N-glycosylation Pathway Research Articles

N-glycoproteomic and proteomic alterations in SRD5A3-deficient fibroblasts.

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P<0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.

Exploring protein N-glycosylation in the green microalga Dunaliella salina

N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum where an oligosaccharide precursor is added to the newly synthesized protein and continues in the Golgi apparatus where the N-linked carbohydrate sequences are processed. Importantly, the most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly currently produced in mammalian cells which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered as efficient and eco-friendly alternative platforms for the production of biologics. However, unlike for C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man5GlcNAc2 oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii that gives rise to the synthesis of a non-canonical oligomannoside Man5GlcNAc2. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man5GlcNAc2.

Galectins induced from hemocytes bridge phosphatidylserine and N-glycosylated Drpr/CED-1 receptor during dendrite pruning

During neuronal pruning, phagocytes engulf shed cellular debris to avoid inflammation and maintain tissue homeostasis. How phagocytic receptors recognize degenerating neurites had been unclear. Here, we identify two glucosyltransferases Alg8 and Alg10 of the N-glycosylation pathway required for dendrite fragmentation and clearance through genetic screen. The scavenger receptor Draper (Drpr) is N-glycosylated with complex- or hybrid-type N-glycans that interact specifically with galectins. We also identify the galectins Crouching tiger (Ctg) and Hidden dragon (Hdg) that interact with N-glycosylated Drpr and function in dendrite pruning via the Drpr pathway. Ctg and Hdg are required in hemocytes for expression and function, and are induced during dendrite injury to localize to injured dendrites through specific interaction with exposed phosphatidylserine (PS) on the surface membrane of injured dendrites. Thus, the galectins Ctg and Hdg bridge the interaction between PS and N-glycosylated Drpr, leading to the activation of phagocytosis.

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Perturbed N-glycosylation of Halobacterium salinarum archaellum filaments leads to filament bundling and compromised cell motility

The swimming device of archaea—the archaellum—presents asparagine (N)-linked glycans. While N-glycosylation serves numerous roles in archaea, including enabling their survival in extreme environments, how this post-translational modification contributes to cell motility remains under-explored. Here, we report the cryo-EM structure of archaellum filaments from the haloarchaeon Halobacterium salinarum, where archaellins, the building blocks of the archaellum, are N-glycosylated, and the N-glycosylation pathway is well-resolved. We further determined structures of archaellum filaments from two N-glycosylation mutant strains that generate truncated glycans and analyzed their motility. While cells from the parent strain exhibited unidirectional motility, the N-glycosylation mutant strain cells swam in ever-changing directions within a limited area. Although these mutant strain cells presented archaellum filaments that were highly similar in architecture to those of the parent strain, N-linked glycan truncation greatly affected interactions between archaellum filaments, leading to dramatic clustering of both isolated and cell-attached filaments. We propose that the N-linked tetrasaccharides decorating archaellins act as physical spacers that minimize the archaellum filament aggregation that limits cell motility.

Integrated multi-omics analyses identify anti-viral host factors and pathways controlling SARS-CoV-2 infection

Host anti-viral factors are essential for controlling SARS-CoV-2 infection but remain largely unknown due to the biases of previous large-scale studies toward pro-viral host factors. To fill in this knowledge gap, we perform a genome-wide CRISPR dropout screen and integrate analyses of the multi-omics data of the CRISPR screen, genome-wide association studies, single-cell RNA-Seq, and host-virus proteins or protein/RNA interactome. This study uncovers many host factors that are currently underappreciated, including the components of V-ATPases, ESCRT, and N-glycosylation pathways that modulate viral entry and/or replication. The cohesin complex is also identified as an anti-viral pathway, suggesting an important role of three-dimensional chromatin organization in mediating host-viral interaction. Furthermore, we discover another anti-viral regulator KLF5, a transcriptional factor involved in sphingolipid metabolism, which is up-regulated, and harbors genetic variations linked to COVID-19 patients with severe symptoms. Anti-viral effects of three identified candidates (DAZAP2/VTA1/KLF5) are confirmed individually. Molecular characterization of DAZAP2/VTA1/KLF5-knockout cells highlights the involvement of genes related to the coagulation system in determining the severity of COVID-19. Together, our results provide further resources for understanding the host anti-viral network during SARS-CoV-2 infection and may help develop new countermeasure strategies.

Subcellular localization of core beta(1,2)-xylosylated N-glycoproteins in the green microalgae Chlamydomonas reinhardtii

The green microalgae Chlamydomonas reinhardtii synthesizes a N-glycan precursor within the endoplasmic reticulum (ER) that is then transferred to specific asparagine residues of the protein N-glycosylation consensus sites. Maturation of this precursor in the ER and then in the Golgi apparatus results in N-glycans composed of a non-canonical linear Man5GlcNAc2 that is partially O-methylated and carries out one or two xylose residues. One xylose residue was demonstrated to be a core beta(1,2)-xylose. Recently, two xylosyltransferases, named A (XTA) and B (XTB) respectively, were demonstrated to be responsible for the addition of this core beta(1,2)-xylose. Nowadays, even if information is available regarding the protein N-glycosylation pathway and especially the core beta(1,2)-xylosylation in C. reinhardtii, no data regarding the subcellular localization of this subset of glycosylated proteins is available. Therefore, in this work, subcellular immunolocalization of N-glycoproteins bearing the core beta(1,2)-xylose epitope was carried out using an optimized protocol of transmission electron microscopy of CC-5325 and CC-5155 reference strains (wild types) compared to the MXTAxIMXTB double insertional mutant strain in which the core beta(1,2)-xylosylation is fully-lacking. Such a study gives a first cartography of specific glycoepitopes in microalgae and new insights on the distribution of core beta(1,2)-xylosylated glycoproteins in C. reinhardtii organelles.

Impact of PpSpi1, a glycosylphosphatidylinositol-anchored cell wall glycoprotein, on cell wall defects of N-glycosylation-engineered Pichia pastoris.

Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Molecular and Cellular Insights: A Focus on Glycans and the HNK1 Epitope in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a synaptic disorder with a GABA/glutamate imbalance in the perineuronal nets and structural abnormalities such as increased dendritic spines and decreased long distance connections. Specific pregnancy disorders significantly increase the risk for an ASD phenotype such as preeclampsia, preterm birth, hypoxia phenomena, and spontaneous miscarriages. They are associated with defects in the glycosylation-immune placental processes implicated in neurogenesis. Some glycans epitopes expressed in the placenta, and specifically in the extra-villous trophoblast also have predominant functions in dendritic process and synapse function. Among these, the most important are CD57 or HNK1, CD22, CD24, CD33 and CD45. They modulate the innate immune cells at the maternal-fetal interface and they promote foeto-maternal tolerance. There are many glycan-based pathways of immunosuppression. N-glycosylation pathway dysregulation has been found to be associated with autoimmune-like phenotypes and maternal-autoantibody-related (MAR) autism have been found to be associated with central, systemic and peripheric autoimmune processes. Essential molecular pathways associated with the glycan-epitopes expression have been found to be specifically dysregulated in ASD, notably the Slit/Robo, Wnt, and mTOR/RAGE signaling pathways. These modifications have important effects on major transcriptional pathways with important genetic expression consequences. These modifications lead to defects in neuronal progenitors and in the nervous system's implementation specifically, with further molecular defects in the GABA/glutamate system. Glycosylation placental processes are crucial effectors for proper maternofetal immunity and endocrine/paracrine pathways formation. Glycans/ galectins expression regulate immunity and neurulation processes with a direct link with gene expression. These need to be clearly elucidated in ASD pathophysiology.

The N-mannosyltransferase MoAlg9 plays important roles in the development and pathogenicity of Magnaporthe oryzae

Magnaporthe oryzae is the causal agent of rice blast. Glycosylation plays key roles in vegetative growth, development, and infection of M. oryzae. However, several glycosylation-related genes have not been characterized. In this study, we identified a Glyco_transf_22 domain-containing protein, MoAlg9, and found that MoAlg9 is localized to the endoplasmic reticulum (ER). Deletion of MoALG9 significantly affected conidial production, normal appressorium formation, responses to stressors, and pathogenicity of M. oryzae. We also found that the ΔMoalg9 mutant was defective in glycogen utilization, appressorial penetration, and invasive growth in host cells. Moreover, we further demonstrated that MoALG9 regulates the transcription of several target genes involved in conidiation, appressorium formation, and cell-wall integrity. In addition, we found that the Glyco_transf_22 domain is essential for normal MoAlg9 function and localization. We also provide evidence that MoAlg9 is involved in N-glycosylation pathway in M. oryzae. Taken together, these results show that MoAlg9 is important for conidiation, appressorium formation, maintenance of cell wall integrity, and the pathogenesis of M. oryzae.

Plasma glycoproteomics delivers high-specificity disease biomarkers by detecting site-specific glycosylation abnormalities

IntroductionThe human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease. ObjectivesHere, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics. MethodsComparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data. ResultsGlycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors. ConclusionOur work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease.

RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/β subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHβ subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHβ results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHβ subunit in pituitaries of women.

In vivo deglycosylation of recombinant glycoproteins in tobacco BY-2 cells.

Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines.

SLC35A2 deficiency reduces protein levels of core 1 β-1,3-galactosyltransferase 1 (C1GalT1) and its chaperone Cosmc and affects their subcellular localization

Nucleotide sugar transporters (NSTs) are multitransmembrane proteins, localized in the Golgi apparatus and/or endoplasmic reticulum, which provide glycosylation enzymes with their substrates. It has been demonstrated that NSTs may form complexes with functionally related glycosyltransferases, especially in the N-glycosylation pathway. However, potential interactions of NSTs with enzymes mediating the biosynthesis of mucin-type O-glycans have not been addressed to date. Here we report that UDP-galactose transporter (UGT; SLC35A2) associates with core 1 β-1,3-galactosyltransferase 1 (C1GalT1; T-synthase). This provides the first example of an interaction between an enzyme that acts exclusively in the O-glycosylation pathway and an NST. We also found that SLC35A2 associated with the C1GalT1-specific chaperone Cosmc, and that the endogenous Cosmc was localized in both the endoplasmic reticulum and Golgi apparatus of wild-type HEK293T cells. Furthermore, in SLC35A2-deficient cells protein levels of C1GalT1 and Cosmc were decreased and their Golgi localization was less pronounced. Finally, we identified SLC35A2 as a novel molecular target for the antifungal agent itraconazole. Based on our findings we propose that NSTs may contribute to the stabilization of their interaction partners and help them to achieve target localization in the cell, most likely by facilitating their assembly into larger functional units.

The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis BVirus antigen and the virus-neutralizing antibody response in vaccinated mice.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of β-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.

Limited N-Glycan Processing Impacts Chaperone Expression Patterns, Cell Growth and Cell Invasiveness in Neuroblastoma.

Enhanced N-glycan branching is associated with cancer, but recent investigations supported the involvement of less processed N-glycans. Herein, we investigated how changes in N-glycosylation influence cellular properties in neuroblastoma (NB) using rat N-glycan mutant cell lines, NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3), as well as the parental cell line NB_1. The two earlier mutant cells have compromised N-acetylglucosaminyltransferase-I (GnT-I) and GnT-II activities. Lectin blotting showed that NB_1(-Mgat3) cells had decreased activity of GnT-III compared to NB_1. ESI-MS profiles identified N-glycan structures in NB cells, supporting genetic edits. NB_1(-Mgat1) had the most oligomannose N-glycans and the greatest cell invasiveness, while NB_1(-Mgat2) had the fewest and least cell invasiveness. The proliferation rate of NB_1 was slightly slower than NB_1(-Mgat3), but faster than NB_1(-Mgat1) and NB_1(-Mgat2). Faster proliferation rates were due to the faster progression of those cells through the G1 phase of the cell cycle. Further higher levels of oligomannose with 6-9 Man residues indicated faster proliferating cells. Human NB cells with higher oligomannose N-glycans were more invasive and had slower proliferation rates. Both rat and human NB cells revealed modified levels of ER chaperones. Thus, our results support a role of oligomannose N-glycans in NB progression; furthermore, perturbations in the N-glycosylation pathway can impact chaperone systems.

Agl28 and Agl29 are key components of a Halobacterium salinarum N-glycosylation pathway.

Although Halobacterim salinarum provided the first example of N-glycosylation outside the Eukarya, only recently has attention focused on delineating the pathway responsible for the assembly of the N-linked tetrasaccharide decorating selected proteins in this haloarchaeon. In the present report, the roles of VNG1053G and VNG1054G, two proteins encoded by genes clustered together with a set of genes demonstrated to encode N-glycosylation pathway components, were considered. Relying on both bioinformatics and gene deletion and subsequent mass spectrometry analysis of known N-glycosylated proteins, VNG1053G was determined to be the glycosyltransferase responsible for addition of the linking glucose, while VNG1054G was deemed to be the flippase that translocates the lipid-bound tetrasaccharide across the plasma membrane to face the cell exterior, or to contribute to such activity. As observed with Hbt. salinarum lacking other components of the N-glycosylation machinery, both cell growth and motility were compromised in the absence of VNG1053G or VNG1054G. Thus, given their demonstrated roles in Hbt. salinarum N-glycosylation, VNG1053G and VNG1054G were re-annotated as Agl28 and Agl29, according to the nomenclature used to define archaeal N-glycosylation pathway components.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

Whole exome sequencing reveals several novel variants in congenital disorders of glycosylation and glycogen storage diseases in seven patients from Iran.

Congenital disorder of glycosylation (CDG) and Glycogen storage diseases (GSDs) are inborn metabolic disorders caused by defects in some metabolic pathways. These disorders are a heterogeneous group of diseases caused by impaired O- as well as N-glycosylation pathways. CDG patients show a broad spectrum of clinical presentations; many GSD types (PGM1-CDG) have muscle involvement and hypoglycemia. We applied WES for all seven patients presenting GSD and CDG symptoms. Then we analyzed the data using various tools to predict pathogenic variants in genes related to the patients' diseases. In the present study, we identified pathogenic variants in Iranian patients suffering from GSD and CDG, which can be helpful for patient management, and family counseling. We detected seven pathogenic variants using whole exome sequencing (WES) in known AGL (c.1998A>G, c.3635T>C, c.3682C>T), PGM1 (c.779G>A), DPM1 (c.742T>C), RFT1 (c.127A>G), and GAA (c.1314C>A) genes. The suspected clinical diagnosis of CDG and GSD patients was confirmed by identifying missense and or nonsense mutations in PGM1, DPM1, RFT1, GAA, and AGL genes by WES of all 7 cases. This study helps us understand the scenario of the disorder causes and consider the variants for quick disease diagnosis.

Glycosyltransferase-Related Protein GtrA Is Essential for Localization of Type IX Secretion System Cargo Protein Cellulase Cel9A and Affects Cellulose Degradation in Cytophaga hutchinsonii.

The Gram-negative bacterium Cytophaga hutchinsonii digests cellulose through a novel cellulose degradation mechanism. It possesses the lately characterized type IX secretion system (T9SS). We recently discovered that N-glycosylation of the C-terminal domain (CTD) of a hypothetical T9SS substrate protein in the periplasmic space of C. hutchinsonii affects protein secretion and localization. In this study, green fluorescent protein (GFP)-CTDCel9A recombinant protein was found with increased molecular weight in the periplasm of C. hutchinsonii. Site-directed mutagenesis studies on the CTD of cellulase Cel9A demonstrated that asparagine residue 900 in the D-X-N-X-S motif is important for the processing of the recombinant protein. We found that the glycosyltransferase-related protein GtrA (CHU_0012) located in the cytoplasm of C. hutchinsonii is essential for outer membrane localization of the recombinant protein. The deletion of gtrA decreased the abundance of the outer membrane proteins and affected cellulose degradation by C. hutchinsonii. This study provided a link between the glycosylation system and cellulose degradation in C. hutchinsonii. IMPORTANCE N-Glycosylation systems are generally limited to some pathogenic bacteria in prokaryotes. The disruption of the N-glycosylation pathway is related to adherence, invasion, colonization, and other phenotypic characteristics. We recently found that the cellulolytic bacterium Cytophaga hutchinsonii also has an N-glycosylation system. The cellulose degradation mechanism of C. hutchinsonii is novel and mysterious; cellulases and other proteins on the cell surface are involved in utilizing cellulose. In this study, we identified an asparagine residue in the C-terminal domain of cellulase Cel9A that is necessary for the processing of the T9SS cargo protein. Moreover, the glycosyltransferase-related protein GtrA is essential for the localization of the GFP-CTDCel9A recombinant protein. Deletion of gtrA affected cellulose degradation and the abundance of outer membrane proteins. This study enriched the understanding of the N-glycosylation system in C. hutchinsonii and provided a link between N-glycosylation and cellulose degradation, which also expanded the role of the N-glycosylation system in bacteria.