Identification Of Glycoproteins Research Articles

Stretchable Photonic Crystal-Assisted Glycoprotein Identification for Ovarian Cancer Diagnosis.

Photonic crystals with specific wavelengths can realize surface-enhanced excitation and emission intensities of fluorophores and enhance the fluorescence signals of fluorescent molecules. Herein, stretchable photonic crystals with good mechanochromic properties provide continuously adjustable forbidden wavelengths by stretching to change the lattice spacing, with reflectance peaks blue-shifted up to 110 nm to match indicators of different wavelengths and produce differentiated optical enhancement effects. Glycoproteins are significantly identified as clinical markers. However, the wide participation of glycoproteins in various life processes poses enormous complexity and critical challenges for rapid, facile, high-throughput, and accurate clinical analysis or health assessment. In this work, we proposed a stretchable photonic crystal-assisted glycoprotein identification approach for early ovarian cancer diagnosis. Stretchable photonic crystals can provide rich optical information to efficiently identify glycoproteins in complex matrices. A double-indicator fluorescence sensor was designed to respond to the protein trunk and oligosaccharide segment of glycoproteins separately for improved recognition accuracy. Seven typical glycoproteins could be discriminated from proteins, saccharides, or mixture interferents. Clinical ovarian cancer samples for early, intermediate, and advanced ovarian cancer and healthy subjects were verified with 100% accuracy. This strategy of stretchable photonic crystal-assisted glycoprotein identification provides an effective method for accurate, rapid ovarian cancer diagnosis and timely clinical treatment.

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells.

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Identification of Core-Fucosylated Glycoproteins by Single-Step Truncation of N-Glycans.

Alpha-1,6 core fucosylation (CF) is a unique glycoform of N-glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N-glycans. Although the detection ability of low-abundance glycoproteins is improved, sample loss, high cost, and the time-consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single-step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Single-step truncation strategy for core fucosylation glycoproteome analysis in human serum Basic Protocol 2: Liquid chromatography-tandem mass spectrometry quantification of site-specific core fucosylation glycopeptides Alternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single-step truncation strategy.

ProGlycProt V3.0: updated insights into prokaryotic glycoproteins and their glycosyltransferases.

ProGlycProt is a comprehensive database of experimentally validated information about protein glycosylation in prokaryotes, including the glycoproteins, glycosyltransferases, and their accessory enzymes. The first release of ProGlycProt featured experimentally validated information on glycoproteins only. For the second release in 2019, the size and scope of the database were expanded twofold, and experimental data on cognate glycosyltransferases and their accessory proteins was incorporated. The growing research and technology interest in microbial glycoproteins and their enzymes is evident from the steady rise in academic publications and patents in this area. Accordingly, the third update comprises a new section on patents related to glycosylation methods, novel glycosyltransferases, and technologies developed therefrom. The structure gallery is reorganized, wherein the number and quality of the models are upgraded with the help of AlphaFold2. Over the years, the influx of experimental proteomics data into public repositories like PRIDE has surged. Harnessing this legacy data for in-silico glycoprotein identification is a smart approach. Version 3.0 adds 45N-glycoprotein entries annotated from MS datasets available on PRIDE and reviewed by independent research groups. With a 67% rise in entries corresponding to 119 genera of prokaryotes, the ProGlycProt continues to be the exclusive database of experimentally validated comprehensive information about protein glycosylation in prokaryotes.

Recombinant lectin Gg for brain imaging of glycan structure and formation in the CNS node of Ranvier.

The nodes of Ranvier are unmyelinated gaps in the axon, important for the efficient transmission of action potentials. Despite the identification of several glycoproteins involved in node formation and maintenance, glycans' structure and formation in the node remain unclear. Previously, we developed a recombinant lectin from the Clostridium botulinum neurotoxin complex, specific to the galactose and N-acetylgalactosamine terminal epitopes (Gg). Gg stained Neuro2a cells. Here, we show Gg punctuate staining in mouse brain cryosections. Thus, we hypothesized that Gg could help study glycans in the node of Ranvier. Lectin histochemistry on mouse brain cryosections confirmed that Gg binds specifically to the node of Ranvier in the central nervous system (CNS). Using a combination of lectin blotting, glycosidase treatment on tissue sections, and lectin histochemistry, Gg ligands were identified as α-galactose terminal glycoproteins in the perinodal extracellular matrix. Furthermore, we detected the spatiotemporal distribution of galactosylated glycans in the CNS node of Ranvier in mouse brain tissues at different postnatal times. Finally, we observed impaired clustering of galactosylated glycans in the nodes during demyelination and remyelination in cuprizone-induced demyelination and remyelination mouse model. In conclusion, Gg can serve as a novel brain imaging tool in glycobiology and report glycoprotein formation and alterations in the CNS node of Ranvier. Our findings might serve as a first step to establish the role of glycans in the node of Ranvier.

COYOTE: Sequence-derived structural descriptors-based computational identification of glycoproteins.

Glycoproteins play an important and ubiquitous role in many biological processes such as protein folding, cell-to-cell signaling, invading microorganism infection, tumor metastasis, and leukocyte trafficking. The key mechanism of glycoproteins must be revealed to model and refine glycosylated protein recognition, which will eventually assist in the design and discovery of carbohydrate-derived therapeutics. Experimental procedures involving wet-lab experiments to reveal glycoproteins are very time-consuming, laborious, and highly costly. However, costly and tedious experimental procedures can be assisted by ranking the most probable glycoproteins through computational methods with improved accuracy. In this study, we have proposed a novel machine learning-based predictive model for glycoproteins identification. Our proposed model is based on sequence-derived structural descriptors (SDSD) that fill the gap of unavailability of protein 3D structures and lack of accuracy in sequence information alone. Through a series of simulation studies, we have shown that our proposed model gives state-of-the-art generalization performance verified through various machine learning-centric and biologically relevant techniques and metrics. Through data mining in this study, we have also identified the role of descriptors in determining glycoproteins. Python-based standalone code together with a webserver implementation of our proposed model (COYOTE: identifiCation Of glYcoprOteins Through sEquences) is available at the URL: https://sites.google.com/view/wajidarshad/software.

Enrichment of IgG and HRP glycoprotein by dipeptide-based polymeric material

Separation, purification, and identification of glycoproteins are essential for understanding their vital roles in biological and pathological processes. However, glycoproteins are difficult to be captured due to their low abundance, strong interference from non-glycosylated proteins. Here, we report a promising dipeptide-based saccharide recognition platform to selectively enrich two typical glycoproteins, named immunoglobin G (IgG) and horseradish peroxidase (HRP). Different from the conventional glycoprotein enrichment method based on boronic acid affinity or hydrophilic interaction with glycans, the present method was established based on affinity between Pro-Glu (PE) dipeptide and mannose, which is a key unit in the pentasaccharide core of the IgG and HRP glycans. The prepared PE homopolymer surface was proved to selectively bind IgG and HRP superior to that of bovine serum albumin (BSA). Benefiting from this feature, selective enrichment of IgG and HRP was achieved from a protein mixture containing 200-fold BSA interference by using polyPE@SiO2 under a dispersive solid-phase extraction (dSPE) mode. High adsorption capacity, controllable and selective adsorption behaviors, as well as satisfactory recovery demonstrated the high potential of the dipeptide-based polymeric material in IgG and HRP enrichment. This study might provide a new insight to solve the challenging problem of glycoprotein separation.

Recent Strategies for Using Monolithic Materials in Glycoprotein and Glycopeptide Analysis

There is continuous effort towards developing monolithic materials as solid supports for the separation, enrichment, and digestion of glycoproteins. The intention of this review is to discuss and summarize work reported in this area during the period 2015–2021 as a follow-up to our prior review. Reports from the past three decades have already proven the advantages of monolithic materials, such as the ease with which they can be prepared and functionalized, their high permeability and low resistance to mass transfer, and their stability over a wide range of pH. Recent works on glycoprotein analysis introduce different strategies in using monolithic materials specifically in separation, enrichment, and identification of glycoproteins, glycopeptides, and free glycans. A majority of these are focused on boronic acid affinity-based technique and others on lectin affinity and HILIC-based techniques. There are also newly developed ligands that utilize different interactions with glycans, such as encapsulation into β-cyclodextrin vesicles, CH- or OH-π interactions with fullerenes, immunoaffinity with monoclonal antibodies, H-bonding interactions with metallophthalocyanines, coordination interactions with cobalt phthalocyanine tetracarboxylic acid, and hydrophilic interaction with cyclodextrin molecular tubes, zwitterionic iminodiacetic acid, and boric acid. Recent strategies for developing on-line, multidimensional systems use immobilized monolithic enzyme reactors (IMERs) for high-throughput glycoprotein analysis. These works serve as contributions to better understand glycan structure-function relationship, as glycoproteins are now widely accepted disease biomarkers.

The Hitchhiker's guide to glycoproteomics.

Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This ‘Hitchhiker's guide to glycoproteomics’ is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins — a class of biomolecules essential in regulating cell function.

Identification of N-linked Glycoproteins in Silkworm Serum Using Con A Lectin Affinity Chromatography and Mass Spectrometry.

Glycosylation is one of the most common post-translational modifications to occur during protein biosynthesis, but remains poorly understood in insects. In this study, we collected serum proteins from two silkworm developmental stages, namely day 7 of the fifth instar larval stage and day 2 of the pupal stage. Results of SDS–PAGE and periodic acid-Schiff staining revealed that most serum proteins with high abundance were putative glycoproteins. LC-MS/MS identified 149 larval and 303 pupal serum proteins in the Con A lectin-enriched fractions. GO analysis revealed that many serum proteins were involved in the proteolysis and carbohydrate metabolic process. 82 N-linked glycoproteins with at least one glycosylation site were identified. N-Linked glycosylation occurred at the sequon, Asn-X-Ser/Thr, and the proportions of Ser and Thr glycosylation at the hydroxy position were found 39.6% and 60.3%, respectively. The N-glycan structures found in serum glycoproteins were mainly Man2FucGlcNAc2 (67.9%). Since storage protein 1 and transferrin had a relatively high abundance in the serum and could be significantly enriched by Con A lectin, their glycosylation was analyzed in detail. Glycoside hydrases, serine proteases and serpins were found to form three interacting glycoprotein networks using the website STRING. This study provides important clues for the understanding of the function of N-linked glycosylation in metabolism, immunity, and metamorphosis.

Boronic acid-containing carbon dots array for sensitive identification of glycoproteins and cancer cells

Discrimination of glycoproteins and cell types is a significant but difficult issue. Herein, we presented a novel fluorescence sensor array for the detection and identification of glycoproteins and cancer cells based on the specific affinity between boronic acid-containing carbon dots (BA-CDs) and cis-diol residues of polysaccharides. The differential binding affinity of three BA-CDs to various glycoproteins resulted in a different fluorescence turn-on signal pattern caused by aggregation-enhanced emission (AEE), along with negligible response from other proteins. Therefore, BA-CDs encompassing sensing elements and signal indicator into one can enable a fast and accurate discrimination of glycoproteins with simple and easy operation. Seven glycoproteins could be well discriminated at a very low concentration of 10 nmol/L. The discriminating capability of glycoproteins is not sacrificed in both human urine and serum. Notably, different glycoprotein compositions of cancer cells provide more recognizable features for identification of cancer cells, comparing to the total protein. Five cell types could be identified in 15 min at a low concentration of 1000 cells/mL. This method is fast, accurate, and easy operation, and has a potential application in cancer diagnosis.

Identification of potential glycoprotein biomarkers in oral squamous cell carcinoma using sweet strategies.

The prevalence of oral squamous cell carcinoma (OSCC) is high in South and Southeast Asia regions. Most OSCC patients are detected at advanced stages low 5-year survival rates. Aberrant expression of glycosylated proteins was found to be associated with malignant transformation and cancer progression. Hence, identification of cancer-associated glycoproteins could be used as potential biomarkers that are beneficial for diagnosis or clinical management of patients. This study aims to identify the differentially expressed glycoproteins using lectin-based glycoproteomics approaches. Serum samples of 40 patients with OSCC, 10 patients with oral potentially malignant disorder (OPMD), and 10 healthy individuals as control group were subjected to two-dimensional gel electrophoresis (2-DE) coupled with lectin Concanavalin A and Jacalin that specifically bind to N- and O-glycosylated proteins, respectively. Five differentially expressed N- and O-glycoproteins with various potential glycosylation sites were identified, namely N-glycosylated α1-antitrypsin (AAT), α2-HS-glycoprotein (AHSG), apolipoprotein A-I (APOA1), and haptoglobin (HP); as well as O-glycosylated AHSG and clusterin (CLU). Among them, AAT and APOA1 were further validated using enzyme-linked immunosorbent assay (ELISA) (n = 120). It was found that AAT and APOA1 are significantly upregulated in OSCC and these glycoproteins are independent risk factors of OSCC. The clinical utility of AAT and APOA1 as potential biomarkers of OSCC is needed for further evaluation.

Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins.

Glycosylation events play an invaluable role in regulating cellular processes including enzymatic activity, immune recognition, protein stability, and cell–cell interactions. However, researchers have yet to realize the full range of glycan mediated biological functions due to a lack of appropriate chemical tools. Fortunately, the past 25 years has seen the emergence of modified sugar analogs, termed metabolic chemical reporters (MCRs), which are metabolized by endogenous enzymes to label complex glycan structures. Here, we review the major reporters for each class of glycosylation and highlight recent applications that have made a tremendous impact on the field of glycobiology.

Salivary Carbohydrate-Deficient Transferrin in Alcohol- and Nicotine-Dependent Males.

Serum carbohydrate-deficient transferrin (CDT), an 80 kDa glycoprotein, is one of the most commonly employed biomarkers to detect alcohol dependence. Some salivary glycoproteins such as α-amylase, clusterin, haptoglobin, light/heavy-chain immunoglobulin, and transferrin, which alter glycosylation in alcohol-dependent persons, have been suggested to be potential alcohol markers. However, their identification is based on indirect analysis of lectin glycosidic bonds and molecular weight. We investigated the CDT content in the saliva of alcohol- and nicotine-dependent men. The CDT concentration (ng/mL, ng/mg protein) was determined by an Enzyme-Linked Immunosorbent Assay (ELISA) commercial kit in 55 men: 20 healthy social drinkers (C), 10 chronic cigarette smokers (S), 10 alcohol-dependent non-smokers (A), and 15 alcohol-dependent smokers (AS). Surprisingly, there were no differences in the concentrations of CDT between the studied groups. Salivary pH was the lowest in the AS and the highest in the A group. Therefore, salivary CDT cannot be used as an alcohol dependence marker as measured by ELISA. We suggest that direct identification of glycoproteins is necessary to search for potential salivary alcohol biomarkers. Molecules smaller than 40 kDa, which easily translocate from blood to the saliva, might be preferred as salivary alcohol markers.

Identification and Characterization of Glycoproteins and Their Responsive Patterns upon Ethylene Stimulation in the Rubber Latex.

Natural rubber is an important industrial material, which is obtained from the only commercially cultivated rubber tree, Hevea brasiliensis. In rubber latex production, ethylene has been extensively used as a stimulant. Recent research showed that post-translational modifications (PTMs) of latex proteins, such as phosphorylation, glycosylation and ubiquitination, are crucial in natural rubber biosynthesis. In this study, comparative proteomics was performed to identify the glycosylated proteins in rubber latex treated with ethylene for different days. Combined with Pro-Q Glycoprotein gel staining and mass spectrometry techniques, we provided the first visual profiling of glycoproteomics of rubber latex and finally identified 144 glycosylated protein species, including 65 differentially accumulated proteins (DAPs) after treating with ethylene for three and/or five days. Gene Ontology (GO) functional annotation showed that these ethylene-responsive glycoproteins are mainly involved in cell parts, membrane components and metabolism. Pathway analysis demonstrated that these glycosylated rubber latex proteins are mainly involved in carbohydrate metabolism, energy metabolism, degradation function and cellular processes in rubber latex metabolism. Protein–protein interaction analysis revealed that these DAPs are mainly centered on acetyl-CoA acetyltransferase and hydroxymethylglutaryl-CoA synthase (HMGS) in the mevalonate pathway for natural rubber biosynthesis. In our glycoproteomics, three protein isoforms of HMGS2 were identified from rubber latex, and only one HMGS2 isoform was sharply increased in rubber latex by ethylene treatment for five days. Furthermore, the HbHMGS2 gene was over-expressed in a model rubber-producing grass Taraxacum Kok-saghyz and rubber content in the roots of transgenic rubber grass was significantly increased over that in the wild type plant, indicating HMGS2 is the key component for natural rubber production.

O-Acetylated Chemical Reporters of Glycosylation Can Display Metabolism-Dependent Background Labeling of Proteins but Are Generally Reliable Tools for the Identification of Glycoproteins.

Monosaccharide analogs bearing bioorthogonal functionalities, or metabolic chemical reporters (MCRs) of glycosylation, have been used for approximately two decades for the visualization and identification of different glycoproteins. More recently, proteomics analyses have shown that per-O-acetylated MCRs can directly and chemically react with cysteine residues in lysates and potentially cells, drawing into question the physiological relevance of the labeling. Here, we report robust metabolism-dependent labeling by Ac42AzMan but not the structurally similar Ac44AzGal. However, the levels of background chemical-labeling of cell lysates by both reporters are low and identical. We then characterized Ac42AzMan labeling and found that the vast majority of the labeling occurs on intracellular proteins but that this MCR is not converted to previously characterized reporters of intracellular O-GlcNAc modification. Additionally, we used isotope targeted glycoproteomics (IsoTaG) proteomics to show that essentially all of the Ac42AzMan labeling is on cysteine residues. Given the implications this result has for the identification of intracellular O-GlcNAc modifications using MCRs, we then performed a meta-analysis of the potential O-GlcNAcylated proteins identified by different techniques. We found that many of the proteins identified by MCRs have also been found by other methods. Finally, we randomly selected four proteins that had only been identified as O-GlcNAcylated by MCRs and showed that half of them were indeed modified. Together, these data indicate that the selective metabolism of certain MCRs is responsible for S-glycosylation of proteins in the cytosol and nucleus. However, these results also show that MCRs are still good tools for unbiased identification of glycosylated proteins, as long as complementary methods are employed for confirmation.

NEGATIVE ION MASS SPECTROMETRY FOR THE ANALYSIS OF N-LINKED GLYCANS.

N-glycans from glycoproteins are complex, branched structures whose structural determination presents many analytical problems. Mass spectrometry, usually conducted in positive ion mode, often requires extensive sample manipulation, usually by derivatization such as permethylation, to provide the necessary structure-revealing fragment ions. The newer but, so far, lesser used negative ion techniques, on the contrary, provide a wealth of structural information not present in positive ion spectra that greatly simplify the analysis of these compounds and can usually be conducted without the need for derivatization. This review describes the use of negative ion mass spectrometry for the structural analysis of N-linked glycans and emphasises the many advantages that can be gained by this mode of operation. Biosynthesis and structures of the compounds are described followed by methods for release of the glycans from the protein. Methods for ionization are discussed with emphasis on matrix-assisted laser desorption/ionization (MALDI) and methods for producing negative ions from neutral compounds. Acidic glycans naturally give deprotonated species under most ionization conditions. Fragmentation of negative ions is discussed next with particular reference to those ions that are diagnostic for specific features such as the branching topology of the glycans and substitution positions of moieties such as fucose and sulfate, features that are often difficult to identify easily by conventional techniques such as positive ion fragmentation and exoglycosidase digestions. The advantages of negative over positive ions for this structural work are emphasised with an example of a series of glycans where all other methods failed to produce a structure. Fragmentation of derivatized glycans is discussed next, both with respect to derivatives at the reducing terminus of the molecules, and to methods for neutralization of the acidic groups on sialic acids to both stabilize them for MALDI analysis and to produce the diagnostic fragments seen with the neutral glycans. The use of ion mobility, combined with conventional mass spectrometry is described with emphasis on its use to extract clean glycan spectra both before and after fragmentation, to separate isomers and its use to extract additional information from separated fragment ions. A section on applications follows with examples of the identification of novel structures from lower organisms and tables listing the use of negative ions for structural identification of specific glycoproteins, glycans from viruses and uses in the biopharmaceutical industry and in medicine. The review concludes with a summary of the advantages and disadvantages of the technique. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Rapid and sensitive glycan targeting by lectin-SERS assay.

Glycosylation is an important part of cell signalling that is implicated in many disease states in which glycans play an essential role. Therefore rapid and sensitive differentiation of glycans on proteins is highly desirable. Current technologies for glycan structural analysis normally involve the isolation of glycans from proteins, or enrichment of glycopeptides, and detection by mass spectrometry, which requires relatively large amounts of sample and is not able to be used by non-specialist laboratories. Herein we present a simple and new strategy for targeting the glycans on a protein (with IgG as a model glycoprotein) using surface-enhanced Raman scattering (SERS) coupled to glycan-binding WGA (wheat germ agglutinin) lectin, in a lectin-SERS assay. With one drop (1 μL) of glycoprotein solution, our lectin-SERS assay can detect as low as 10 ng IgG within two hours with high glycan specificity. We extend our technique to examine the surface glycan profiles on two human colorectal cancer cell lines, which show different and unique glycan signatures specific to the target cell lines. Thus, we believe that this method could be potentially used for the real-time and in situ monitoring of glycans on the surface of cells or tissue or in body fluids, and is thus a powerful tool for glycomics research.

MoGT2 Is Essential for Morphogenesis and Pathogenicity of Magnaporthe oryzae.

Magnaporthe oryzae causes the rice blast disease, which is one of the most serious diseases of cultivated rice worldwide. Glycosylation is an important posttranslational modification of secretory and membrane proteins in all eukaryotes, catalyzed by glycosyltransferases (GTs). In this study, we identified and characterized a type 2 glycosyltransferase, MoGt2, in M. oryzae Targeted gene deletion mutants of MoGT2 (mogt2Δ strains) were nonpathogenic and were impaired in vegetative growth, conidiation, and appressorium formation at hyphal tips. Moreover, MoGT2 plays an important role in stress tolerance and hydrophobin function of M. oryzae Site-directed mutagenesis analysis showed that conserved glycosyltransferase domains (DxD and QxxRW) are critical for biological functions of MoGt2. MoGT2 deletion led to altered glycoproteins during M. oryzae conidiation. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified several candidate proteins as potential substrates of MoGt2, including several heat shock proteins, two coiled-coil domain-containing proteins, aminopeptidase 2, and nuclease domain-containing protein 1. On the other hand, we found that a conidiation-related gene, genes involved in various metabolism pathways, and genes involved in cell wall integrity and/or osmotic response were differentially regulated in the mogt2Δ mutant, which may potentially contribute to its condiation defects. Taken together, our results show that MoGt2 is important for infection-related morphogenesis and pathogenesis in M. oryzaeIMPORTANCE The ascomycete fungus Magnapothe oryzae is the causal agent of rice blast disease, leading to severe loss in cultivated rice production worldwide. In this study, we identified a conserved type 2 glycosyltransferase named MoGt2 in M. oryzae The mogt2Δ targeted gene deletion mutants exhibited pleiotropic defects in vegetative growth, conidiation, stress response, hyphal appressorium-mediated penetration, and pathogenicity. Furthermore, conserved glycosyltransferase domains are critical for MoGt2 function. The comparative transcriptome analysis revealed potential target genes under MoGt2 regulation in M. oryzae conidiation. Identification of potential glycoproteins modified by MoGt2 provided information on its regulatory mechanism of gene expression and biological functions. Overall, our study represents the first report of type 2 glycosyltransferase function in M. oryzae infection-related morphogenesis and pathogenesis.

Capture and Analysis of Cell Surface N-Glycans by Hydrazide-Modified Magnetic Beads and CE-LIF

Cell surface proteins are important target molecules for biomarkers discovery. Diseases are often reflected within the glycan profiles of proteins. As such, studying the glycan profiles of cell surface proteins may give rise to new biomarker or drug target discoveries. Focused on cell surface glycoproteins, hydrazine technology had been applied to cell surface protein capturing (CSC) technology. In this study, hydrazide-functionalized magnetic beads were used to capture surface glycoproteins on intact cells and the released N-glycan profiling, except protein profiling, was investigated by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The use of magnetic beads gave rapid separation and enrichment of the captured glycoproteins. The sample processing parameters, including the ratio of cells to hydrazine groups, oxidation time and cell incubation time with magnetic beads, cell lysis and glycopeptide cleavage methods, were evaluated using MALDI-TOF–TOF mass spectrometry analysis. Subsequently, citric acid was applied at optimized temperature for the glycan cleavage from the beads. The technique developed has the potential in sensing, diagnostic and therapeutic applications by speeding up the method development of glycoprotein identification, N-glycan profiling, drug target screening and biomarker discovery.