Analysis Of N-glycans Research Articles

Apo-H (beta-2-glycoprotein) intact N-glycan analysis by MALDI-TOF-MS using sialic acid derivatization.

Apo-H is a plasma glycoprotein. Nearly 19% of the molecular weight of this protein is composed of glycans. Up- and down-regulation and structural changes in protein glycans provide diagnostic value for disease detection. Here, an efficient, sensitive, and optimized method is developed for Apo-H N-glycans analysis by MALDI-TOF-MS in positive mode. This bioanalytical method includes sample preparation, sample purification, and detection. An Apo-H enrichment method is developed using standard proteins by anti-Apo-H beads followed by enrichment from plasma samples. SDS-PAGE confirms the Apo-H protein enrichment, which is further verified by LC-MS/MS analysis. The lower ionization efficiency of sialylated glycan hampers their analysis by MALDI-MS. For this, stabilization of sialic acids is done by selective derivatization of carboxyl groups to differentiate between α(2,3)- and α(2,6)-linked sialic acids. Glycans are further purified by HILIC-SPE and analyzed by MALDI-MS. Several branched bi- and tri-antennary glycans with fucosylation and sialylation are identified. The reproducibility of the developed method is tested by analyzing multiple replicates of human plasma, where the same glycans are consistently identified. This method could be applied for the Apo-H glycan profiling of large clinical cohorts for diagnostic purposes.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger.

The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in Aspergillus niger. EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.

Long-term intrathecal administration of morphine vs. baclofen: Differences in CSF glycoconjugate profiles using multiglycomics

Opioid use for treatment of persistent pain has increased dramatically over the past two decades, but it has not resulted in improved pain management outcomes. To understand the molecular mechanisms of opioids, molecular signatures that arise from opioid exposure are often sought after, using various analytical methods. In this study, we performed proteomics, and multiglycomics via sequential analysis of polysialic acids, glycosaminoglycans, N-glycans and O-glycans, using the same cerebral spinal fluid (CSF) sample from patients that had long-term (>2years), intrathecal morphine or baclofen administered via an indwelling pump. Proteomics and N-glycomics signatures between the two treatment groups were highly conserved, while significant differences were observed in polysialic acid, heparan sulfate glycosaminoglycan and O-glycan profiles between the two treatment groups. This represents the first study to investigate the potential relationships between diverse CSF conjugated glycans and long-term intrathecal drug exposure. The unique changes, observed by a sequential analytical workflow, reflect previously undescribed molecular effects of opioid administration and pain management.

Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

BackgroundSulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans.ResultsOur screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated.ConclusionThe present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

DiLeuPMP: A Multiplexed Isobaric Labeling Method for Quantitative Analysis of O-Glycans.

As one of the most important post-translational modifications, glycosylation plays a pivotal role in many essential physiological functions, including cell recognition, signaling, and immune response. Thus, various qualitative and quantitative analytical strategies for glycomic profiling have been developed in recent decades. However, while extensive efforts have been devoted to the analysis of N-glycans, high-throughput quantitative analysis of O-glycans is often overlooked and underexplored. This is partially due to the lack of a universal enzyme for the release of O-glycans from the protein backbone. Furthermore, the traditional chemical releasing method suffers from severe side reactions and involves tedious sample preparation procedures. Here, a multiplexed isobaric labeling method enabled by N,N-dimethyl leucine containing pyrazolone analogue (DiLeuPMP) is introduced. This method combines the release and labeling of O-glycans in a one-pot reaction and achieves accurate MS2-based relative quantification with the ability to process four samples at a time. The method has been applied to core-1 O-glycan standard and three glycoproteins first, and the results demonstrated its validity. Following this proof-of-principle demonstration, we analyzed more complex biological specimen using human serum samples. Overall, this method provides an effective and reliable approach for the profiling and high-throughput quantitative analysis of O-glycans in complex samples.

Glycoprotein analysis using lectin microcolumns and capillary electrophoresis: Characterization of alpha1-acid glycoprotein by combined separation methods

Separations based on combinations of 2.1 mm I.D. high-performance affinity microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.

Abstract 473: Glycomic characterization of carbohydrate antigen-defined pancreatic cancer tissues using lectins and imaging mass spectrometry

Abstract N-glycosylation alterations in tumors are now recognized as cancer hallmarks for its role in oncogenesis, cancer signaling and metastasis. For pancreatic adenocarcinoma (PDAC), the sole FDA-approved biomarker is the carbohydrate antigen 19-9 (CA19-9). Because of CA19-9's association with pancreatitis and other gastrointestinal cancers, it is used for surveillance of disease progression rather than for diagnosis. A recently identified carbohydrate antigen, sialylated tumor-related antigen (sTRA) recognized by the TRA-1-60 mAb, is expressed in a non-overlapping subset of pancreatic cancer and is under consideration for use alongside CA19-9. To better understand these carbohydrate antigens in relation to PDAC pathogenesis, complementary N-glycan analysis approaches were applied to a cohort of 53 patient-matched tumor and normal tissue samples represented by tissue microarrays (TMA) and whole-tissue FFPE samples. The distribution of N-glycans in these tissues were defined using a matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) workflow. Over 100 individual N-glycan species were mapped to their histopathology locations. The same cohort was characterized by multi-round immunofluorescence for CA19-9 and sTRA expression as well as two lectins, PHA-E and GSL-II,. N-glycan IMS data from the TMAs were evaluated by LASSO-regularized logistic regression for the identification of N-glycan analytes with predictive value in differentiating between tumor and normal tissue. LASSO selected N-glycan masses were incorporated alongside biomarker expression data to construct a model for PDAC tissue classification. N-glycans associated with tumor tissue tended to be bi-, tri- and tetra-antennary complex structures with multople fucose, terminal sialic acids and bisecting GlcNAc. Quantitative analysis of the IMS data by LASSO regression identified 9 differentiating N-glycan masses, 7 from tumor cores and 2 from normal cores. When both IMS glycan masses and biomarker expression data for CA19-9 and sTRA were integrated into a single linear regression model, receiver operator curve (ROC) accuracy and AUC improvements, +0.172 and +0.221 respectively, were observed in comparison to models utilizing either dataset individually. Critically, this model was able to rescue cases where biomarker data was missing or insufficient so that they could be correctly classified as tumor or normal tissue. Analysis of this PDAC tissue cohort using MALDI-IMS illustrated the utility of investigating specific N-glycans and N-glycan structural classes correlated with carbohydrate PDAC biomarkers. Integration of N-glycan IMS and biomarker lectin immunostaining bolsters pancreatic cancer identification and posits expanded utility for each approach for PDAC surveillance and early identification. Citation Format: Colin McDowell, Zachary Klamer, Johnathan Hall, Luke Wisniewski, Peggi Angel, Anand Mehta, Brian Haab, Richard R. Drake. Glycomic characterization of carbohydrate antigen-defined pancreatic cancer tissues using lectins and imaging mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 473.

Robustness and repeatability of GlycoWorks RapiFluor-MS IgG N-glycan profiling in a long-term high-throughput glycomic study.

Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here, we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a 5-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three-fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2%. The highest CVs ranging from 16 to 29% accompanied low abundance glycan peaks with the individual relative peak area below 1% that together made up for <2% of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

Structural analysis of N-glycans in medaka gut exposed to silver and titanium dioxide nanoparticles.

Nanomaterials are in general use in a broad range of industries. However, there are concerns that their intense use leads to heavy damage to the aquatic environment, and their discharge harms many aquatic organisms. N-Glycans are widely distributed in eukaryotic organisms and are intimately involved in most life phenomena. However, little is known about N-glycans in aquatic organisms exposed to nanomaterials. In this study, we investigated how nanomaterials affect N-glycans in the gut of adult female medaka. We found that silver nanoparticles exposure had little effect on gut N-glycans, whereas titanium dioxide nanoparticles (TiO2NPs) exposure increased the relative levels of several N-glycans in comparison with control. Structural analysis showed high levels of N-glycans of the high-mannose type, of which five N-glycans were free N-glycans with one β-N-acetylglucosamine residue on the reducing end. The levels of free N-glycans are closely related to protein quality control in the endoplasmic reticulum and cytosol. Our results suggest that TiO2NPs exposure increases the levels of misfolded glycoproteins, resulting in generation of considerable amounts of free N-glycans. Our findings also suggest that TiO2NPs exposure suppresses cytosolic α-mannosidase trimming. This study provides new evidence for the effect of TiO2NPs on medaka gut from the aspect of environmental glycobiology.

In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans.

Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (β1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

A new method for purifying N-Glycans released from milk glycoprotein

Human milk is the first source of nutrition for infants, which delivers an array of unique bioactive components to offspring. Modern bovine-milk-based infant formulas are good substitutes when mother's milk is not available. As the third most abundant component in human milk, human free oligosaccharides (HMOs) may interference the analysis of total N-glycans on the glycoproteins in human milk. Herein, we combined acetone precipitation protein with the filter aided sample preparation method (FASP) to thoroughly remove HMOs and purify N-glycans. Furthermore, we also compared both N-glycosylation and glycoproteins between human and bovine milk, which may provide new ideas for the composition adjustment of infant formula in the food industry. SignificanceWe described a new method, which can successfully remove HMOs, further extract and purify the N-glycans on glycoproteins from pooled human milk for MALDI-TOF MS analysis by applying acetone precipitation and FASP together. We applied the new method to purify the N-glycans from whey proteins in pooled bovine milk and compared the N-glycosylation differences between pooled human and bovine milk by MALDI-TOF MS. We first reported the difference of N-glycan pattern of glycoproteins between pooled bovine and human milk by lectin blotting, and found significant differences in types and abundance of glycoproteins between the two sourced milk.

High-throughput N-glycan screening method for therapeutic antibodies using a microchip-based DNA analyzer: a promising methodology for monitoring monoclonal antibody N-glycosylation.

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.

Negative-mode mass spectrometry in the analysis of invertebrate, fungal, and protist N-glycans.

The approaches for analysis of N-glycans have radically altered in the last 20 years or so. Due to increased sensitivity, mass spectrometry has become the predominant method in modern glycomics. Here, we summarize recent studies showing that the improved resolution and detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has contributed greatly to the discovery of a large range of anionic and zwitterionic N-glycan structures across the different kingdoms of life, whereby MALDI-TOF MS in negative mode is less widely performed than in positive mode. However, its use enables the detection of key fragments indicative of certain sugar modifications such as sulfate, (methyl) phosphate, phosphoethanolamine, (methyl)aminoethylphosphonate, glucuronic, and sialic acid, thereby enabling certain isobaric glycan variations to be distinguished. As we also discuss in this review, complementary approaches such as negative-mode electrospray ionization-MS/MS, Fourier-transform ion cyclotron resonance MS, and ion mobility MS yield, respectively, cross-linkage fragments, high accuracy masses, and isomeric information, thus adding other components to complete the jigsaw puzzle when defining unusual glycan modifications from lower organisms.

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

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

Expanding the phenotype, genotype and biochemical knowledge of ALG3-CDG.

Congenital disorders of glycosylation (CDGs) are a continuously expanding group of monogenic disorders of glycoprotein and glycolipid biosynthesis that cause multisystem diseases. Individuals with ALG3-CDG frequently exhibit severe neurological involvement (epilepsy, microcephaly, and hypotonia), ocular anomalies, dysmorphic features, skeletal anomalies, and feeding difficulties. We present 10 unreported individuals diagnosed with ALG3-CDG based on molecular and biochemical testing with 11 novel variants in ALG3, bringing the total to 40 reported individuals. In addition to the typical multisystem disease seen in ALG3-CDG, we expand the symptomatology of ALG3-CDG to now include endocrine abnormalities, neural tube defects, mild aortic root dilatation, immunodeficiency, and renal anomalies. N-glycan analyses of these individuals showed combined deficiencies of hybrid glycans and glycan extension beyond Man5 GlcNAc2 consistent with their truncated lipid-linked precursor oligosaccharides. This spectrum of N-glycan changes is unique to ALG3-CDG. These expanded features of ALG3-CDG facilitate diagnosis and suggest that optimal management should include baseline endocrine, renal, cardiac, and immunological evaluation at the time of diagnosis and with ongoing monitoring.

Site-specific analysis of N-glycans from different sheep prion strains

Prion diseases are a group of neurodegenerative diseases affecting a wide range of mammalian species, including humans. During the course of the disease, the abnormally folded scrapie prion protein (PrPSc) accumulates in the central nervous system where it causes neurodegeneration. In prion disorders, the diverse spectrum of illnesses exists because of the presence of different isoforms of PrPSc where they occupy distinct conformational states called strains. Strains are biochemically distinguished by a characteristic three-band immunoblot pattern, defined by differences in the occupancy of two glycosylation sites on the prion protein (PrP). Characterization of the exact N-glycan structures attached on either PrPC or PrPSc is lacking. Here we report the characterization and comparison of N-glycans from two different sheep prion strains. PrPSc from both strains was isolated from brain tissue and enzymatically digested with trypsin. By using liquid chromatography coupled to electrospray mass spectrometry, a site-specific analysis was performed. A total of 100 structures were detected on both glycosylation sites. The N-glycan profile was shown to be similar to the one on mouse PrP, however, with additional 40 structures reported. The results presented here show no major differences in glycan composition, suggesting that glycans may not be responsible for the differences in the two analyzed prion strains.