Polysialic acid (polySia) is a glycan polymer composed of a linear chain of sialic acid residues detected on a small set of proteins in mammalian cells. This negatively charged glycan modulates various cellular functions and has potential biomedical applications. However, the targeted synthesis of polySia remains challenging due to biosynthetic and structural complexity. In this study, we investigated three polysialyltransferases from the salmonid Coregonus maraena (Cma-ST8Sia) for their application in glycoengineering. For this, the corresponding genes Cmast8Sia2-R1, Cmast8Sia2-R2, and Cmast8Sia4 were transiently expressed in Nicotiana benthamiana, and confocal laser scanning microscopy of GFP-fused Cma-ST8Sia exhibited trans-Golgi localization. Co-expression of Cma-ST8Sia with genes from the mammalian sialic acid biosynthesis pathway resulted in autopolysialylation of CmaST8SiaII-R1 and CmaST8SiaIV, but not of CmaST8SiaII-R2. Furthermore, co-expression with glycoproteins demonstrated polysialylation of recombinant proteins with distinct efficiencies and chain lengths. Inactive EndoN-coupled resin was used to enrich polysialylated proteins, and by the application of HPLC/DMB labeling, a degree of polymerization up to 45 was determined. Finally, a microglia inhibition assay demonstrated the biological activity of the engineered polySia. Collectively, these findings advance the capacity to engineer high-quality polySia in an eco-friendly, sustainable system, facilitating in-depth studies and the rational design of complex glycans.

Asparagine (N)-linked glycosylation is a fundamental co- and post-translational modification of proteins, playing a crucial role in protein folding, stability and function, protein-protein interactions, biotic and abiotic stress response as well as glycan-dependent quality control processes in the endoplasmic reticulum (ER). Protein N-glycosylation is initiated in the ER and continued in the Golgi apparatus by N-glycan-processing glycosyltransferases and glycosidases, which are compartmentalized in a highly organized manner reflecting their function in the sequential modification of glycans. Therefore, the precise localization of these enzymes is crucial for the optimal functioning of the glycosylation process and the secretory pathway and hence must be tightly regulated to maintain protein function, cellular health, and overall organismal development. Here, we highlight recent developments that contribute to a better understanding of the localization mechanisms of this important class of Golgi residents and discuss future directions to move the field forward.

The endoplasmic reticulum (ER) utilizes ER-associated degradation (ERAD), a highly conserved eukaryotic pathway, to eliminate misfolded or unassembled proteins and maintain protein homeostasis in cells. The clearance of misfolded glycoproteins involves several distinct steps, including the recognition of a specific glycan signal, retrotranslocation to the cytosol, and subsequent degradation of the misfolded protein by the ubiquitin proteasome system. Confocal microscopy was used to track the fate of a well-characterized ERAD substrate via a self-complementing split fluorescent protein assay. The results demonstrate that a misfolded variant of the STRUBBELIG (SUB) extracellular protein domain (SUBEX-C57Y) is retrotranslocated to the cytosol when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Retrotranslocation requires a protein domain with a lesion that is exposed in the lumen of the ER, N-glycan trimming by α-mannosidases, HRD1-mediated ubiquitination, and the ATPase function of CDC48. The retrotranslocated SUBEX-C57Y ERAD substrate undergoes deglycosylation, and proteasomal degradation is blocked by a catalytically inactive cytosolic peptide N-glycanase. These findings define distinct aspects of ERAD that have been elusive until now and may represent the default pathway for degrading misfolded glycoproteins in plants.

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of 'humanising' proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems. For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae. Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins. Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells. In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

IntroductionThe Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly understood.MethodsHere, we examined the role of the stem region of the cis/medial Golgi enzyme N-acetylglucosaminyltransferase I (GNTI) in homomeric complex formation in the Golgi of Nicotiana benthamiana using biochemical approaches and confocal microscopy.ResultsTransient expression of the N-terminal cytoplasmic, transmembrane, and stem (CTS) regions of GNTI leads to a block in N-glycan processing on a co-expressed recombinant glycoprotein. Overexpression of the CTS region from Golgi α-mannosidase I, which can form in planta complexes with GNTI, results in a similar block in N-glycan processing, while GNTI with altered subcellular localization or N-glycan processing enzymes located further downstream in the Golgi did not affect complex N-glycan processing. The GNTI-CTS-dependent alteration in N-glycan processing is caused by a specific nine-amino acid sequence motif in the stem that is required for efficient GNTI-GNTI interaction.DiscussionTaken together, we have identified a conserved motif in the stem region of the key N-glycan processing enzyme GNTI. We propose that the identified sequence motif in the GNTI stem region acts as a dominant negative motif that can be used in transient glycoengineering approaches to produce recombinant glycoproteins with predominantly mannosidic N-glycans.

In plants, the endoplasmic reticulum (ER) and Golgi bodies are not only in close proximity, but are also physically linked. This unique organization raises questions about the nature of the transport vectors carrying cargo between the two organelles. Same as in metazoan and yeast cells, it was suggested that cargo is transported from the ER to Golgi cisternae via COPII-coated vesicles produced at ribosome-free ER exit sites (ERES). Recent developments in mammalian cell research suggest, though, that COPII helps to select secretory cargo, but does not coat the carriers leaving the ER. Furthermore, it was shown that mammalian ERES expand into a tubular network containing secretory cargo, but no COPII components. Because of the close association of the ER and Golgi bodies in plant cells, it was previously proposed that ERES and the Golgi comprise a secretory unit that travels over or with a motile ER membrane. In this study, we aimed to explore the nature of ERES in plant cells and took advantage of high-resolution confocal microscopy and imaged ERES labelled with canonical markers (Sar1a, Sec16, Sec24). We found that ERES are dynamically connected to Golgi bodies and most likely represent pre-cis-Golgi cisternae. Furthermore, we showed fine tubular connections from the ER to Golgi compartments (ERGo tubules) as well as fine protrusions from ERES/Golgi cisternae connecting with the ER. We suggest that these tubules observed between the ER and Golgi as well as between the ER and ERES are involved in stabilizing the physical connection between ER and ERES/Golgi cisternae, but may also be involved in cargo transport from the ER to Golgi bodies.

The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves interactions between multiple interconnecting proteins. Where subcellularly and how different proteins interact need to be preserved during investigation. Furthermore, these complex events occurring simultaneously may lead to a single or multiple end point or cell function such as protein synthesis, cell cytoskeleton formation, DNA damage repair, or autophagy. There is therefore a need of real-time noninvasive methods for protein assays to enable direct visualization of the interactions in their natural environment and hence overcome the limitations of methods that rely on invasive cell disruption techniques. Forster resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is an advanced imaging method to observe protein-protein interactions at nanometer scale inside single living cells in real-time. Here we describe the development and use of two-channel pulsed interleave excitation (PIE) for multiple protein interactions in the mTORC1 pathway. The proteins were first tagged with multiple color fluorescent protein derivatives. The FRET-FLIM combination means that the information gained from using standard steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction.

The Golgi apparatus consists of stacked cisternae filled with enzymes that facilitate the sequential and highly controlled modification of glycans from proteins that transit through the organelle. Although the glycan processing pathways have been extensively studied, the underlying mechanisms that concentrate Golgi-resident glycosyltransferases and glycosidases in distinct Golgi compartments are poorly understood. The single-pass transmembrane domain (TMD) of n-acetylglucosaminyltransferaseI (GnTI) accounts for its steady-state distribution in the cis/medial-Golgi. Here, we investigated the contribution of individual amino acid residues within the TMD of Arabidopsis (Arabidopsis thaliana) and Nicotiana tabacum GnTI toward Golgi localization and n-glycan processing. Conserved sequence motifs within the TMD were replaced with those from the established trans-Golgi enzyme α2,6-sialyltransferase and site-directed mutagenesis was used to exchange individual amino acid residues. Subsequent subcellular localization of fluorescent fusion proteins and n-glycan profiling revealed that a conserved Gln residue in the GnTI TMD is essential for its cis/medial-Golgi localization. Substitution of the crucial Gln residue with other amino acids resulted in mislocalization to the vacuole and impaired n-glycan processing in vivo. Our results suggest that sequence-specific features of the GnTI TMD are required for its interaction with a Golgi-resident adaptor protein or a specific lipid environment that likely promotes coat protein complexI-mediated retrograde transport, thus maintaining the steady-state distribution of GnTI in the cis/medial-Golgi of plants.

The Arabidopsis ER-α-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. The enzyme itself has so far not been detected in the ER. Here, we provide evidence that in plants MNS3 exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is an amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. This retention is a means to spatially separate MNS3 from ER-localized mannose trimming steps that generate the glycan signal required for flagging terminally misfolded glycoproteins for ERAD. The physiological importance of the very specific MNS3 localization is demonstrated here by means of a structurally impaired variant of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1.

Glycosylation is an important protein modification in all eukaryotes. Whereas the early asparagine-linked glycosylation (N-glycosylation) and N-glycan processing steps in the endoplasmic reticulum are conserved between mammals and plants, the maturation of complex N-glycans in the Golgi apparatus differs considerably. Due to a restricted number of Golgi-resident N-glycan processing enzymes and the absence of nucleotide sugars such as CMP-N-acetylneuraminic acid, plants produce only a limited repertoire of different N-glycan structures. Moreover, mammalian mucin-type O-glycosylation of serine or threonine residues has not been described in plants and the required machinery is not encoded in their genome which enables de novo build-up of the pathway. As a consequence, plants are very well-suited for the production of homogenous N- and O-glycans and are increasingly used for the production of recombinant glycoproteins with custom-made glycans that may result in the generation of biopharmaceuticals with improved therapeutic potential.

Protein N-glycosylation is an essential posttranslational modification which is initiated in the endoplasmic reticulum. In plants, the N-glycans play a pivotal role for protein folding and quality control. Through the interaction of glycan processing and binding reactions mediated by ER-resident glycosidases and specific carbohydrate binding proteins, the N-glycans contribute to the adoption of a native protein conformation. Properly folded glycoproteins are released from these processes and allowed to continue their transit to the Golgi where further processing and maturation of N-glycans leads to the formation of more complex structures with different functions. Incompletely folded glycoproteins are removed from the ER by a highly conserved degradation process to prevent the accumulation or secretion of misfolded proteins and maintain ER homeostasis. Here, we describe methods to analyze the N-glycosylation status and the glycan-dependent ER-associated degradation process in plants.

Plants are attractive expression hosts for the production of recombinant glycoprotein therapeutics. The quality and efficiency of these biopharmaceuticals are very often influenced by the glycosylation profile. Consequently, approaches are needed that enable the production of recombinant glycoproteins with customized and homogenous N- and O-glycan structures. Here, we describe convenient tools that allow targeting and retention of glycan-modifying enzymes in the early secretory pathway of plants. These protocols can be used to fine-tune the subcellular localization of glycosyltransferases and glycosidases in plants and consequently to increase the homogeneity of glycosylation on recombinant glycoproteins.

SummaryGolgi‐resident type–II membrane proteins are asymmetrically distributed across the Golgi stack. The intrinsic features of the protein that determine its subcompartment‐specific concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of Nicotiana benthamiana N–acetylglucosaminyltransferase I (GnTI) for its cis/medial‐Golgi localization and for protein–protein interaction in the Golgi. The individual GnTI protein domains were replaced with those from the well‐known trans‐Golgi enzyme α2,6–sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co‐localization analysis and N–glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial‐Golgi localization. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub‐Golgi localization was not able to complement the GnTI‐dependent glycosylation defect. Our results suggest that sequence‐specific features in the transmembrane domain of GnTI account for its steady‐state distribution in the cis/medial‐Golgi in plants, which is a prerequisite for efficient N–glycan processing in vivo.

Real-time noninvasive fluorescence-based protein assays enable a direct access to study interactions in their natural environment and hence overcome the limitations of other methods that rely on invasive cell disruption techniques. The determination of Forster resonance energy transfer (FRET) by means of fluorescence lifetime imaging microscopy (FLIM) is currently the most advanced method to observe protein-protein interactions at nanometer resolution inside single living cells and in real-time. In the FRET-FLIM approach, the information gained using steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction. The combination of confocal laser scanning microscopy with the sensitive advanced technique of time-correlated single photon counting allows the mapping of the spatial distribution of fluorescence lifetimes inside living cells on a pixel-by-pixel basis that is the same as the fluorescence image. Moreover, the use of multiphoton excitation particularly for plant cells provides further advantages such as reduced phototoxicity and photobleaching. In this protocol, we briefly describe the instrumentation and experimental design to study protein interactions within the plant endomembrane system, with a focus on the imaging of plant cells expressing fluorescent proteins and acquisition and analysis of fluorescence lifetime resolved data.

N-glycosylation of proteins plays an important role in the determination of the fate of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Specific oligosaccharide structures recruit molecular chaperones that promote folding or mannose-binding lectins that assist in the clearance of improperly-folded glycoproteins by delivery to ER-associated degradation (ERAD). In plants, the mechanisms and factors that recognize non-native proteins and sort them to ERAD are poorly understood. In the present study, we provide evidence that a misfolded variant of the STRUBBELIG (SUB) extracellular domain (SUBEX-C57Y) is degraded in a glycan-dependent manner in plants. SUBEX-C57Y is an ER-retained glycoprotein with three N-glycans that is stabilized in the presence of kifunensine, a potent inhibitor of α-mannosidases. Stable expression in Arabidopsis thaliana knockout mutants revealed that SUBEX-C57Y degradation is dependent on the ER lectin OS9 and its associated ERAD factor SEL1L. SUBEX-C57Y was also stabilized in plants lacking the α-mannosidases MNS4 and MNS5 that generate a terminal α1,6-linked mannose on the C-branch of N-glycans. Notably, the glycan signal for degradation is not constrained to a specific position within SUBEX-C57Y. Structural analysis revealed that SUBEX-C57Y harbours considerable amounts of Glc1Man7GlcNAc2N-glycans suggesting that the ER-quality control processes involving calnexin/calreticulin (CNX/CRT) and ERAD are tightly interconnected to promote protein folding or disposal by termination of futile folding attempts.

To ensure that aberrantly folded proteins are cleared from the endoplasmic reticulum (ER), all eukaryotic cells possess a mechanism known as endoplasmic reticulum-associated degradation (ERAD). Many secretory proteins are N-glycosylated, and despite some recent progress, little is known about the mechanism that selects misfolded glycoproteins for degradation in plants. Here, we investigated the role of Arabidopsis thaliana class I alpha-mannosidases (MNS1 to MNS5) in glycan-dependent ERAD. Our genetic and biochemical data show that the two ER-resident proteins MNS4 and MNS5 are involved in the degradation of misfolded variants of the heavily glycosylated brassinosteroid receptor, BRASSINOSTEROID INSENSITIVE1, while MNS1 to MNS3 appear dispensable for this ERAD process. By contrast, N-glycan analysis of different mns mutant combinations revealed that MNS4 and MNS5 are not involved in regular N-glycan processing of properly folded secretory glycoproteins. Overexpression of MNS4 or MNS5 together with ER-retained glycoproteins indicates further that both enzymes can convert Glc(0-1)Man(8-9)GlcNAc(2) into N-glycans with a terminal alpha 1,6-linked Man residue in the C-branch. Thus, MNS4 and MNS5 function in the formation of unique N-glycan structures that are specifically recognized by other components of the ERAD machinery, which ultimately results in the disposal of misfolded glycoproteins.

Asparagine-linked glycosylation of proteins is an essential cotranslational and posttranslational protein modification in plants. The central step in this process is the transfer of a preassembled oligosaccharide to nascent proteins in the endoplasmic reticulum by the oligosaccharyltransferase (OST) complex. Despite the importance of the catalyzed reaction, the composition and the function of individual OST subunits are still ill defined in plants. Here, we report the function of the highly conserved OST subunit OST3/6. We have identified a mutant in the OST3/6 gene that causes overall underglycosylation of proteins and affects the biogenesis of the receptor kinase EF-TU RECEPTOR involved in innate immunity and the endo-β-1,4-glucanase KORRIGAN1 required for cellulose biosynthesis. Notably, the ost3/6 mutation does not affect mutant variants of the receptor kinase BRASSINOSTEROID-INSENSITIVE1. OST3/6 deficiency results in activation of the unfolded protein response and causes hypersensitivity to salt/osmotic stress and to the glycosylation inhibitor tunicamycin. Consistent with its role in protein glycosylation, OST3/6 resides in the endoplasmic reticulum and interacts with other subunits of the OST complex. Together, our findings reveal the importance of Arabidopsis (Arabidopsis thaliana) OST3/6 for the efficient glycosylation of specific glycoproteins involved in different physiological processes and shed light on the composition and function of the plant OST complex.

N-Glycan processing is one of the most important cellular protein modifications in plants and as such is essential for plant development and defense mechanisms. The accuracy of Golgi-located processing steps is governed by the strict intra-Golgi localization of sequentially acting glycosidases and glycosyltransferases. Their differential distribution goes hand in hand with the compartmentalization of the Golgi stack into cis-, medial-, and trans-cisternae, which separate early from late processing steps. The mechanisms that direct differential enzyme concentration are still unknown, but the formation of multienzyme complexes is considered a feasible Golgi protein localization strategy. In this study, we used two-photon excitation-Förster resonance energy transfer-fluorescence lifetime imaging microscopy to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Following the coexpression of fluorescent protein-tagged amino-terminal Golgi-targeting sequences (cytoplasmic-transmembrane-stem [CTS] region) of enzyme pairs in leaves of tobacco (Nicotiana spp.), we observed that all tested cis- and medial-Golgi enzymes, namely Arabidopsis (Arabidopsis thaliana) Golgi α-mannosidase I, Nicotiana tabacum β1,2-N-acetylglucosaminyltransferase I, Arabidopsis Golgi α-mannosidase II (GMII), and Arabidopsis β1,2-xylosyltransferase, form homodimers and heterodimers, whereas among the late-acting enzymes Arabidopsis β1,3-galactosyltransferase1 (GALT1), Arabidopsis α1,4-fucosyltransferase, and Rattus norvegicus α2,6-sialyltransferase (a nonplant Golgi marker), only GALT1 and medial-Golgi GMII were found to form a heterodimer. Furthermore, the efficiency of energy transfer indicating the formation of interactions decreased considerably in a cis-to-trans fashion. The comparative fluorescence lifetime imaging of several full-length cis- and medial-Golgi enzymes and their respective catalytic domain-deleted CTS clones further suggested that the formation of protein-protein interactions can occur through their amino-terminal CTS region.

In the endoplasmic reticulum, immature polypeptides coincide with terminally misfolded proteins. Consequently, cells need a well-balanced quality control system, which decides about the fate of individual proteins and maintains protein homeostasis. Misfolded and unassembled proteins are sent for destruction via the endoplasmic reticulum-associated degradation (ERAD) machinery to prevent the accumulation of potentially toxic protein aggregates. Here, we report the identification of Arabidopsis thaliana OS9 as a component of the plant ERAD pathway. OS9 is an ER-resident glycoprotein containing a mannose-6-phosphate receptor homology domain, which is also found in yeast and mammalian lectins involved in ERAD. OS9 fused to the C-terminal domain of YOS9 can complement the ERAD defect of the corresponding yeast Δyos9 mutant. An A. thaliana OS9 loss-of-function line suppresses the severe growth phenotype of the bri1-5 and bri1-9 mutant plants, which harbour mutated forms of the brassinosteroid receptor BRI1. Co-immunoprecipitation studies demonstrated that OS9 associates with Arabidopsis SEL1L/HRD3, which is part of the plant ERAD complex and with the ERAD substrates BRI1-5 and BRI1-9, but only the binding to BRI1-5 occurs in a glycan-dependent way. OS9-deficiency results in activation of the unfolded protein response and reduces salt tolerance, highlighting the role of OS9 during ER stress. We propose that OS9 is a component of the plant ERAD machinery and may act specifically in the glycoprotein degradation pathway.