Protein N-glycosylation Pathway Research Articles

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.

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.

Glycotherapy: A New Paradigm in Breast Cancer Research.

Breast cancer is an ancient disease recognized first by the Egyptians as early as 1600 BC. The first cancer-causing gene in a chicken tumor virus was found in 1970. The United States signed the National Cancer Act in 1971, authorizing federal funding for cancer research. Irrespective of multi-disciplinary approaches, diverting a great deal of public and private resources, breast cancer remains at the forefront of human diseases, affecting as many as one in eight women during their lifetime. Because of overarching challenges and changes in the breast cancer landscape, five-year disease-free survival is no longer considered adequate. The absence of a cure, and the presence of drug resistance, severe side effects, and destruction of the patient’s quality of life, as well as the fact that therapy is often expensive, making it unaffordable to many, have created anxiety among patients, families, and friends. One of the reasons for the failure of cancer therapeutics is that the approaches do not consider cancer holistically. Characteristically, all breast cancer cells and their microenvironmental capillary endothelial cells express asparagine-linked (N-linked) glycoproteins with diverse structures. We tested a small biological molecule, Tunicamycin, that blocks a specific step of the protein N-glycosylation pathway in the endoplasmic reticulum (ER), i.e., the catalytic activity of N-acetylglusosaminyl 1-phosphate transferase (GPT). The outcome was overwhelmingly exciting. Tunicamycin quantitatively inhibits angiogenesis in vitro and in vivo, and inhibits the breast tumor progression of multiple subtypes in pre-clinical mouse models with “zero” toxicity. Mechanistic details support ER stress-induced unfolded protein response (upr) signaling as the cause for the apoptotic death of both cancer and the microvascular endothelial cells. Additionally, it interferes with Wnt signaling. We therefore conclude that Tunicamycin can be expected to supersede the current therapeutics to become a glycotherapy for treating breast cancer of all subtypes.

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum.

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

A New Bacmid for Customized Protein Glycosylation Pathway Engineering in the Baculovirus-Insect Cell System.

One attractive feature of the baculovirus-insect cell system (BICS) is the baculoviral genome has a large capacity for genetic cargo. This enables construction of viral vectors designed to accept multigene insertions, which has facilitated efforts to produce recombinant multisubunit protein complexes. However, the large genetic capacity of baculovirus vectors has not yet been exploited for multistep pathway engineering. Therefore, we created PolyBac, which is a novel baculovirus shuttle vector, or bacmid, that can be used for this purpose. PolyBac was designed to accept multiple transgene insertions by three different mechanisms at three different sites within the baculovirus genome. After constructing and characterizing PolyBac, we used it to isolate nine derivatives encoding various combinations of up to eight different protein N-glycosylation pathway functions, or glycogenes. We then used these derivatives, which were designed to progressively extend the endogenous insect cell pathway, to assess PolyBac's utility for protein glycosylation pathway engineering. This assessment was enabled by engineering each derivative to produce a recombinant influenza hemagglutinin (rH5), which was used to probe the impact of each glycoengineered PolyBac derivative on the endogenous insect cell pathway. Genetic analyses of these derivatives confirmed PolyBac can accept large DNA insertions. Biochemical analyses of the rH5 products showed each had distinct N-glycosylation profiles. Finally, the major N-glycan on each rH5 product was the predicted end product of the engineered N-glycosylation pathways encoded by each PolyBac derivative. These results generally indicate that PolyBac has utility for multistep metabolic pathway engineering and directly demonstrate that this new bacmid can be used for customized protein glycosylation pathway engineering in the BICS.

Abstract 2442: Identification of a novel role for the β-catenin/CBP signaling in epigenetic regulation of the N-glycosylation gene, DPAGT1, in head and neck cancer

Abstract Aberrant activation of the DPAGT1 gene, encoding an essential enzyme in the metabolic pathway of protein N-glycosylation, has been shown to be associated with head and neck squamous cell carcinoma (HNSCC). We have shown that DPAGT1 inhibits intercellular adhesion and functions in a positive feedback loop with Wnt/β-catenin signaling, and that the nuclear β-catenin/CBP signaling underlies the progression of HNSCC to advanced disease. However, the tumor promoting effects of DPAGT1 and its molecular links to the nuclear β-catenin/CBP axis are not well defined. We carried out genomic and functional analyses of DPAGT1 perturbation in indolent (CAL27) and metastatic (HSC-3) HNSCC cells, and in orthotopic HSC-3-derived xenografts in mice. We further generated and annotated DPAGT1 inhibition signature in HSC-3 cells and interrogated it in TCGA HNSCC. We then examined the effects of inhibition of β-catenin-CBP interaction with E7386 on DPAGT1 expression using ChIP-seq and computational approaches. E7386, a novel β-catenin/CBP modulator displays activity profile that closely overlaps with that of ICG-001, but exhibits ~50 - 100-fold lower EC50 values. Ectopic expression of DPAGT1 in indolent CAL27 cells induced epithelial-to-mesenchymal transition (EMT) which coincided with increased abundance of active β-catenin. Partial knockdown of DPAGT1 with siRNA in metastatic HSC-3 cells inhibited EMT, diminished cell migration and enhanced intercellular adhesion. Inhibition of the DPAGT1 enzyme activity using tunicamycin interfered with orthotopic tongue tumor growth and metastasis. DPAGT1 knockdown in HSC-3 cells defined DPAGT1-activated gene signature as enriched in pro-tumorigenic signaling pathways, including stem cell-like genes. Integrative analysis of the DPAGT1-activated genes in TCGA validated the association of DPAGT1 activity with the EMT transcription factors, ZEB1, Twist1/2 and Slug. ChIP-seq analyses without and with the E7386 treatment revealed reduced occupancy of H3K4me3 at two DPAGT1 transcription start sites following the E7386 treatment. In conclusion, our studies align aberrant activation of DPAGT1 with the induction of EMT and stem cell associated genes and suggest a novel role of β-catenin/CBP/MLL1 in the epigenetic regulation DPAGT1 and protein N-glycosylation in HNSCC. Citation Format: Khalid A. Alamoud, Vinay Kartha, Huamei Yang, Andrew Tilston-Lunel, Anthony Federico, Manish Bais, Takachi Owa, Kenichi Nomoto, Xalarabos Varelas, Stefano Monti, Maria A. Kukuruzinska. Identification of a novel role for the β-catenin/CBP signaling in epigenetic regulation of the N-glycosylation gene, DPAGT1, in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2442.

Influence of Protein Glycosylation on Campylobacter fetus Physiology.

Campylobacter fetus is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that C. fetus infection can result from the consumption or handling of contaminated food products, but C. fetus is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, Campylobacter jejuni, the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two C. fetus subsp. fetus protein glycosylation (pgl) mutants (pglX and pglJ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H2-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H2 and increased hydrogenase activity, suggesting that N-glycosylation in C. fetus is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the C. fetus pgl pathway and its enzymes, heterologous expression in Escherichia coli followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the C. fetus hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in C. jejuni. In addition, the pgl mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of C. fetus, but also expands our knowledge on the influence of protein N-glycosylation on Campylobacter cell physiology.

Exome sequence identified a c.320A > G ALG13 variant in a female with infantile epileptic encephalopathy with normal glycosylation and random X inactivation: Review of the literature

Congenital Disorders of Glycosylation (CDG) are new and rapidly expanding neurometabolic disorders with multisystem involvements, broad phenotypic manifestations, and variable severity. The majority results from a defect of one of the steps involved with protein or lipid N-glycosylation pathway. Almost all are inherited in autosomal recessive patterns with a few exceptions such as the X-linked ALG13. Mutations of ALG13 are reported, so far in only 10 patients, all were ascertained through exome/genome sequencing. Specifically, the ALG13 c.320A > G (p.Asn107Ser) variant was reported only in females and in all were de novo mutations. These findings may suggest an X-linked dominant inheritance of this mutation with embryonic male lethality. These patients presented with severe infantile epileptic encephalopathy, global developmental delay, and multisystem abnormalities. Only two of these females had glycosylation studies done, and both showed normal pattern of glycosylated serum transferrin isoforms, and none had their X-chromosome inactivation patterns studied.Here, we report on another female patient who is heterozygous for the same ALG13 c.320A > G (p.Asn107Ser) variant. She presented with infantile spasms, epileptic encephalopathy, hypsarrhythmia, hypotonia, developmental delay, intellectual disability, abnormal coagulation profile, feeding problems, hypotonia, and dysmorphic features. The diagnosis of CGD was suspected clinically, but glycosylation studies were done twice and showed normal patterns on both occasions. Her X-inactivation study was also done and, surprisingly, showed a random pattern of X-inactivation, with no evidence of skewness.

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D100), IV (F220) and V (F264) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Mass spectrometry of transferrin and apolipoprotein C-III for diagnosis and screening of congenital disorder of glycosylation.

Congenital disorder of glycosylation (CDG), formerly representing a group of diseases due to defects in the biosynthetic pathway of protein N-glycosylation, currently covers a wide range of disorders affecting glycoconjugates. Since its first application to serum transferrin from a CDG patient with phosphomannomutase-2 deficiency in 1992, mass spectrometry (MS) has been playing a key role in identification and characterization of glycosylation defects affecting glycoproteins. MS of native transferrin detects a lack of glycans characteristic to the classical CDG-I type of molecular abnormality. Electrospray ionization MS of native transferrin, especially, allows glycoforms to be analyzed precisely but requires basic knowledge regarding deconvolution of multiply-charged ions which may generate ghost signals upon transformation into a singly-charged form. MS of glycopeptides from tryptic digestion of transferrin delineates site-specific glycoforms and reveals a delicate balance of donor/acceptor substrates or the conformational effect of nascent proteins in cells. Matrix-assisted laser desorption ionization MS of apolipoprotein C-III is a simple method of elucidating the profiles of mucin-type core 1 O-glycans including site occupancy and glycoforms. In this technological review, the principle and pitfalls of MS for CDG are discussed and mass spectra of various types of CDG are presented.

Targeted glycoengineering extends the protein N-glycosylation pathway in the silkworm silk gland

The silkworm silk glands are powerful secretory organs that can produce and secrete proteins at high levels. As such, it has been suggested that the biosynthetic and secretory power of the silk gland can be harnessed to produce and secrete recombinant proteins in tight or loose association with silk fibers. However, the utility of the silkworm platform is constrained by the fact that it has a relatively primitive protein N-glycosylation pathway, which produces relatively simple insect-type, rather than mammalian-type N-glycans. In this study, we demonstrate for the first time that the silk gland protein N-glycosylation pathway can be glycoengineered. We accomplished this by using a dual piggyBac vector encoding two distinct mammalian glycosyltransferases under the transcriptional control of a posterior silk gland (PSG)-specific promoter. Both mammalian transgenes were expressed and each mammalian N-glycan processing activity was induced in transformed silkworm PSGs. In addition, the transgenic animals produced endogenous glycoproteins containing significant proportions of mammalian-type, terminally galactosylated N-glycans, while the parental animals produced none. This demonstration of the ability to glycoengineer the silkworm extends its potential utility as a recombinant protein production platform.

An enzyme-based screening system for the rapid assessment of protein N-glycosylation efficiency in yeast.

N-Glycosylation efficiency is a key parameter when studying components of the protein N-glycosylation pathway, but was recently also recognized as an important factor in the production of glycosylated proteins. We have developed a novel assay to quantify N-glycosylation efficiency of proteins. This assay is based on the secreted activity of yeast acid phosphatase, the proper folding and hence secretion of which is strongly dependent on its N-glycosylation status. The results show that the reporter yields a quantitative measure for protein N-glycosylation in yeast, which is in good agreement with classically used assay based on protein migration patterns on SDS-PAGE. However, the assay is less laborious and is adaptable to high-throughput screening approaches as exemplified.

N-Glycosylation Induces the CTHRC1 Protein and Drives Oral Cancer Cell Migration

Oral squamous cell carcinoma (OSCC) is one of the most pernicious malignancies, but the mechanisms underlying its development and progression are poorly understood. One of the key pathways implicated in OSCC is the canonical Wnt/β-catenin signaling pathway. Previously, we reported that canonical Wnt signaling functions in a positive feedback loop with the DPAGT1 gene, a principal regulator of the metabolic pathway of protein N-glycosylation, to hyperglycosylate E-cadherin and reduce intercellular adhesion. Here, we show that in OSCC, DPAGT1 and canonical Wnt signaling converge to up-regulate CTHRC1 (collagen triple helix repeat containing 1), an N-glycoprotein implicated in tumor invasion and metastasis. We found that in human OSCC specimens, amplification of the levels of CTHRC1 was associated with its hyperglycosylation. Partial inhibition of DPAGT1 expression in OSCC CAL27 cells reduced CTHRC1 abundance by increasing protein turnover, indicating that N-glycosylation stabilizes CTHRC1. Additionally, canonical Wnt signaling promoted β-catenin/T-cell factor transcriptional activity at the CTHRC1 promoter to further elevate CTHRC1 levels. We demonstrate that DPAGT1 promotes cell migration and drives the localization of CTHRC1 to cells at the leading edge of a wound front coincident with drastic changes in cell morphology. We propose that in OSCC, dysregulation of canonical Wnt signaling and DPAGT1-dependent N-glycosylation induces CTHRC1, thereby driving OSCC cell migration and tumor spread.

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

Post-translational modification of proteins with glycosylation is of key importance in many biological systems in eukaryotes, influencing fundamental biological processes and regulating protein function. Changes in glycosylation are therefore of interest in understanding these processes and are also useful as clinical biomarkers of disease. The presence of glycosylation can also inhibit protease digestion and lower the quality and confidence of protein identification by mass spectrometry. While deglycosylation can improve the efficiency of subsequent protease digest and increase protein coverage, this step is often excluded from proteomic workflows. Here, we performed a systematic analysis that showed that deglycosylation with peptide-N-glycosidase F (PNGase F) prior to protease digestion with AspN or trypsin improved the quality of identification of the yeast cell wall proteome. The improvement in the confidence of identification of glycoproteins following PNGase F deglycosylation correlated with a higher density of glycosylation sites. Optimal identification across the proteome was achieved with PNGase F deglycosylation and complementary proteolysis with either AspN or trypsin. We used this combination of deglycosylation and complementary protease digest to identify changes in the yeast cell wall proteome caused by lack of the Alg3p protein, a key component of the biosynthetic pathway of protein N-glycosylation. The cell wall of yeast lacking Alg3p showed specifically increased levels of Cis3p, a protein important for cell wall integrity. Our results showed that deglycosylation prior to protease digestion improved the quality of proteomic analyses even if protein glycosylation is not of direct relevance to the study at hand.

Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

The mevalonate pathway regulates microRNA activity in Caenorhabditis elegans

The mevalonate pathway is highly conserved and mediates the production of isoprenoids, which feed into biosynthetic pathways for sterols, dolichol, ubiquinone, heme, isopentenyl adenine, and prenylated proteins. We found that in Caenorhabditis elegans, the nonsterol biosynthetic outputs of the mevalonate pathway are required for the activity of microRNAs (miRNAs) in silencing their target mRNAs. Inactivation of genes that mediate multiple steps of the mevalonate pathway causes derepression of several miRNA target genes, with no disruption of the miRNA levels, suggesting a role in miRNA-induced silencing complex activity. Dolichol phosphate, synthesized from the mevalonate pathway, functions as a lipid carrier of the oligosaccharide moiety destined for protein N-linked glycosylation. Inhibition of the dolichol pathway of protein N-glycosylation also causes derepression of miRNA target mRNAs. The proteins that mediate miRNA repression are therefore likely to be regulated by N-glycosylation. Conversely, drugs such as statins, which inhibit the mevalonate pathway, may compromise miRNA repression as well as the more commonly considered cholesterol biosynthesis.

Aberrant amplification of the crosstalk between canonical Wnt signaling and N-glycosylation gene DPAGT1 promotes oral cancer

Oral cancer is one of the most aggressive epithelial malignancies, whose incidence is on the rise. Previous studies have shown that in a subset of human oral squamous cell carcinoma (OSCC) tumor specimens, overexpression of the DPAGT1 gene, encoding the dolichol-P-dependent N-acetylglucoseamine-1-phosphate transferase, a key regulator of the metabolic pathway of protein N-glycosylation, drives tumor cell discohesion by inhibiting E-cadherin adhesive function. Recently, we reported that DPAGT1 was a target of the canonical Wnt signaling pathway. Here, we link overexpression of DPAGT1 in human OSCC tumor specimens to aberrant activation of canonical Wnt signaling. We report dramatic increases in β- and γ-catenins at the DPAGT1 promoter and correlate them with reduced expression of a Wnt inhibitor, Dickkopf-1 (Dkk-1). Using human squamous carcinoma cell lines of the head and neck, we show that partial inhibition of DPAGT1 reduces canonical Wnt signaling, indicating that DPAGT1 and canonical Wnt signaling function in a positive feedback loop. We provide evidence that E-cadherin inhibits DPAGT1, canonical Wnt signaling and the OSCC cancer phenotype by depleting nuclear β- and γ-catenins, with hypoglycosylated E-cadherin being the most effective. This suggests that in human OSCC, extensive N-glycosylation of E-cadherin compromises its ability to inhibit canonical Wnt signaling and DPAGT1 expression. Our studies reveal a novel interplay between DPAGT1/N-glycosylation and canonical Wnt signaling and suggest that dysregulation of this crosstalk is a key mechanism underlying OSCC. They also suggest that partial inhibition of DPAGT1 may represent an effective way to restore normal interactions among these essential pathways in oral cancer.

Coordinate regulation of N-glycosylation gene DPAGT1, canonical Wnt signaling and E-cadherin adhesion

The metabolic pathway of protein N-glycosylation influences intercellular adhesion by affecting the composition and cytoskeletal association of E-cadherin protein complexes, or adherens junctions (AJs). In sparse cells, E-cadherin is modified extensively with complex N-glycans and forms nascent AJs, while in dense cultures, hypoglycosylated E-cadherin drives the assembly of mature AJs with increased levels of γ- and α-catenins. N-glycosylation of E-cadherin is controlled by the DPAGT1 gene, a key regulator of the N-glycosylation pathway. DPAGT1 is a target of the canonical Wnt signaling pathway, with both β- and γ-catenins binding to Tcf at its promoter. We now report that DPAGT1 senses cell density through canonical Wnt signaling. In dense cells, depletion of β-catenin from the DPAGT1 promoter correlated with downregulation of its cellular abundance, while loss of nuclear γ-catenin reflected its greater recruitment to AJs. DPAGT1 itself affected canonical Wnt signaling, with forced changes in its expression resulting in corresponding changes in transcriptionally active β-catenin and canonical Wnt activity. Remarkably, a 2.4-fold increase in the DPAGT1 mRNA level resulted in increased N-glycosylation and reduced membrane localization of E-cadherin, coincident with dramatic changes in cell morphology. Lastly, we present evidence that N-glycosylation status of E-cadherin controls its antagonism of canonical Wnt signaling. Transfection of hypoglycosylated E-cadherin mutant, V13, but not fully N-glycosylated E-cadherin, into sparse cells inhibited canonical Wnt activity by depleting nuclear β- and γ-catenins. Collectively, our studies show that cells coordinate DPAGT1 expression and protein N-glycosylation with canonical Wnt signaling and E-cadherin adhesion via positive and negative feedback mechanisms.

A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway

The inability to produce recombinant glycoproteins with authentic N-glycans is a limitation of many heterologous protein expression systems. In the baculovirus-insect cell system, this limitation has been addressed by glycoengineering insect cell lines with mammalian genes encoding protein N-glycosylation functions ("glycogenes") under the transcriptional control of constitutive promoters. However, a potential problem with this approach is that the metabolic load imposed by the expression of multiple transgenes could adversely impact the growth and/or stability of glycoengineered insect cell lines. Thus, we created a new transgenic insect cell line (SfSWT-5) with an inducibly mammalianized protein N-glycosylation pathway. Expression of all six glycogenes was induced when uninfected SfSWT-5 cells were cultured in growth medium containing doxycycline. Higher levels of expression and induction were observed when SfSWT-5 cells were cultured with doxycycline and infected with a baculovirus. Interestingly, there were no major differences in the short-term growth properties of SfSWT-5 cells cultured with or without doxycycline. Furthermore, there were no major differences in the phenotypic stability of these cells after continuous culture for over 300 passages with or without doxycycline. Baculovirus-infected Sf9 and SfSWT-5 cells produced about the same amounts of a model recombinant glycoprotein, but only the latter sialylated this product and sialylation was more pronounced when the cells were treated with doxycycline. In summary, this is the first report of a lower eukaryotic system with an inducibly mammalianized protein N-glycosylation pathway and the first to examine how the presumed metabolic load imposed by multiple transgene expression impacts insect cell growth and stability.