosidase (α-fucosidase Os, 58kDa) that is active for α1-4 fucosyl linkage in Lewis a unit of plant N-glycans was purified to homogeneity. α-fucosidase Os showed activity against α1-3 fucosyl linkage in Lacto-N-fucopentaose III but not α1-3 fucosyl linkage in the core of plant N-glycans. The N-terminal sequence of α-fucosidase Os was identified as A-A-P-T-P-P-P-L-, and this sequence was found in the amino acid sequence of the putative rice α-fucosidase 1 (Os04g0560400).