Glycomic Profiling Research Articles

The Value of Serum Immunoglobulin G Glycome in the Preoperative Discrimination of Peritoneal Metastasis from Advanced Gastric Cancer.

Background: Peritoneal metastasis, associated with poor prognosis in gastric cancer, is difficult to discriminate from advanced gastric cancer preoperatively. However, operative diagnosis could bring both mental and physical trauma and economic burden for patients. Consequently, a non-invasive biomarker is necessary to reduce the burden of operative diagnosis and improve survival quality of patients. This study aims to elucidate the correlation between Immunoglobulin G (IgG) N-glycome and peritoneal metastasis and find potential biomarkers in preoperative discrimination of peritoneal metastasis from advanced gastric cancer based on the comprehensive sample set.Methods: A total of 373 gastric cancer patients were enrolled and randomly sorted into training cohort (n=249) and validation cohort (n=124). The IgG N-glycome composition was analyzed by ultra-performance liquid chromatography.Results: Twenty-four glycan peaks were directly detected and 15 traits based on the same structures were evaluated between peritoneal metastasis group and advanced gastric cancer group. Several differences in IgG glycosylation were found: sialylation and fucosylation were increased in peritoneal metastasis, while neutral glycosylation, monogalacosylation and bisecting GlcNAc were decreased. Based on the significant glycomics profile, a glyco-model composed of five glycan peaks (GP6, GP9, GP11, GP21 and GP23) was established with area under the receiver operating characteristic curve (AUC) value of 0.80 (training cohort) and 0.77 (validation cohort), which showed good potential in discriminating peritoneal metastasis from advanced gastric cancer. The diagnostic performance of this model was further validated in a combined cohort (AUC=0.79). Two patients with gastric cancer were selected to perform and demonstrate the usage of the diagnostic workflow.Conclusions: Here we firstly present IgG glycome profiles in a large number of preoperative peritoneal metastasis serums. The IgG glycan was highly associated with peritoneal metastasis. These findings enhance the understanding of peritoneal metastasis. Besides, our results suggested that the newly established glyco-model could be a reliable predictor of the presence of peritoneal metastasis in patients with advanced gastric cancer.

Blueberry cell wall fractionation, characterization and glycome profiling

Blueberry powder was sequentially fractionated to water (WSF), chelator (CSF) and sodium carbonate (NSF) soluble fractions. Uronic acid content was above 22 μg/mg AIS. The 4% potassium hydroxide (4KSF) and 24% potassium hydroxide (24KSF) soluble fractions were hemicellulose rich fractions. Cell wall glycan-directed monoclonal antibodies (mAbs) based glycome profiling analyses revealed that WSF, CSF and NSF fractions had epitopes of homogalacturonan (HG), arabinogalactan (AG), and xyloglucan (XG), but no xylan. 4KSF and 24KSF fractions had higher abundance of xyloglucan and xylan epitopes in addition to pectic epitopes. Arabinose and galactose were the two major neutral sugars in the WSF, CSF, and NSF. Xylose represented about 70 mol% neutral sugar in 4KSF and 24KSF fractions. Increased flexibility and presence of protein likely influence the propensity of pectin to bind anthocyanins.

Structural Analysis on the Effect of Base-Catalysed Delignification Process Parameters on Palm Oil Empty Fruit Bunches Fibres using Glycome Profiling

Malaysian Oil Palm Empty Fruit Bunches (EFB) fibre was used as a model substrate and was subjected to base-catalysed delignification technique as a function of different temperature, time and base concentration. The resultant substrates were analysed using glycome profiling technique to ascertain the degree of delignification effectiveness and position of the residual lignin in the structure of the pre-treated biomass. The study indicated that the base-catalysed delignification technique removes all the outer lignin structure of the fibre due to the accessibility of the base to the surface of the biomass. Base wash concentration using sodium hydroxide (NaOH) and temperature are the two main factors that determine the effectiveness of lignin removal and increment of surface area accessibility (SAA). The pre-treated biomass that contains the lowest amount of residual lignin content of 6.19 wt.% which corresponds to 74.47% lignin removal shows complete removal of the outer layer of lignin structure but leaves the inner lignin structure intact. This study suggests that the complex interaction between different cell wall constituents plays a much bigger role in order to achieve efficient delignification.

Enhancing FVIII Expression By Modifying B-Domain Sequence for Hemophilia A Gene Therapy

Background: We have previously shown that conserving six N-linked glycosylation sites of the human factor VIII (FVIII) B-domain sequences in a synthetic linker, V3 (31aa/N6), increased FVIII expression by 3-fold over B-domain deleted FVIII (BDD-FVIII) following AAV mediated gene transfer in mice (McIntosh et. al. Blood 2013). These N-linked glycosylation consensus sequences (N-X-T/S) are highly conserved across different species but their role in the context of transgenic FVIII expression following gene transfer remains unknown. We, therefore, undertook systemic B-domain linker engineering studies to elucidate the mechanism by which the V3-linker improves FVIII expression following AAV mediated gene transfer.Methods: A series of FVIII variants were made by modifying the B-domain linker size and amino acid sequence and then cloned into AAV or lentiviral vectors. The latter were used for making stable FVIII producer cell lines to facilitate biochemical characterization including glycome profile using MALDI-TOF mass spectrometry. The AAV vectors, in which expression of the FVIII gene was directed by a small synthetic liver specific promoter (HLP) were used to assess transgene expression in-vitro and in murine models.Results: Purified recombinant FVIII-V3 had the same biological activity as that of full-length and BDD FVIII protein concentrates when assessed by thrombin generation and vWF/FVIII binding affinity assays (Dissociation constant (KD) <1nM) suggesting that V3 linker did not interfere with the potency of FVIII. Replacement of the asparagine residues with alanine (FVIII-V5) within the six N-linked glycosylation motifs resulted in a >20 reduction in glycan structures but the expression profile and biological properties of FVIII-V5 were comparable to those of FVIII-V3. In contrast, FVIII-V1 (44aa), in which the N-linked glycosylation triplets in FVIII-V3 were flanked by two additional amino acids that are normally adjacent to these moieties in the native FVIII B-domain, was expressed at 3-fold low levels when compared to FVIII-V3 and V5 in mice. Similarly, randomly scrambling the amino acid sequence of the V3-linker (FVIII-V2) without modifying size resulted in a 7-fold decrease in FVIII expression levels in mice. Notably, FVIII-V3 and V5 mRNA levels were 4-fold higher than the levels observed with BDD-FVIII and FVIII-V1 variants. Deletion analysis of the V3 B-domain linker identified a shorter form (FVIII-N2 19aa/N2) that exhibited similar levels of FVIII secretion to those observed with FVIII-V3.Conclusion: We have critically evaluated the role of the FVIII B domain V3 linker on human FVIII expression in human cell lines and mice following AAV mediated gene transfer. In all cases, the FVIII-V3 variant performed better than the BDD-FVIII, in large part due to an increase in FVIII-V3 mRNA levels. Although the FVIII-V3 molecule is fully glycosylated, our studies show that glycan content did not significantly influence FVIII expression but increasing size and modifying amino-acid content of the linker, markedly reduced FVIII expression following AAV mediated gene transfer in mice. Therefore, the results presented here support the use of FVIII-V3 for gene therapy for hemophilia A in our on-going investigator lead Phase 1 trial. DisclosuresMcIntosh:Freeline: Consultancy, Equity Ownership; BioMarin: Patents & Royalties. Dane:Freeeline: Employment, Equity Ownership. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties.

Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns

The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.

Glycomic Profiling Highlights Increased Fucosylation in Pseudomyxoma Peritonei

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma that most often originates from the appendix, and grows in the peritoneal cavity filling it with mucinous ascites. KRAS and GNAS mutations are frequently found in PMP, but other common driver mutations are infrequent. As altered glycosylation can promote carcinogenesis, we compared N-linked glycan profiles of PMP tissues to those of normal appendix. Glycan profiles of eight normal appendix samples and eight low-grade and eight high-grade PMP specimens were analyzed by mass spectrometry. Our results show differences in glycan profiles between PMP and the controls, especially in those of neutral glycans, and the most prominent alteration was increased fucosylation. We further demonstrate up-regulated mRNA expression of four fucosylation-related enzymes, the core fucosylation performing fucosyltransferase 8 and three GDP-fucose biosynthetic enzymes in PMP tissues when compared with the controls. Up-regulated protein expression of the latter three enzymes was further observed in PMP cells by immunohistochemistry. We also demonstrate that restoration of fucosylation either by salvage pathway or by introduction of an expression of intact GDP-mannose 4,6-dehydratase enhance expression of MUC2, which is the predominant mucin molecule secreted by the PMP cells, in an intestinal-derived adenocarcinoma cell line with defective fucosylation because of deletion in the GDP-mannose 4,6-dehydratase gene. Thus, altered glycosylation especially in the form of fucosylation is linked to the characteristic mucin production of PMP. Glycomic data are available via ProteomeXchange with identifier PXD010086.

Glycomics in rare diseases: from diagnosis tomechanism

The National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP) studies rare genetic disorders not only to achieve diagnoses, but to understand human biology. To ascertain the contribution of protein glycosylation to rare diseases, the NIH UDP used mass spectrometry to agnostically identify abnormalities of N-linked and O-linked glycans in plasma and free oligosaccharides in the urine of 207 patients. 60% of UDP patients had a glycome profile that deviated from control values in at least 1 fluid. Additional evaluation of the fibroblast glycome in 66 patients with abnormalities in plasma and/or urine revealed a consistent glycome phenotype in 83% of these cases. Many of these patients may have secondary glycosylation defects, since it is unlikely that they all have congenital disorders of glycosylation (CDGs). In fact, whole exome sequencing revealed only a few patients with CDGs, along with several others having disorders indirectly altering glycosylation. In summary, we describe a biochemical phenotyping screen to identify defects in protein glycosylation that can elucidate mechanisms of disease among NIH UDP patients.

Current Technologies for Complex Glycoproteomics and Their Applications to Biology/Disease-Driven Glycoproteomics.

Glycoproteomics is an important recent advance in the field of glycoscience. In glycomics, glycan structures are comprehensively analyzed after glycans are released from glycoproteins. However, a major limitation of glycomics is the lack of insight into glycoprotein functions. The Biology/Disease-driven Human Proteome Project has a particular focus on biological and medical applications. Glycoproteomics technologies aimed at obtaining a comprehensive understanding of intact glycoproteins, i.e., the kind of glycan structures that are attached to particular amino acids and proteins, have been developed. This Review focuses on the recent progress of the technologies and their applications. First, the methods for large-scale identification of both N- and O-glycosylated proteins are summarized. Next, the progress of analytical methods for intact glycopeptides is outlined. MS/MS-based methods were developed for improving the sensitivity and speed of the mass spectrometer, in parallel with the software for complex spectrum assignment. In addition, a unique approach to identify intact glycopeptides using MS1-based accurate masses is introduced. Finally, as an advance of glycomics, two approaches to provide the spatial distribution of glycans in cells are described, i.e., MS imaging and lectin microarray. These methods allow rapid glycomic profiling of different types of biological samples and thus facilitate glycoproteomics.

Characterization of plant carbon substrate utilization by Auxenochlorella protothecoides

Algae hold great potential as a source of renewable fuel due to their ability to produce refinery-compatible diesel and jet fuel precursors. Significant effort has been made to maximize productivity under photoautotrophic growth conditions; however, little progress has been made to discover and understand reduced carbon assimilation pathways or enzymatic degradation of complex carbon substrates in algae. We purport that utilization of plant-based carbon substrates in addition to photosynthesis (mixotrophic growth) for biochemical assimilation into biomass, biofuels, and bioproducts, can increase cultivation productivity and improve the economic viability of algal-derived biofuels. Herein we report that a freshwater production strain of microalgae, Auxenochlorella protothecoides UTEX 25, is capable of directly degrading and utilizing non-food plant substrates, such as switchgrass, for cell growth. Glycome profiling of plant substrates before and after addition to A. protothecoides cultures demonstrates the utilization of xyloglucans. Genomic, proteomic and transcriptomic analyses revealed the identity of many enzymes that are hypothesized to be involved in complex carbohydrate degradation, including several family 5 and 9 glycosyl hydrolases. This work paves the way for future designer engineering of plant‑carbon utilization to further improve productivity of algal production strains.

Elicitors and defense gene induction in plants with altered lignin compositions.

A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositions led to differential extractability of a range of heterogeneous oligosaccharide epitopes, with elicitor activity originating from different cell wall polymers. Alteration of lignin composition affects interactions with plant cell wall matrix polysaccharides to alter the sequestration of multiple latent defense signal molecules with an impact on biotic stress responses.

Mining the acidic serum proteome utilizing off-gel isoelectric focusing and label free quantitative liquid chromatography mass spectrometry

Serum remains an attractive source for the discovery of disease related biomarkers due to its intimate interaction with the majority of tissues within the body. The dynamic range of proteins present within serum has long complicated the ability to detect low level tissue leakage proteins that offer more promise as potential biomarkers due to their higher degree of specificity. Depletion strategies, using affinity based sorbents, to remove the most abundant serum proteins are routinely used for matrix simplification during discovery strategies focused on the serum proteome or glycoproteome. Glycoproteins bearing glycans with terminal sialic acid residues have been shown to be differentially expressed in a variety of cancers and are of interest due to the varied involvement of sialic acid in biological functions related to cancer development and metastasis. Herein, we describe the use of 14P serum depletion with subsequent off-gel isoelectric focusing using narrow pH range acidic strips, to facilitate simplification and enrichment of the acidic proteome and glycoproteome prior to label free liquid chromatography mass spectrometry (LC–MS) based proteomic analysis. The developed platform enabled the detection of proteins present within serum at sub-nanomolar concentrations while data analysis using a variety of bioinformatics resources suggested that many of the detected proteins were tissue leakage proteins or proteins associated with specific cellular compartments, rather than traditional secreted proteins. Comparison with theoretical isoelectric point (pI) values also revealed that some detected proteins had higher pI values than the separation range applied during the IEF step, suggesting that their presence may be due to glycans with high degrees of sialylation being attached to the protein backbone, as well as the presence of other post translational modifications. Such findings agree with offline glycomic profiling of the IEF fractions wherein increasing degrees of sialylation were detected across the pH strip from the basic towards the acidic end. Application of the platform using pooled serum from patients bearing gastric cancer, both before and after surgical intervention for tumour removal, revealed a number of differentially expressed proteins associated with cell recognition and cell signalling. The developed platform enabled excellent sensitivity and offers strong potential for application within biomarker discovery studies focused on the acidic proteome.

Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples.

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS2 and pseudo-MS3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

Towards automation of glycomic profiling of complex biological materials.

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.

Intact transferrin and total plasma glycoprofiling for diagnosis and therapy monitoring in phosphoglucomutase-I deficiency

Phosphoglucomutase 1 (PGM1) deficiency results in a mixed phenotype of a Glycogen Storage Disorder and a Congenital Disorder of Glycosylation (CDG). Screening for abnormal glycosylation has identified more than 40 patients, manifesting with a broad clinical and biochemical spectrum which complicates diagnosis. Together with the availability of D-galactose as dietary therapy, there is an urgent need for specific glycomarkers for early diagnosis and treatment monitoring. We performed glycomics profiling by high-resolution QTOF mass spectrometry in a series of 19 PGM1-CDG patients, covering a broad range of biochemical and clinical severity. Bioinformatics and statistical analysis were used to select glycomarkers for diagnostics and define glycan-indexes for treatment monitoring. Using 3 transferrin glycobiomarkers, all PGM1-CDG patients were diagnosed with 100% specificity and sensitivity. Total plasma glycoprofiling showed an increase in high mannose glycans and fucosylation, while global galactosylation and sialylation were severely decreased. For treatment monitoring, we defined 3 glycan-indexes, reflecting normal glycosylation, a lack of complete glycans (LOCGI) and of galactose residues (LOGI). These indexes showed improved glycosylation upon D-galactose treatment with a fast and near-normalization of the galactose index (LOGI) in 6 out of 8 patients and a slower normalization of the LOCGI in all patients. Total plasma glycoprofiling showed improvement of the global high mannose glycans, fucosylation, sialylation, and galactosylation status on D-galactose treatment. Our study indicates specific glycomarkers for diagnosis of mildly and severely affected PGM1-CDG patients, and to monitor the glycan-specific effects of D-galactose therapy.

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells

Increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4-5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration.

Differential recognition of natural and remodeled glycotopes by three Diocleae lectins.

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I1 and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIβ glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.

Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed Alkaline-Oxidative Pretreatment

Both untransformed poplar and genetically modified “zip-lignin” poplar, in which additional ester bonds were introduced into the lignin backbone, were subjected to mild alkaline and copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment. Our hypothesis was that the lignin in zip-lignin poplar would be removed more easily than lignin in untransformed poplar during this alkaline pretreatment, resulting in higher sugar yields following enzymatic hydrolysis. We observed improved glucose and xylose hydrolysis yields for zip-lignin poplar compared to untransformed poplar following both alkaline-only pretreatment (56% glucose yield for untransformed poplar compared to 67% for zip-lignin poplar) and Cu-AHP pretreatment (77% glucose yield for untransformed poplar compared to 85% for zip-lignin poplar). Compositional analysis, glycome profiling, and microscopy all supported the notion that the ester linkages increase delignification and improve sugar yields. Essentially no differences were noted in the m...

Use of Mass Spectrometry to Screen Glycan Early Markers in Hepatocellular Carcinoma.

Association between altered glycosylation patterns and poor prognosis in cancer points glycans as potential specific tumor markers. Most proteins are glycosylated and functionally arranged on cell surface and extracellular matrix, mediating interactions and cellular signaling. Thereby, aberrant glycans may be considered a pathological phenotype at least as important as changes in protein expression for cancer and other complex diseases. As most serum glycoproteins have hepatic origin, liver disease phenotypes, such as hepatocellular carcinoma (HCC), may present altered glycan profile and display important modifications. One of the prominent obstacles in HCC is the diagnostic in advanced stages when patients have several liver dysfunctions, limiting treatment options and life expectancy. The characterization of glycomic profiles in pathological conditions by means of mass spectrometry (MS) may lead to the discovery of early diagnostic markers using non-invasive approaches. MS is a powerful analytical technique capable of elucidating many glycobiological issues and overcome limitations of the serological markers currently applied in clinical practice. Therefore, MS-based glycomics of tumor biomarkers is a promising tool to increase early detection and monitoring of disease.

Structural analysis of N-/O-glycans assembled on proteins in yeasts.

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.