Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Complexity and modification of the bull sperm glycocalyx during epididymal maturation.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.

Proteomic and phosphoproteomic reveal immune-related function of milk fat globule membrane in bovine milk of different lactation periods.

Information regarding protein expression and phosphorylation modifications in the bovine milk fat globule membrane is scarce, particularly throughout various lactation periods. This study employed a complete proteome and phosphoproteome between bovine colostrum and mature milk. A total of 11 proteins were seen in both protein expression and phosphorylation levels. There were 400 proteins identified in only protein expression, and 104 phosphoproteins identified in only phosphorylation levels. A total of 232 significant protein characteristics were identified within the proteome and significant phosphorylation sites within 86 phosphoproteins of the phosphoproteome. Biological activities and pathways primarily exhibited associations with the immune system. Simultaneously, a comprehensive analysis of proteins and phosphorylation sites using a multi-omics approach. Hence, the data we have obtained has the potential to expand our understanding of how the bovine milk fat globule membrane might be utilized as a beneficial component in dairy products.

The Exploitation of the Glycosylation Pattern in Asthma: How We Alter Ancestral Pathways to Develop New Treatments.

Asthma has reached epidemic levels, yet progress in developing specific therapies is slow. One of the main reasons for this is the fact that asthma is an umbrella term for various distinct subsets. Due to its high heterogeneity, it is difficult to establish biomarkers for each subset of asthma and to propose endotype-specific treatments. This review focuses on protein glycosylation as a process activated in asthma and ways to utilize it to develop novel biomarkers and treatments. We discuss known and relevant glycoproteins whose functions control disease development. The key role of glycoproteins in processes integral to asthma, such as inflammation, tissue remodeling, and repair, justifies our interest and research in the field of glycobiology. Altering the glycosylation states of proteins contributing to asthma can change the pathological processes that we previously failed to inhibit. Special emphasis is placed on chitotriosidase 1 (CHIT1), an enzyme capable of modifying LacNAc- and LacdiNAc-containing glycans. The expression and activity of CHIT1 are induced in human diseased lungs, and its pathological role has been demonstrated by both genetic and pharmacological approaches. We propose that studying the glycosylation pattern and enzymes involved in glycosylation in asthma can help in patient stratification and in developing personalized treatment.

Genetic association of inflammatory marker GlycA with lung function and respiratory diseases.

Association of circulating glycoprotein acetyls (GlycA), a systemic inflammation biomarker, with lung function and respiratory diseases remain to be investigated. We examined the genetic correlation, shared genetics, and potential causality of GlycA (N = 115,078) with lung function and respiratory diseases (N = 497,000). GlycA showed significant genetic correlation with FEV1 (rg = -0.14), FVC (rg = -0.18), asthma (rg = 0.21) and COPD (rg = 0.31). We consistently identified ten shared loci (including chr3p21.31 and chr8p23.1) at both SNP and gene level revealing potential shared biological mechanisms involving ubiquitination, immune response, Wnt/ß-catenin signaling, cell growth and differentiation in tissues or cells including blood, epithelium, fibroblast, fetal thymus, and fetal intestine. Genetically elevated GlycA was significantly correlated with lung function and asthma susceptibility (354.13 ml decrement of FEV1, 442.28 ml decrement of FVC, and 144% increased risk of asthma per SD increment of GlycA) from MR analyses. Our findings provide insights into biological mechanisms of GlycA in relating to lung function, asthma, and COPD.

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.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites.

BACKGROUND: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated. METHODS: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC-MS/MS. RESULTS: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated. CONCLUSIONS: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Induction of acrosome reaction by 4-Br-A23187 alters the glycoproteomic profile of boar spermatozoa.

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 µM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion.

Sweet complexity: O-linked protein glycosylation in pathogenic Neisseria.

The genus Neisseria, which colonizes mucosal surfaces, includes both commensal and pathogenic species that are exclusive to humans. The two pathogenic Neisseria species are closely related but cause quite different diseases, meningococcal sepsis and meningitis (Neisseria meningitidis) and sexually transmitted gonorrhea (Neisseria gonorrhoeae). Although obvious differences in bacterial niches and mechanisms for transmission exists, pathogenic Neisseria have high levels of conservation at the levels of nucleotide sequences, gene content and synteny. Species of Neisseria express broad-spectrum O-linked protein glycosylation where the glycoproteins are largely transmembrane proteins or lipoproteins localized on the cell surface or in the periplasm. There are diverse functions among the identified glycoproteins, for example type IV biogenesis proteins, proteins involved in antimicrobial resistance, as well as surface proteins that have been suggested as vaccine candidates. The most abundant glycoprotein, PilE, is the major subunit of pili which are an important colonization factor. The glycans attached can vary extensively due to phase variation of protein glycosylation (pgl) genes and polymorphic pgl gene content. The exact roles of glycosylation in Neisseria remains to be determined, but increasing evidence suggests that glycan variability can be a strategy to evade the human immune system. In addition, pathogenic and commensal Neisseria appear to have significant glycosylation differences. Here, the current knowledge and implications of protein glycosylation genes, glycan diversity, glycoproteins and immunogenicity in pathogenic Neisseria are summarized and discussed.

STC1 competitively binding ßPIX enhances melanoma progression via YAP nuclear translocation and M2 macrophage recruitment through the YAP/CCL2/VEGFA/AKT feedback loop.

This study investigates the role of Stanniocalcin-1 (STC1) in melanoma progression, with a focus on its impact on metastasis, angiogenesis, and immune evasion. Systematic bioinformatics analysis revealed the potential influence of STC1 dysregulation on prognosis, immune cell infiltration, response to immune therapy, and cellular functions. In vitro assays were conducted to assess the proliferation, invasion, migration, and angiogenesis capabilities of A375 cells. In vivo experiments utilizing C57BL/6 J mice established a lung metastasis model using B16-F10 cells to evaluate macrophage infiltration and M2 polarization. A Transwell co-culture system was employed to explore the crosstalk between melanoma and macrophages. Molecular interactions among STC1, YAP, ßPIX, and CCL2 are investigated using mass spectrometry, Co-Immunoprecipitation, Dual-Luciferase Reporter Assay, and Chromatin Immunoprecipitation experiments. STC1 was found to enhance lung metastasis by promoting the recruitment and polarization of M2 macrophages, thereby fostering an immunosuppressive microenvironment. Mechanistically, STC1 competes with YAP for binding to ßPIX within the KER domain in melanoma cells, leading to YAP activation and subsequent CCL2 upregulation. CCL2-induced M2 macrophages secrete VEGFA, which enhances tumor vascularization and increases STC1 expression via the AKT signaling pathway in melanoma cells, establishing a pro-metastatic feedback loop. Notably, STC1-induced YAP activation increases PD-L1 expression, promoting immune evasion. Silencing STC1 enhances the efficacy of PD-1 immune checkpoint therapy in mice. This research elucidates STC1's role in melanoma metastasis and its complex interactions with tumor-associated macrophages, proposing STC1 as a potential therapeutic target for countering melanoma metastasis and augmenting the efficacy of PD-1 immunotherapy.

Aß42 and ROS dual-targeted multifunctional nanocomposite for combination therapy of Alzheimer's disease.

Amyloid-ß (Aß) readily misfolds into neurotoxic aggregates, generating high levels of reactive oxygen species (ROS), leading to progressive oxidative damage and ultimately cell death. Therefore, simultaneous inhibition of Aß aggregation and scavenging of ROS may be a promising therapeutic strategy to alleviate Alzheimer's disease pathology. Based on the previously developed antibody 1F12 that targets all forms of Aß42, we developed an Aß42 and ROS dual-targeting nanocomposite using biodegradable mesoporous silica nanoparticles as carriers to load ultra-small cerium oxide nanocrystals (bMSNs@Ce-1F12). By modifying the brain-targeted rabies virus glycoprotein 29 (RVG29-bMSNs@Ce-1F12), this intelligent nanocomposite can efficiently target brain Aß-rich regions. Combined with peripheral and central nervous system treatments, RVG29-bMSNs@Ce-1F12 can significantly alleviate AD symptoms by inhibiting Aß42 misfolding, accelerating Aß42 clearance, and scavenging ROS. Furthermore, this synergistic effect of ROS scavenging and Aß clearance exhibited by this Aß42 and ROS dual-targeted strategy also reduced the burden of hyperphosphorylated tau, alleviated glial cell activation, and ultimately improved cognitive function in APP/PS1 mice. Our findings indicate that RVG29-bMSNs@Ce-1F12 is a promising nanodrug that can facilitate multi-target treatment of AD.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

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.

Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

Human brain glycoform coregulation network and glycan modification alterations in Alzheimer's disease.

Despite the importance of protein glycosylation to brain health, current knowledge of glycosylated proteoforms or glycoforms in human brain and their alterations in Alzheimer's disease (AD) is limited. Here, we report a proteome-wide glycoform profiling study of human AD and control brains using intact glycopeptide-based quantitative glycoproteomics coupled with systems biology. Our study identified more than 10,000 human brain N-glycoforms from nearly 1200 glycoproteins and uncovered disease signatures of altered glycoforms and glycan modifications, including reduced sialylation and N-glycan branching and elongation as well as elevated mannosylation and N-glycan truncation in AD. Network analyses revealed a higher-order organization of brain glycoproteome into networks of coregulated glycoforms and glycans and discovered glycoform and glycan modules associated with AD clinical phenotype, amyloid-ß accumulation, and tau pathology. Our findings provide valuable insights into disease pathogenesis and a rich resource of glycoform and glycan changes in AD and pave the way forward for developing glycosylation-based therapies and biomarkers for AD.

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.

Glycoprotein-glycoprotein Receptor Binding Detection Using Bioluminescence Resonance Energy Transfer.

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.