Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Multi-epitope vaccine design using in silico analysis of glycoprotein and nucleocapsid of NIPAH virus.

According to the 2018 WHO R&D Blueprint, Nipah virus (NiV) is a priority disease, and the development of a vaccine against NiV is strongly encouraged. According to criteria used to categorize zoonotic diseases, NiV is a stage III disease that can spread to people and cause unpredictable outbreaks. Since 2001, the NiV virus has caused annual outbreaks in Bangladesh, while in India it has caused occasional outbreaks. According to estimates, the mortality rate for infected individuals ranges from 70 to 91%. Using immunoinformatic approaches to anticipate the epitopes of the MHC-I, MHC-II, and B-cells, they were predicted using the NiV glycoprotein and nucleocapsid protein. The selected epitopes were used to develop a multi-epitope vaccine construct connected with linkers and adjuvants in order to improve immune responses to the vaccine construct. The 3D structure of the engineered vaccine was anticipated, optimized, and confirmed using a variety of computer simulation techniques so that its stability could be assessed. According to the immunological simulation tests, it was found that the vaccination elicits a targeted immune response against the NiV. Docking with TLR-3, 7, and 8 revealed that vaccine candidates had high binding affinities and low binding energies. Finally, molecular dynamic analysis confirms the stability of the new vaccine. Codon optimization and in silico cloning showed that the proposed vaccine was expressed to a high degree in Escherichia coli. The study will help in identifying a potential epitope for a vaccine candidate against NiV. The developed multi-epitope vaccine construct has a lot of potential, but they still need to be verified by in vitro & in vivo studies.

Glycoproteomics: Charting new territory in mass spectrometry and glycobiology.

Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.

Ultrasound modification on milk fat globule membrane and soy lecithin to improve the physicochemical properties, microstructure and stability of mimicking human milk fat emulsions.

Starting from the consideration of the structure of human milk fat globule (MFG), this study aimed to investigate the effects of ultrasonic treatment on milk fat globule membrane (MFGM) and soy lecithin (SL) complexes and their role in mimicking human MFG emulsions. Ultrasonic power significantly affected the structure of the MFGM-SL complex, further promoting the unfolding of the molecular structure of the protein, and then increased solubility and surface hydrophobicity. Furthermore, the microstructure of mimicking MFG emulsions without sonication was unevenly distributed, and the average droplet diameter was large. After ultrasonic treatment, the droplets of the emulsion were more uniformly dispersed, the particle size was smaller, and the emulsification properties and stability were improved to varying degrees. Especially when the ultrasonic power was 300 W, the mimicking MFG emulsion had the highest encapsulation rate and emulsion activity index and emulsion stability index were increased by 60.88 % and 117.74 %, respectively. From the microstructure, it was observed that the spherical droplets of the mimicking MFG emulsion after appropriate ultrasonic treatment remain well separated without obvious flocculation. This study can provide a reference for the screening of milk fat globules mimicking membrane materials and the further utilization and development of ultrasound in infant formula.

N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Enrichment methods of N-linked glycopeptides from human serum or plasma: A mini-review.

Human diseases often correlate with changes in protein glycosylation, which can be observed in serum or plasma samples. N-glycosylation, the most common form, can provide potential biomarkers for disease prognosis and diagnosis. However, glycoproteins constitute a relatively small proportion of the total proteins in human serum and plasma compared to the non-glycosylated protein albumin, which constitutes the majority. The detection of microheterogeneity and low glycan abundance presents a challenge. Mass spectrometry facilitates glycoproteomics research, yet it faces challenges due to interference from abundant plasma proteins. Therefore, methods have emerged to enrich N-glycans and N-linked glycopeptides using glycan affinity, chemical properties, stationary phase chemical coupling, bioorthogonal techniques, and other alternatives. This review focuses on N-glycans and N-glycopeptides enrichment in human serum or plasma, emphasizing methods and applications. Although not exhaustive, it aims to elucidate principles and showcase the utility and limitations of glycoproteome characterization.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Generally applicable circulating tumor cell enrichment and identification through a membrane glycoprotein-targeting strategy combining magnetic isolation and biological orthogonality labeling.

Although the importance of circulating tumor cells (CTCs) has been widely recognized, it is still a challenge to realize high-efficiency and accurate enrichment and identification of highly heterogeneous CTCs derived from various types of tumors in complex cancer processes. Currently, the most widely used methods follow the general idea of sequential immunoaffinitive capture and immunostaining to achieve the abovementioned goal. However, different organ/tissue origins as well as the inherent heterogeneity of CTCs would lead to the missed detection of certain CTC subtypes using such methods. Further, immunocytochemistry (ICC) immunostaining disrupts the physiological structure of cells, severely limiting the detection and application scenarios that require the participation of live cells. To address these limitations, we have developed a generally applicable strategy for the isolation and labeling of CTCs. This strategy focuses on targeting the universal characteristics of all tumor cells, specifically the abnormally expressed cell membrane glycoproteins, such as the transferrin receptor and sialic acid. Strategically, transferrin-functionalized magnetic beads (TMBs) were applied to enrich CTCs, and azide-based bioorthogonal chemistry was employed to label target CTCs. Accordingly, the membrane glycoprotein-targeting strategy achieved unbiased enrichment and labeling of broad-spectrum CTCs that were both epithelial and non-epithelial phenotypic populations with varied organ/tissue origins (MCF-7, HepG2, A549, Jurkat, and B16), with a capture efficiency of >95% and a detection limit as low as 5 cells per mL in artificial blood. In particular, our developed strategy displayed excellent specificity, and the CTCs under capture and fluorescence labelling remained with good viability and could be further cultivated and analyzed. Finally, the membrane glycoprotein-targeting strategy successfully detected and identified 33-223 CTCs from 1 mL patient blood samples.

An XIC-Centric Strategy for Improved Identification and Quantification in Proteomic Data Analyses.

Reproducibility is a "proteomic dream" yet to be fully realized. A typical data analysis workflow utilizing extracted ion chromatograms (XICs) often treats the information path from identification to quantification as a one-way street. Here, we propose an XIC-centric approach in which the data flow is bidirectional: identifications are used to derive XICs whose information is in turn applied to validate the identifications. In this study, we employed liquid chromatography-mass spectrometry data from glycoprotein and human hair samples to illustrate the XIC-centric concept. At the core of this approach was XIC-based monoisotope repicking. Taking advantage of the intensity information for all detected isotopes across the whole range of an XIC peak significantly improved the accuracy and uncovered misidentifications originating from monoisotope assignment mistakes. It could also rescue non-top-ranked glycopeptide hits. Identification of glycopeptides is particularly susceptible to precursor mass errors for their low abundances, large masses, and glycans differing by 1 or 2 Da easily confused as isotopes. In addition, the XIC-centric strategy significantly reduced the problem of one XIC peak associated with multiple unique identifications, a source of quantitative irreproducibility. Taken together, the proposed approach can lead to improved identification and quantification accuracy and, ultimately, enhanced reproducibility in proteomic data analyses.

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Site-Specific Analysis of Core and Antenna Fucosylation on Serum Glycoproteins.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N-glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N-glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N-glycans, 37% by CF N-glycans, and 13% by dual-fucosylated N-glycans. Interestingly, CF and AF N-glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N-glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N-glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.

Novel post-translationally cleaved Ara h 2 proteoforms: Purification, characterization and IgE-binding properties.

The 2S albumins Ara h 2 and Ara h 6 have been shown to be the most important source of allergenicity in peanut. Several isoforms of these allergens have been described. Using extraction and liquid chromatography we isolated proteins with homology to Ara h 2 and characterized hitherto unknown Ara h 2 proteoforms with additional post-translational cleavage. High-resolution mass spectrometry located the cleavage site on the non-structured loop of Ara h 2 while far UV CD spectroscopy showed a comparable structure to Ara h 2. The cleaved forms of Ara h 2 were present in genotypes of peanut commonly consumed. Importantly, we revealed that newly identified Ara h 2 cleaved proteoforms showed comparable IgE-binding using sera from 28 peanut-sensitized individuals, possessed almost the same IgE binding potency and are likely similarly allergenic as intact Ara h 2. This makes these newly identified forms relevant proteoforms of peanut allergen Ara h 2.

Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices.

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.

Integrated landscape of chicken egg chalaza proteomics.

Chicken egg chalaza (CLZ) is a natural colloidal structure in eggs that exists as an egg yolk stabilizer and is similar in composition to egg white. In this study, the proteome, phosphoproteome, and N-glycoproteome of CLZ were characterized in depth. We hydrolyzed the CLZ proteins and enriched the phosphopeptides and glycopeptides. We identified 45 phosphoproteins and 80 N-glycoproteins, containing 59 phosphosites and 203 N-glycosylation sites, respectively. Typically, the ovalbumin in CLZ was both phosphorylated and N-glycosylated, with 4 phosphosites and 4 N-glycosylation sites. Moreover, we identified 2 N-glycosylated subunits of ovomucin, mucin-5B and mucin-6, with 32 and nine N- glycosylation sites, respectively. Analysis of the phosphorylation and N-glycosylation status of CLZ proteins could provide novel insights into the structural and functional characteristics of CLZ.

CSF N-Glycomics Using High-Throughput UPLC-ESI Techniques in Alzheimer's Disease.

In this chapter, we will present a high-throughput method applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. This methodology is based on a commercial equipment developed by WATERS™ to speed up N-deglycosylation and N-glycan labeling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies. This analytical kit is sold under the trade name of RapiFluor-MS (RFMS). We have slightly modified the methodology, increasing the glycosylation time and using a high-resolution mass analyzer for the analysis of CSF N-glycans, thus obtaining a high-throughput method (up to 96 samples simultaneously), mass accuracy better than 5 ppm, and the ability to separate and identify isomers.

CSF N-Glycomics Using MALDI MS Techniques.

In this chapter, we will present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease (AD).

Updates of the current strategies of labeling for N-glycan analysis.

This mini review summarizes the current methods used for screening N-glycosylation of glycoproteins, with a specific focus on therapeutic proteins and on techniques involving the release of N-glycans. With the continuous development of biopharmaceuticals, particularly monoclonal antibodies (mAbs), which are N-glycosylated proteins, monitoring has gained importance in recent decades. Glycosylation of therapeutic glycoproteins is considered a critical quality attribute because it can impact the efficacy and safety of these therapeutic drugs. The protocols and instrumentation have evolved with the advancement of technologies. Nowadays, methods are becoming increasingly robust, rapid, and sensitive. For the release of N-glycans, the most commonly used method is enzymatic release using PNGase F. The latter is discussed in light of the advent of rapid release that is now possible. The strategy for separating N-glycans using either liquid chromatography (LC) with hydrophilic interaction liquid chromatography (HILIC) chemistry or capillary electrophoresis will be discussed. The selection of the labeling agent is a crucial step in sample preparation for the analysis of released N-glycans. This review also discusses labeling agents that are compatible with and dependent on the separation and detection techniques employed. The emergence of multiplex labeling agents is also summarized. The latter enables the analysis of multiple samples in a single run, but it requires MS analysis.

Structure, Biological Functions, Separation, Properties, and Potential Applications of Milk Fat Globule Membrane (MFGM): A Review.

BACKGROUND: The milk fat globule membrane (MFGM) is a thin film that exists within the milk emulsion, suspended on the surface of milk fat globules, and comprises a diverse array of bioactive components. Recent advancements in MFGM research have sparked a growing interest in its biological characteristics and health-related functions. Thorough exploration and utilization of MFGM as a significant bioactive constituent in milk emulsion can profoundly impact human health in a positive manner. Scope and approach: This review comprehensively examines the current progress in understanding the structure, composition, physicochemical properties, methods of separation and purification, and biological activity of MFGM. Additionally, it underscores the vast potential of MFGM in the development of additives and drug delivery systems, with a particular focus on harnessing the surface activity and stability of proteins and phospholipids present on the MFGM for the production of natural emulsifiers and drug encapsulation materials. KEY FINDINGS AND CONCLUSIONS: MFGM harbors numerous active substances that possess diverse physiological functions, including the promotion of digestion, maintenance of the intestinal mucosal barrier, and facilitation of nerve development. Typically employed as a dietary supplement in infant formula, MFGM's exceptional surface activity has propelled its advancement toward becoming a natural emulsifier or encapsulation material. This surface activity is primarily derived from the amphiphilicity of polar lipids and the stability exhibited by highly glycosylated proteins.

Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.

Modification of N-Linked Glycan Sites in Viral Glycoproteins.

Post-translational modification of proteins by the addition of sugar chains, or glycans, is a functionally important hallmark of proteins trafficked through the secretory system. These proteins are termed glycoproteins. Glycans are known to be important for initiating signaling through binding of cell surface receptors, facilitating protein folding, and maintaining protein stability. For pathogens, glycans can also mask vulnerable protein regions from neutralizing antibodies. Thus, there is a need to develop methods to decipher the role of specific glycans attached to proteins in order to understand their biological role. Here, we describe established methods for identifying glycosylated residues and understanding their role in protein synthesis and function using viral glycoproteins as a model.