The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites

Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man₅GlcNAc₂). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.Entamoeba histolytica causes amebic dysentery and liver abscess in the developing world (10, 20, 29). We are interested in E. histolytica glycoproteins containing Asn-linked glycans (N-glycans) for numerous reasons. E. histolytica makes an N-glycan precursor that contains 7 sugars (Man₅GlcNAc₂-PP-dolichol) rather than 14 sugars (Glc₃Man₉GlcNAc₂-PP-dolichol) made by most animals, plants, and fungi (21, 31, 44). E. histolytica N-glycans are used for quality control of glycoprotein folding in the endoplasmic reticulum (ER) lumen, and there is positive selection for sites of N-linked glycosylation in secreted and membrane proteins of E. histolytica (5, 11, 53).Unprocessed Man₅GlcNAc₂, by far the most abundant E. histolytica N-glycan, is present on the plasma membrane and vesicular membranes (31). The antiretroviral lectin cyanovirin-N, which is specific for α-1,2-linked mannose present on unprocessed N-glycans, binds E. histolytica N-glycans and forms aggregates or caps on the surface of E. histolytica trophozoites (1, 25, 31, 44, 45). E. histolytica glycoproteins are also capped by the plant lectin concanavalin A (ConA), which has a broader carbohydrate specificity (mannose and glucose) than cyanovirin-N (3, 16, 18, 19). Heavy subunits of the Gal/GalNAc lectin, the most important E. histolytica vaccine candidate, have 7 to 10 potential sites for N-linked glycosylation (32, 39, 43). Inhibition of N-glycan synthesis results in Gal/GalNAc lectins that are unable to bind to sugars on host epithelial cells.Carbohydrates appear to be an important target on the surface of E. histolytica as anti-proteophosphoglycan (PPG) monoclonal antibodies bind to O-phosphodiester-linked glycans and protect animal models from amebic infection (6, 33, 35, 40, 48). Lectin affinity columns are a powerful method for enriching unique parasite glycoproteins that may be identified by mass spectrometry (MS) of tryptic fragments (17, 55). For example, we recently used the plant lectin wheat germ agglutinin to dramatically enrich glycoproteins with short N-glycans of Giardia (42).The goal of the present studies was to explore further the interaction of the antiretroviral lectin cyanovirin-N with E. histolytica trophozoites in vitro. Questions asked included the following: Are E. histolytica glycoproteins with N-glycans replenished on the plasma membrane after capping with cyanovirin-N? What is the effect of cyanovirin-N capping on other amebic virulence factors and/or vaccine candidates (e.g., the Gal/GalNAc lectin and PPG)? Is capping by cyanovirin-N mediated by actin, as described for capping by the Gal/GalNAc lectin and ConA? What is the effect of the cyanovirin-N on amebic phagocytosis of mucin-coated beads, a surrogate assay for virulence? Which trophozoite glycoproteins are potential targets of cyanovirin-N (identified by mass spectrometry of lectin-enriched E. histolytica proteins)? Are any of them potential vaccine candidates?     

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of α1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER α1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.Recognition and sorting of improperly folded proteins is essential to cell survival, and hence, an elaborate quality control system is found in cells. ER⁴ quality control is well characterized with respect to the N-linked oligosaccharides regulating the folding and degradation of newly synthesized proteins in the ER (1). Immediately after polypeptides enter the ER, Glc₃Man₉GlcNAc₂ (G3M9) precursor oligosaccharides are covalently attached and subsequently processed. Terminally misfolded proteins are removed from the ER by the ERAD machinery (1–4). Aberrant conformers are recognized, retrotranslocated to the cytosol, and degraded by the ubiquitin-proteasome system (5, 6). Processing of mannose residues from the N-linked oligosaccharides acts as a timer for the recognition of misfolded glycoproteins in the ER lumen (1, 7). ER α1,2-mannosidase I (ER ManI) in mammals and ER α-mannosidase in yeast preferentially trim mannose residues from the middle branch of N-glycans, generating the Man₈GlcNAc₂ (M8) isomer B (M8B) (8). In mammals, further mannose processing is required as a signal for degradation (1, 9, 10), whereas the presence of M8B is sufficient to signal degradation in yeast (11). The postulated lectin EDEMs in mammals, their yeast homolog Htm1p/Mnl1p, and the yeast MRH domain-containing lectin Yos9p have all been proposed to recognize glycoproteins targeted for degradation (12).The role of Yos9p in glycoprotein ERAD was identified using a genetic screen in Saccharomyces cerevisiae (13). Yos9p, a homolog of hOS-9, contains an MRH domain (14) and functions as a lectin. Yos9p recognizes substrates of the ERAD-lumenal pathway (15–17), generating a large ER membrane complex containing the Hrd1p-Hrd3p ubiquitin ligase core complex (18–20). The M8B and Man₅GlcNAc₂ (M5) N-glycans are predicted to function as ligands for Yos9p (17). Bipartite recognition of both glycan and polypeptide by Yos9p has also been reported (15).Recent studies revealed that two mammalian MRH domain-containing lectins, OS-9 and XTP3-B, are ER luminal proteins involved in ER quality control and form a large complex containing the HRD1-SEL1L ubiquitin-ligase in the ER membrane (21–24). The components of the complex are similar to yeast, suggesting evolutionary conservation, although the molecular mechanisms underlying the role of OS-9 and XTP3-B remain elusive. Studies using lectin mutants have suggested that the MRH domains are required not for binding to ERAD substrates but for interactions with SEL1L (21), which has multiple N-glycans (25, 26). Additionally, lectin activity appears to be dispensable for hOS-9 binding to misfolded glycoproteins (21, 24). Thus, to understand the role of hOS-9 in the ER quality control pathway, the specific carbohydrate structures recognized by the hOS-9 MRH domain need to be identified, and the requirement of the lectin domain in substrate recognition needs to be determined.In the present study we demonstrate that the lectin activity of hOS-9 is required for enhancement of glycoprotein ERAD. We identified the N-glycan structures recognized by the recombinant hOS-9 MRH domain in vitro by frontal affinity chromatography (FAC). Using a model ERAD substrate, NHK (27), we show that the ability of hOS-9 to enhance ERAD in vivo depends on the oligosaccharides present on NHK, consistent with the FAC results.     

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1–4). In fact, clinical biomarkers and therapeutic targets are often glycoproteins (5–9). Comprehensive glycoprotein characterization, involving glycosylation site identification, glycan structure determination, site occupancy, and glycan isoform distribution, is a technical challenge particularly for quantitative profiling of complex protein mixtures (10–13). Both N- and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS¹ signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11–13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them. However, when peptides and glycopeptides co-elute, parent ion scanning is required to selectively detect the glycopeptides (14). This can be problematic in terms of sensitivity, especially for detecting glycopeptides in digests of complex protein extracts.Isolation of glycopeptides from proteolytic digests of complex protein mixtures can greatly enhance the MS signals of glycopeptides using reversed-phase LC-ESI-MS (RPLC-ESI-MS) or MALDI-MS (15–24). Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15–17). Capturing glycopeptides with lectins has been widely used, but restricted specificities and unspecific binding are major drawbacks of this method (18–21). Under reversed-phase LC conditions, glycopeptides from tryptic digests of gel-separated glycoproteins have been enriched using graphite powder medium (22). In this case, however, a second digestion with proteinase K is required for trimming down the peptide moieties of tryptic glycopeptides so that the glycopeptides (typically <5 amino acid residues) essentially resemble the glycans with respect to hydrophilicity for subsequent separation. Moreover, the short peptide sequences of the proteinase K digest are often inadequate for de novo sequencing of the glycopeptides.Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23–28). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZIC®-HILIC column (23–27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g. the five high mannose type glycopeptides modified by 7–11 monosaccharide units from a tryptic digest of ribonuclease b (RNase B), nor for enriching O-linked glycopeptides of bovine fetuin using a ZIC-HILIC column (23). The use of Sepharose medium for enriching glycopeptides yielded only modest recovery of glycopeptides (28). In addition, binding of hydrophilic non-glycopeptides with these hydrophilic media contaminates the enriched glycopeptides (23, 28).We have recently developed an ion-pairing normal-phase LC (IP-NPLC) method to enrich glycopeptides from complex tryptic digests using Sepharose medium and salts or bases as ion-pairing reagents (29). Though reasonably effective the technique still left room for significant improvement. For example, the method demonstrated relatively modest glycopeptide selectivity, providing only 16% recovery for high mannose type glycopeptides (29). Here we report on a new IP-NPLC method using acids as ion-pairing reagents and polyhydroxyethyl aspartamide (A) as the stationary phase for the effective isolation of tryptic glycopeptides. The method was developed and evaluated using a tryptic digest of RNase B and fetuin mixture. In addition, we demonstrate that O-linked glycopeptides can be effectively isolated from a fetuin tryptic digest by IP-NPLC after removal of the N-linked glycans by PNGase F.The new IP-NPLC method was used to enrich N-linked glycopeptides from the tryptic digests of protein extracts of wild-type (wt) and PglD mutant strains of Campylobacter jejuni NCTC 11168. C. jejuni has a unique N-glycosylation system that glycosylates periplasmic and inner membrane proteins containing the extended N-linked sequon, D/E-X-N-X-S/T, where X is any amino acid other than proline (30–32). The N-linked glycan of C. jejuni has been previously determined to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 (BacGalNAc₅Glc residue mass: 1406 Da), where Bac is 2,4-diacetamido-2,4,6-trideoxyglucopyranose (30). In addition, the glycan structure of C. jejuni is conserved, unlike in eukaryotic systems (30–32). IP-NPLC recovered close to 100% of the bacterial N-linked glycopeptides with virtually no contamination of non-glycopeptides. Furthermore, we demonstrate for the first time that acetylation of bacillosamine is incomplete in the wt using IP-NPLC and label-free MS.     

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of ¹⁸O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.Chlamydomonas reinhardtii is a green alga that is used as a model organism for studying a number of biological processes such as photosynthesis, flagellar assembly and function, organelle biosynthesis, phototaxis, and circadian rhythms (1). Studies on glycosylation pathways in C. reinhardtii have been mostly focused on O-glycosylation processing, as the cell wall of this organism consists of a vast framework of O-glycosylated hydroxyproline-rich glycoproteins (2, 3). More recently, Bollig et al. even demonstrated that O-glycans from C. reinhardtii cell wall glycoproteins contain arabinose and galactose, the latter being in the furanose form (4). In contrast, the N-glycosylation pathway, although a major post-translational modification step in the maturation of secreted proteins in eukaryotes, has received very little attention so far. In N-glycan processing, a Man₅GlcNAc₂-PP-dolichololigosaccharide intermediate is first assembled onto a dolichol pyrophosphate on the cytosolic face of the endoplasmic reticulum (ER).¹ After translocation of this intermediate by a flippase, the biosynthesis continues in the lumen of the ER until a Glc₃Man₉GlcNAc₂-PP-dolichol N-glycan precursor is completed (5). This precursor is then transferred by the oligosaccharyltransferase (OST) multisubunit complex onto the asparagine residues of the consensus Asn-X-Ser/Thr sequences of a protein (5). The precursor is then deglucosylated/reglucosylated to ensure the quality control of the neosynthesized protein through the interaction with ER-resident chaperones such as calnexin and calreticulin. These ER events are crucial for the proper folding of secreted proteins (6), conserved in eukaryotes investigated so far, and involve a limited number of oligomannoside N-glycans. In contrast, the evolutionary adaptation of N-glycan processing in the Golgi apparatus gives rise to a large variety of organism-specific complex structures (7). Type I mannosidases located in this compartment first degrade the oligosaccharide precursor into oligomannoside N-glycans ranging from Man₉GlcNAc₂ (Man-9) to Man₅GlcNAc₂ (Man-5). N-acetylglucosaminyltransferase I (GnT I) then transfers a first GlcNAc residue on the α(1,3)-mannose arm of Man-5 to initiate the synthesis of polyantennary complex-type N-glycans (7).To date, a few studies carried out in Chlorophycaea using on-blot affinodetection or a combination of exoglycosidase digestions and two-dimensional HPLC separation have suggested that proteins secreted by these microalgae harbor mainly oligomannosides or mature N-glycans having a core xylose residue (8–10). Deeper insight into the structure of glycans N-linked to proteins secreted by two algal species, Porphyridium sp. and Phaeodactylum tricornutum, has been recently reported. A cell wall glycoprotein from the red microalgae Porphyridium sp. was found to carry Man-8 and Man-9 oligomannosides containing 6-O-methyl mannose and substituted by one or two xylose residues (11). In contrast, glycans N-linked to proteins secreted by the diatom P. tricornutum can be processed through a GnT I-dependent pathway into paucimannosidic oligosaccharides (12).In contrast to glycomic analysis, which focuses on the structure of N-linked oligosaccharides irrespective of the carrier proteins, glycoproteomics is used to characterize and determine the cell localization of individual proteins carrying these carbohydrate post-translational modifications. Whereas mammalian N-glycoproteomes have been studied extensively down to tissue- and cell-type-specific levels (13–17), less information is available regarding the N-glycoproteomes of plants and green algae (18, 19). The use of glycoproteomic approaches could help unravel the identity of endogenous glycoproteins from C. reinhardtii. As this green alga possesses many animal-like features (20), glycoproteomic analyses will help provide information concerning similarities and differences relative to not only mammalian but also vascular plant N-glycosylation pathways and glycoprotein trafficking.Recently, microalgae have emerged as an alternative system for the production of biopharmaceuticals, which represents a multibillion-dollar industry worldwide (21). The high expense and complicating factor of potential virus contamination encountered with commonly used expression systems have driven scientists to seek alternatives such as C. reinhardtii cells. Actually, they are cheap, easy to grow, safe, and scalable for the production of a high amount of proteins, making them ideal hosts for industrial production (22). Several studies have already demonstrated that the green alga C. reinhardtii is a convenient platform for producing recombinant proteins, including those of human origin (23). For example, a large single-chain antibody directed against glycoprotein D of the herpes simplex virus (24) and full-length IgG1 monoclonal antibodies directed against anthrax protective antigen 83 (25) have been successfully expressed in the chloroplast of transgenic C. reinhardtii cells. The production of secreted therapeutic proteins such as erythropoietin has also been evaluated (26). In contrast to the expression of proteins in the chloroplast, protein post-translational modifications such as N-glycosylation acquired by the secreted recombinant protein are a major concern for biopharmaceuticals, as more than half of the approved ones are glycosylated (27). Moreover, glycosylation is a critical quality attribute for biopharmaceuticals, because the presence and structures of the N-glycans are required for their biological activity, stability, and half-life (28, 29). However, given that unsuitable N-glycan structures can induce immune responses in humans (30–32) and generate adverse reactions, as reported for α(1,3)-Gal epitope on therapeutic drugs like cetuximab (33), it is essential to take into account the N-glycosylation capacity for an optimal expression system. Therefore, a suitable expression system should allow the production of glycomolecules harboring N-glycans and/or O-glycans compatible with human therapeutical applications and better efficacy of the therapeutic drug (34).In this study, we used a comprehensive approach including genomic, glycomic, and glycoproteomic analyses to investigate the N-glycosylation pathway occurring in C. reinhardtii. Our results revealed that the biosynthesis and maturation of N-glycans occur in the ER and Golgi apparatus through a GnT I-independent pathway and yield novel complex structures in addition to oligomannoside N-glycans.     

Clustered O-Glycans of IgA1: DEFINING MACRO- AND MICROHETEROGENEITY BY USE OF ELECTRON CAPTURE/TRANSFER DISSOCIATION*

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1–5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1)1 contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9–11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).Open in a separate windowFig. 1.IgA1 structural elements. IgA1 has N-linked glycans (filled circles) and O-linked glycans (open circles). The O-glycosylated sites are in the HR between the first and second constant region domains of the heavy chains. The HR is a Pro-rich segment with nine possible sites of O-glycan attachment. Underlined serine and threonine residues are usually glycosylated (31). Arrows show cleavage sites of trypsin and IgA-specific proteases.An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13–15). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16–21), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20, 22). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23–26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23, 26, 27).IgAN is the most common primary glomerulonephritis worldwide (28) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1, 10, 14–16, 29, 30). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13, 15). This distinctive IgA1 is in circulating immune complexes (8, 10, 15) and in the glomerular deposits of IgAN patients (16, 29). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9, 10). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9, 10, 15) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8, 9). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.     

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.Colorectal cancer (CRC)¹ is a very prevalent and heterogeneous pathology with highly variable disease progression and clinical outcome among patients. It is the third most common cancer in men and the second most common in women (1) with a highly stage-specific patient survival (2). Treatments are often curative for patients with local disease stages (stage I-II), whereas a 5-year survival of only 13% is observed in patients with distant metastasis (stage IV) (2). As CRC is often asymptomatic in the first years, unfortunately, only 40% of the patients are diagnosed at stage I-II, thus pointing to the urgent need of sensitive diagnostic tools for early detection and consequently effective, curative treatment (3). In this context, understanding the complex mechanisms of CRC is an overriding condition for the development of new, more efficient means of detection, treatment, and prognosis of the disease.Altered glycosylation is a hallmark of cancer (4) and is known to occur with cancer progression (4, 5) as glycans are involved in many cancer-associated events such as adhesion, invasion, and cell signaling (6). As a result of altered glycan structures, cellular processes can be affected due to a change of interactions with glycan-binding proteins (7–9). Several CRC tissue-associated changes in N-glycans, O-glycans, and glycosphingolipid glycans have been reported and recently reviewed (7). For instance, N-glycans extracted from colorectal tumor tissues are characterized by an increase of sulfated glycans, (truncated) high-mannose-type glycans, and glycans containing sialylated Lewis type epitopes, while showing a decrease of bisection as compared with glycans from nontumor colorectal tissue of the same individuals (10). In accordance, elevated expression of sialyl Lewis A (NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc-R; NeuAc = N-acetylneuraminic acid, Gal = galactose, Fuc = fucose, GlcNAc = N-acetylglucosamine, R = rest) and pauci-mannosidic N-glycans (truncated high-mannose-type, Man1–4GlcNAc1–4GlcNAc; Man = mannose) was recently found to be correlated with poor prognosis in (advanced) colon carcinomas and N-glycomic profiling was successfully applied to distinguish colorectal adenomas from carcinomas (11).Due to limitations in accessibility of tumor materials and possibilities of in vivo studies on a large scale, cancer cell lines represent a relevant alternative and are widely used as model systems for studying the molecular mechanisms associated with cancer outcome and progression. Since the early 1960s, colorectal cancer cell lines have been established with HT29, LoVo, LS-180, LS-174T, and Co115 representing the first continuous cell lines derived from colon tumors and xenografts (12–14). Major benefits of cancer cell lines are their continuous availability, their fast growth, and relatively easy handling, making them suitable also for high-throughput screenings (15) and a large range of experimental possibilities (16). Of note, advantages and limitations of cell lines have been recently reviewed (15).In order to select suitable in vitro models, the characterization of molecular features and their comparison to tumor tissues are needed. A detailed Cancer Cell Line Encyclopedia was recently established containing a genomic dataset for 947 human cancer cell lines, from which 58 are colorectal cancer lineages (17). The Cancer Cell Line Encyclopedia includes data collections on genomic characterization, point mutation frequencies, DNA copy number, and mRNA expression levels. Comparison of these features between cell lines and primary tumors showed a high correlation in most cancer types, especially for colorectal cancer, suggesting that cell lines do represent tumor tissues quite reasonably at least on the genetic level. However, the number of publications characterizing cancer cell lines at a molecular level is far behind the number of articles using cancer cell lines as model systems (18), and only few studies have been conducted on whether in vitro cultured cell lines can serve as suitable models for human tumors (19–22). Furthermore, cell lines are well characterized genetically, but they are largely understudied with regard to their glycosylation profiles.Here, we developed and optimized a new analytical method for the more sensitive and higher throughput N-glycome profiling of cells. This method is based on the release of N-glycans in a 96-well plate format from a PVDF-membrane (23) starting from a low number of cells (250,000 cells), the chemical derivatization of released N-glycans enabling the stabilization and discrimination of α2,3- and α2,6-linked N-acetylneuraminic acids (24), followed by registration of the N-glycans by MALDI-TOF(/TOF)-MS. The method was applied to characterize the N-glycome of 25 different colorectal cell lines in a fast and robust manner, including biological and technical replicates for all the cell lines. We obtained the comprehensive N-glycan profiles of 21 cell lines derived from primary tumors, two from lymph node metastases, one from a lung metastasis, and one from ascites fluid to assess their potential as glycobiological tumor model systems. Cancer cell line glycosylation features were then correlated with cancer cell markers and phenotypes as well as glycosyltransferase expressions. This study provides new insights into colon-cancer-associated glycan changes and sets a basis for studies into the functions of N-glycans in CRC with cell lines as model systems.     

A Comparative Study of Lectin Affinity Based Plant N-Glycoproteome Profiling Using Tomato Fruit as a Model

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be determined whether certain lectins, or combinations of lectins are optimal for plant N-glycoproteome profiling; or whether specific lectins show preferential association with particular N-glycosylation sites or N-glycan structures. We describe here a comparative study of three mannose-binding lectins, concanavalin A, snowdrop lectin, and lentil lectin, to profile the N-glycoproteome of mature green stage tomato (Solanum lycopersicum) fruit pericarp. Through coupling lectin affinity chromatography with a shotgun proteomics strategy, we identified 448 putative N-glycoproteins, whereas a parallel lectin affinity chromatography plus hydrophilic interaction chromatography analysis revealed 318 putative N-glycosylation sites on 230 N-glycoproteins, of which 100 overlapped with the shotgun analysis, as well as 17 N-glycan structures. The use of multiple lectins substantially increased N-glycoproteome coverage and although there were no discernible differences in the structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that differentially bound to each lectin, differences were observed in the amino acid frequency at the −1 and +1 subsites of the N-glycosylation sites. We also demonstrated an alternative and complementary in planta recombinant expression strategy, followed by affinity MS analysis, to identify the putative N-glycan structures of glycoproteins whose abundance is too low to be readily determined by a shotgun approach, and/or combined with deglycosylation for predicted deamidated sites, using a xyloglucan-specific endoglucanase inhibitor protein as an example.N-glycosylation is one of the most heterogeneous and common post-translational modifications of eukaryotic proteins and one that affects many aspects of protein targeting, enzymatic properties, stability and intermolecular interactions (1–3). There is therefore considerable interest in developing robust and sensitive high throughput analytical pipelines to isolate and structurally characterize N-glycoprotein populations (2, 4–8), allowing glycoprotein identification and analysis of the glycosylation site occupancy and N-glycan structure. To this end, lectin affinity chromatography (LAC)¹ is increasingly popular: specifically, various lectins are known to have different binding affinities for N-glycans and so the selective binding of N-glycoproteins in complex protein extracts to these lectins and their subsequent release allows a critical enrichment step before sequencing and glycan analysis by MS (9).As a refinement of this approach, the use of multiple lectin affinity chromatography (MLAC) in yeast and animal studies (4, 6, 10), using different proteomic platforms, has been shown to increase the numbers of isolated N-glycoproteins or N-glycopeptides. Collectively, these studies of taxonomically diverse eukaryotic N-glycoproteomes suggest a general conservation of the glycosylation site (N-X-S/T, where X can be any amino acid except proline), as well as conserved features of three-dimensional protein structure (4, 6, 10). Although there have been several studies to determine the structural basis of the binding specificity of specific lectins to yeast and animal N-glycoproteins (11), larger scale N-glycoproteomic analyses have typically not attempted to determine whether a particular combination of lectins provides optimal enrichment, or whether specific features, such as N-glycan structure or amino acid sequence at and around the N-glycosylation site, are associated with different lectins. Therefore, systematic comparative studies are essential to determine whether particular lectins can be optimal for specific tissues, organs, and organisms.LAC has also been used in plant N-glycoprotein analyses to enrich for populations of cell wall localized proteins (8, 12, 13) and a recent report (13) described the application of MLAC to map substantial numbers of N-glycosylation sites in a range of key experimental model organisms, included the plant Arabidopsis thaliana. Using a LTQ-Orbitrap Velos mass spectrometer, the authors identified 2186 unique N-glycosylation sites in proteins extracted from five different arabidopsis organs (13), which represents a substantial increase in the number of identified N-glycosylation sites that have resulted from previous plant N-glycoprotein studies. However, the particular analytical platform that was used did not allow the structural characterization of the N-glycans or N-glycopeptides (13). Indeed, certain features of N-glycopeptides, such as poor fragmentation, heterogeneity and a large dynamic range in most complex mixtures often limits the structural analysis of N-glycans in high throughput systematic analyses (2). It is also important to note that the structures of plant N-glycans differ from those of animals and yeast, as exemplified by the presence of β-1, 2-xylose and α-1, 3-fucose and the complete absence of multiantennary N-glycans and sialic acid in plant N-glycoproteins (1, 14). Therefore, assumptions that are made with regard to the lectin binding of animal and yeast proteins do not necessarily apply to those from plants. Consequently, there is a need to investigate the structural basis of lectin binding to plant N-glycoproteins. Moreover, the limitations of typical shotgun based profiling approaches in identifying and characterizing low abundance N-glycoproteins in complex protein extracts need to be addressed to allow more comprehensive plant N-glycoproteome profiling.In the present study we address both these issues using mature green stage tomato (Solanum lycopersicum) fruit pericarp as an experimental model to carry out a comparative analysis of N-glycoproteins associated with each of three mannose-binding lectins: concanavalin A (ConA), snowdrop lectin (GNA), and lentil lectin (LCH). Fruit development is associated with substantial cell wall metabolism and the expression of many wall localized N-glycoproteins (8) and tomato in particular represents an excellent model for studies of fleshy fruits and cell wall N-glycoproteins (15). We established an MLAC analytical pipeline that included shotgun proteomic profiling and deglycosylation and deamidation analysis, to allow the determination of N-glycoprotein protein identity, N-glycosylation site and N-glycan structures. This information was then used to establish whether a combination of lectins is indeed advantageous for the study of plant N-glycoproteomes, and to assess whether any of these structural characteristics result in predictable preferential binding to specific lectins. From these studies it became evident that large dynamic range of N-glycoprotein abundance was a significant limiting factor in the structural determination of the tomato, and that there was a bias toward the detection of highly abundant N-glycopeptides. We therefore evaluated the use of an in planta recombinant expression strategy, combined with affinity purification MS (AP-MS), as a means to characterize the N-glycan structures of glycoproteins whose abundance is too low to be readily determined via the primary shotgun pipeline, using a tomato xyloglucan-specific endoglucanase inhibitor protein (XEGIP) as a test case.     

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.Human blood plasma harbors arguably the most complex yet also the most informative proteome present in the human body (1). A significant impact on its clinical relevance and diagnostic potential is attributed to the features and functions of a plethora of proteins (60–80 mg protein per ml plasma), covering a dynamic concentration range of more than ten orders of magnitude (2). The majority, that is 99%, of these proteins are classical blood plasma proteins, like albumins, (immuno)globulins, clotting factors, and proteins of the complement system; however, also a lower abundant but—no less meaningful—fraction of nonclassical proteins is present that comprises a multitude of cytokines as well as tissue leakage proteins. Several clinical studies could show that qualitative and quantitative alterations of these proteins (and peptides)—analyzed individually or in their entirety as a proteome (or peptidome)—can directly reflect pathophysiological states, and can serve as biomarkers for the onset and progression of a number of diseases (3–5). In recent years the focus of in-depth analyses of the human blood plasma proteome has evolved from the identification and quantification of the entire proteome (or peptidome) (6–10) toward the analysis of subproteomes like the interactome (11), phosphoproteome (12, 13) or the glycoproteome (14). The latter has received particular interest in recent years, because the majority of blood plasma proteins is N- and/or O-glycosylated (2). Although the comprehensive analysis of the N-glycoproteome is already quite advanced (15), even in complex samples like human blood plasma (16, 17), similar analyses of the O-glycoproteome - though arguably equally important and relevant - are still lagging behind. The most ubiquitously found and functionally relevant form of O-glycosylation, as shown by a number of O-glycan-related (clinical) studies (18–23), is the mucin-type O-glycosyation (O-GalNAc), in particular the core-1 and core-2 types (24, 25). The predominantly clustered occurrence of mucin-type O-glycans on proteins is described to confer overall stability and proteolytic protection (26). Apart from this global impact, recent studies could link the presence of O-glycans in the proximity of regulatory domains to proteolysis events involved in protein maturation (proprotein-convertase-processing) (27). To better understand these protective and regulatory capabilities and to move the mucin-type O-glycoproteome from form to function comprehensive site-specific O-glycosylation analyses are required.One of the main obstacles in site-specific mucin-type O-glycosylation analyses relates to the lack of a predictable O-glycan consensus-motif within the peptide backbone as it can be found for N-glycans (28). The initial attachment of the N-acetylgalactosamine monosaccharide to the hydroxyl group of either serine or threonine, but also to tyrosine or hydroxylysine, is governed by a family of 20 distinct polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with different but partially overlapping peptide specificities and tissue expression patterns. This dynamic regulation, in turn, contributes to the complexity of the mucin-type O-glycoproteome. However, previous studies could show that mucin-type O-glycans are primarily attached to serine or threonine in regions with a high content of serine, threonine and proline (Ser/Thr-X-X-Pro, Ser/Thr-P and Pro-Ser/Thr) (29, 30). As O-glycosylation is a postfolding event, taking place in the Golgi apparatus, the attachment is depended on protein surface accessibility and is thus predominantly found in coil, turn, and linker regions (31). Additional confounding factors during mucin-type O-glycosylation analyses are the clustered occurrence of O-glycans and the lack of a universal endo-O-glycosidase that enables the release of intact O-glycans from the proteins; though, chemical O-glycan release methods do exist (28).Mass spectrometry has proven to be the core technique in site-specific N- and O-glycosylation analyses. A generic O-glycoproteomic workflow usually starts with the isolation, enrichment or prefractionation of a single glycoprotein or a group of glycoproteins. In subsequent steps, (glyco)peptides are generated by proteolytic digestion primarily using specific proteases like trypsin. Apart from this, also broad- and nonspecific proteases like Proteinase K or Pronase E were successfully employed in recent years (32–34). Essential to nearly every glycoproteomic approach is the removal of high-abundant and interfering nonglycosylated peptides by selective enrichment of the usually lower abundant glycopeptides. The repertoire of glycopeptide enrichment and separation techniques covers different solid phase extraction and chromatography based methods such as hydrophilic liquid interaction chromatography (HILIC) (35, 36). The most frequently used setup for the measurement of enriched (glyco)peptides is liquid chromatography (LC)¹ coupled online to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Recent advances in instrumentation, in particular the development of electron-transfer/electron-capture dissociation (ETD/ECD) (37, 38), and high resolution orbital mass analyzers, have paved the way for the mapping of thousands of occupied N- and O-glycosylation sites as recently shown (17, 27). Combined workflows using ETD/ECD fragmentation along with (multistage, MSⁿ) fragmentation with high- and/or low collisional induced dissociation energy (HCD/CID) can provide compositional (structural) information on the glycan moiety as well as information on the peptide sequence and the glycosylation site (39, 40). Recent advances in mass spectrometry driven O-glycoproteomics have been reviewed in detail elsewhere (41, 42). Owing to the amount and complexity of O-glycoproteomic data a number of bioinformatic software tools for the prediction of mucin-type O-glycosylation sites (27) as well as for the database assisted interpretation and annotation of glycan and glycopeptide fragment spectra have been developed (43, 44). Moreover, reporting guidelines for collecting, sharing, integrating, and interpreting mass spectrometry based glycomics data have been specified by the MIRAGE consortium (minimum information required for a glycomics experiment) (45, 46).The aim of our study was to develop a glycoproteomic workflow that allows the explorative nontargeted analysis of O-glycosylated human blood plasma proteins, which are known to carry mainly short mono- and disialylated mucin-type core-1 and -2 O-glycans. To achieve this, we have combined O-glycopeptide selective offline-HILIC fractionation of Proteinase K digested peptides with nano-reversed-phase liquid chromatography coupled online to multistage ion-trap mass spectrometry (nanoRP-LC-ESI-IT-MS: CID-MS²/-MS³, ETD-MS²). The workflow has been applied to investigate the mucin-type O-glycoproteome of a pooled blood plasma sample derived from 20 healthy donors. Based on the mass spectrometric analysis of intact O-glycopeptides, we were able to characterize the O-glycosylation (i.e. peptide, site, and attached O-glycans) of a number of major human blood glycoproteins, including many acute phase proteins such as fibrinogen and plasminogen. Overall, the site-specific glycosylation analysis of human blood plasma glycopeptides revealed exclusively mono- and disialylated core-1 mucin-type O-glycopeptides. Interestingly, also a few novel O-glycosylation sites could be identified.     

A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by Mass Spectrometry (MS)

Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.Glycosylation is an extremely important protein modification that frequently regulates protein folding, trafficking, and stability. It is also involved in a wide range of cellular events (1) such as immune response (2, 3), cell proliferation (4), cell-cell interactions (5), and signal transduction (6). Aberrant protein glycosylation is believed to have a direct correlation with the development of several diseases, including diabetes, infectious diseases, and cancer (7–11). Secretory proteins frequently get glycosylated, including those in body fluids such as blood, saliva, and urine (12, 13). Samples containing these proteins can be easily obtained and used for diagnostic and therapeutic purposes. Several glycoproteins have previously been identified as biomarkers, including Her2/Neu in breast cancer (14), prostate-specific antigen (PSA) in prostate cancer (15), and CA125 in ovarian cancer (16, 17), which highlights the clinical importance of identifying glycoproteins as indicators or biomarkers of diseases. Therefore, effective methods for systematic analysis of protein glycosylation are essential to understand the mechanisms of glycobiology, identify drug targets and discover biomarkers.Approximately half of mammalian cell proteins are estimated to be glycosylated at any given time (18). There have been many reports regarding identification of protein glycosylation sites and elucidation of glycan structures (19–30). Glycan structure analysis can lead to potential therapeutic and diagnostic applications (31, 32), but it is also critical to identify which proteins are glycosylated as well as the sites at which the modification occurs. Despite progress in recent years, the large-scale analysis of protein glycosylation sites using MS-based proteomics methods is still a challenge. Without an effective enrichment method, the low abundance of glycoproteins prohibits the identification of the majority of sites using the popular intensity-dependent MS sequence method.About a decade ago, a very beautiful and elegant method based on hydrazide chemistry was developed to enrich glycopeptides. Hydrazide conjugated beads reacted with aldehydes formed from the oxidation of cis-diols in glycans (33). This method has been extensively applied to many different types of biological samples (34–41). Besides the hydrazide-based enrichment method, lectins have also been frequently used to enrich glycopeptides or glycoproteins before MS analysis (28, 29, 42–46). However, there are many different types of lectins, and each is specific to certain glycans (47, 48). Therefore, no combination of lectins can bind to all glycosylated peptides or proteins, which prevents comprehensive analysis of protein glycosylation. Because of the complexity of biological samples, effective enrichment methods are critical for the comprehensive analysis of protein glycosylation before MS analysis.One common feature of all glycoproteins and glycopeptides is that they contain multiple hydroxyl groups in their glycans. From a chemistry point of view, this can be exploited to effectively enrich them. Ideally, chemical enrichment probes must have both strong and specific interactions with multiple hydroxyl groups. The reaction between boronic acids and 1,2- or 1,3-cis-diols in sugars has been extensively studied (49–52) and applied for the small-scale analysis of glycoproteins (53–55). Furthermore, boronate affinity chromatography has been employed for the analysis of nonenzymatically glycated peptides (56, 57). Boronic acid-based chemical enrichment methods are expected to have great potential for global analysis of glycopeptides when combined with modern MS-based proteomics techniques. However, the method has not yet been used for the comprehensive analysis of protein N-glycosylation in complex biological samples (58).Yeast is an excellent model biological system that has been extensively used in a wide range of experiments. Last year, two papers reported the large-scale analysis of protein N-glycosylation in yeast (59, 60). In one study, a new MS-based method was developed based on N-glycopeptide mass envelopes with a pattern via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides with the recoded envelopes were specifically targeted for fragmentation, facilitating high confidence site mapping (59). Using this method, 133 N-glycosylation sites were confidently identified in 58 yeast proteins. When combined with an effective enrichment method, this MS-based analysis will provide a more complete coverage of the N-glycoproteome. The other work combined lectin enrichment with digestion by two enzymes (Glu_c and trypsin) to increase the peptide coverage, and 516 well-localized N-glycosylation sites were identified in 214 yeast proteins by MS (60).Here we have comprehensively identified protein N-glycosylation sites in yeast by combining a boronic acid-based chemical enrichment method with MS-based proteomics techniques. Magnetic beads conjugated with boronic acid were systematically optimized to selectively enrich glycosylated peptides from yeast whole cell lysates. The enriched peptides were subsequently treated with Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)¹ in heavy-oxygen water. Finally, peptides were analyzed by an on-line LC-MS system. Over 800 protein N-glycosylation sites were identified in the yeast proteome, which clearly demonstrates that the boronic acid-based chemical method is an effective enrichment method for large-scale analysis of protein glycosylation by MS.     

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.Clinical proteomics focusing on the identification and validation of biomarkers and the discovery of proteins as therapeutic targets is an emerging and highly important area of proteomics. Biomarkers are measurable indicators of a specific biological state (particularly one relevant to the risk of contraction) and the presence or the stage of disease, and are thus expected to be useful for the prediction, detection, and diagnosis of disease as well as to follow the efficacy, toxicology, and side effects of drug treatment, and to provide new functional insights into biological processes.At present, proteomics methods based on mass spectrometry (MS) have emerged as the preferred strategy for discovery of diagnostic, prognostic, and therapeutic protein biomarkers. Most biomarker discovery studies use unbiased, “identified-based” approaches that rely on high performance mass spectrometers and extensive sample processing. Semiquantitative comparisons of protein relative abundance between disease and control patient samples are used to identify proteins that are differentially expressed and, thus, to populate lists of potential biomarkers. De novo proteomics discovery experiments often result in tens to hundreds of candidate biomarkers that must be subsequently verified in serum. However, despite the large numbers of putative biomarkers, only a small number of them are passed through the development and validation process into clinical practice, and their rate of introduction is declining. The first non-standard abbreviation (MS above is standard) must be footnoted the same as the abbreviation footnote, and MRM must be the first abbreviation in the list because it is the one footnoted. After that the order does not matter.Targeted proteomics using multiple reaction monitoring (MRM)1 is emerging as a technology that complements the discovery capabilities of shotgun strategies as well as an alternative powerful novel MS-based approach to measure a series of candidate biomarkers (1–7). Therefore, MRM is expected to provide a powerful high throughput platform for biomarker validation, although clinical validation of novel biomarkers has been traditionally relying on immunoassays (8, 9). MRM exploits the unique capabilities of triple quadrupoles (QQQ) MS for quantitative analysis. In MRM, the first and the third quadrupoles act as filters to specifically select predefined m/z values corresponding to the peptide precursor ion and specific fragment ion of the peptide, whereas the second quadrupole serves as collision cell. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with retention time and signal intensity for a specific transition as coordinates. These measurements have been multiplexed to provide 30 or more specific assays in one run. Such methods are slowly gaining acceptance in the clinical laboratory for the routine measurement of endogenous metabolites (10) (e.g. in screening newborns for a panel of inborn errors of metabolism) some drugs (11) (e.g. immunosuppressants), and the component analysis of sugars (12).One of the profound challenges in clinical proteomics is the need to handle highly complex biological mixtures. This complexity presents unique analytical challenges that are further magnified with the use of clinical serum/plasma samples to search for novel biomarkers of human disease. The serum proteome is composed of tens of thousands of unique proteins, of which concentrations may exceed 10 orders of magnitude. Protein glycosylation, one of the most common post-translational modifications, generates tremendous diversity, complexity, and heterogeneity of gene products. It changes the biological and physical properties of proteins, which include functions as signals or ligands to control their distribution, antigenicity, metabolic fate, stability, and solubility. Protein glycosylation, in particular by N-linked glycans, is prevalent in proteins destined for extracellular environments. These include proteins on the extracellular side of the plasma membrane, secreted proteins, and proteins contained in body fluids (such as blood serum, cerebrospinal fluid, urine, breast milk, saliva, lung lavage fluid, or pancreatic juice). Considering that such body fluids are most easily accessible for diagnostic and therapeutic purposes, it is not surprising that many clinical biomarkers and therapeutic targets are glycoproteins. These include, for example, cancer antigen 125 (CA125) in ovarian cancer, human epidermal growth factor receptor 2 (Her2/neu) in breast cancer, and prostate-specific antigen (PSA) in prostate cancer. In addition, changes in the extent of glycosylation and the structure of N-glycans or O-glycans attached to proteins on the cell surface and in body fluids have been shown to correlate with cancer and other disease states, highlighting the clinical importance of this modification as an indicator or effector of pathologic mechanisms (13–16). Thus, clinical proteomic platforms should have capability to provide protein glycosylation information as well as sufficient analytical depth to reliably detect and quantify specific proteins with sufficient accuracy and throughput.To improve the detection limits to the required sensitivities, one needs to dramatically reduce the complexity of the sera samples. For focused glycoproteomics, several techniques using lectins or antibodies enabling the large-scale identification of glycoproteins have recently been developed (17–19). Notably, Zhang et al. reported a method for the selective isolation of peptides based on chemical oxidation of the carbohydrate moiety and subsequent conjugation to a solid support using hydrazide chemistry (20–26). However, it is not possible to provide any structural information about N-glycans because the MS analysis is performed on peptides of which N-glycans are removed preferentially by treating with peptide N-glycanase (PNGase). In 2007, we developed a method for rapid enrichment analysis of peptides bearing sialylated N-glycans on the MALDI-TOF-MS platform (27). The method involves highly selective oxidation of sialic acid residues of glycopeptides to elaborate terminal aldehyde group and subsequent enrichment by chemical ligation with a polymer reagent, namely, reverse glycoblotting technique inspired from an original concept of glycoblotting method (28). This method, in principle, is capable identifying both glycan and peptide sequences concurrently. Recently, Nilsson et al. reported that glycopeptides from human cerebrospinal fluid can be enriched on the basis of the same principle as the reverse glycoblotting protocol, and captured glycopeptides were analyzed with ESI FT-ICR MS (29). Because it is well known that sialic acids play important roles in various biological processes including cell differentiation, immune response, and oncogenesis (30–34), our attention has been directed toward feasibility of the reverse glycoblotting technique in quantitative analysis of the specific glycopeptides carrying sialic acid(s) by combining with multiplexed MRM-based MS.     

Identification of Sequences in the Polysialyltransferases ST8Sia II and ST8Sia IV That Are Required for the Protein-specific Polysialylation of the Neural Cell Adhesion Molecule, NCAM

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel α-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423–436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg²⁷⁶/Arg²⁷⁷ or Arg²⁶⁵ in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg⁸²/Arg⁹³ in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu⁹² and Asp⁹⁴, surround Arg⁹³. Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.Polysialic acid is a linear homopolymer of α2,8-linked sialic acid that is added to a small subset of mammalian glycoproteins by the polysialyltransferases (polySTs)3 ST8Sia II (STX) and ST8Sia IV (PST) (1–4). Substrates for the polySTs include the neural cell adhesion molecule (NCAM) (5, 6), the α-subunit of the voltage-dependent sodium channel (7, 8), CD36, a scavenger receptor found in milk (9), neuropilin-2 expressed by dendritic cells (10), and the polySTs themselves, which can polysialylate their own N-glycans in a process called autopolysialylation (11, 12). This small number of polysialylated proteins and other evidence from our laboratory (13–15) suggest that polysialylation is a protein-specific modification that requires an initial protein-protein interaction between the polySTs and their glycoprotein substrates.The most abundant polysialylated protein is NCAM. The three major NCAM isoforms consist of five Ig domains, two fibronectin type III repeats, and a transmembrane domain and cytoplasmic tail (NCAM140 and NCAM180) or a glycosylphosphatidylinositol anchor (NCAM120) (16). Polysialylation takes place primarily on two N-linked glycans in the Ig5 domain (17). We have previously shown that a truncated NCAM140 protein consisting of Ig5, the first fibronectin type III repeat (FN1), the transmembrane region, and cytoplasmic tail is fully polysialylated (13). However, a protein consisting of Ig5, the transmembrane region, and cytoplasmic tail is not polysialylated (13). This suggests that the polySTs recognize and bind the FN1 domain to polysialylate N-glycans on the adjacent Ig5 domain. We subsequently identified an acidic patch unique to NCAM FN1, consisting of Asp⁴⁹⁷, Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴ (15).⁴ When three of these residues (Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴) are mutated to alanine or arginine, NCAM polysialylation is reduced or abolished, suggesting that the acidic patch is part of a larger recognition region. We anticipate that within this putative recognition region there will be amino acids required for mediating polyST-NCAM binding, and those that do not mediate binding per se but instead are required for correct positioning of the enzyme-substrate complex for polysialylation. For example, we have identified a novel α-helix in the FN1 domain that when replaced leads to polysialylation of O-glycans found on the FN1 domain rather than N-glycans on the Ig5 domain (14). This helix may mediate an interdomain interaction that positions the Ig5 N-glycans for polysialylation by an enzyme bound to the FN1 domain (14). Alternatively, the helix could act as a secondary interaction site that positions the polyST properly on the substrate.The expression of the polySTs is developmentally regulated with high levels of STX and moderate levels of PST expressed throughout the developing embryo (2, 18, 19). STX levels decline after birth, although PST expression persists in specific regions of the adult brain where polysialylated NCAM is involved in neuronal regeneration and synaptic plasticity (18–23). The large size and negative charge of polysialic acid disrupt NCAM-dependent and NCAM-independent interactions, thereby negatively modulating cell adhesion (24–26). Simultaneous disruption of both PST and STX in mice results in severe neuronal defects and death usually within 4 weeks after birth (27). Interestingly, when NCAM expression is also eliminated in these mice, they have a nearly normal phenotype, suggesting the main function of polysialic acid is to modulate NCAM-mediated cell adhesion during development (27). In addition, re-expression of highly polysialylated NCAM has been associated with several cancers, including neuroblastomas, gliomas, small cell lung carcinomas, and Wilms tumor. The presence of polysialic acid is thought to promote cancer cell growth and invasiveness (28–35).Sialyltransferases, including the polySTs, have three motifs required for catalytic activity (36–38) (see Fig. 1A). Sialyl motif Large (SML) is thought to bind the donor substrate CMP-sialic acid (39), whereas sialyl motif Small (SMS) is believed to bind both donor and carbohydrate acceptor substrates (40). The sialyl motif Very Small (SMVS) has a conserved His residue that is required for catalytic activity (38, 41). However, the precise function of this motif is unknown. An additional 4-amino acid motif, motif III, is conserved in the sialyltransferases (42–44). It was suggested that this motif, and particularly His and Tyr residues within its sequence, may be required for optimal activity and acceptor recognition (42).Open in a separate windowFIGURE 1.PST and STX polybasic regions and mutants generated for this study. A, representation of the polySTs and their polybasic regions and sialyl motifs. The PBR is a 35-amino acid region present in both PST and STX, equidistant from the SML of each enzyme and rich in conserved positively charged amino acids. The PSTD is a region identified by Nakata et al. (47) that is 32 amino acids in length, rich in basic residues, and contiguous with the SMS of the enzymes. The sialyl motifs (SML, SMS, SMVS, and motif III) are regions of homology found in all sialyltransferases that are believed to be involved in substrate and donor interactions. B, PSTD of PST and the mutants made in this region that are used in this study. C, PBR of PST and STX and the mutants made in this region that are used in this study.Angata et al. (45) used chimeric enzymes to identify regions within the polySTs required for catalytic activity and NCAM polysialylation. Sequences from PST, STX, and ST8Sia III were used to construct the chimeric proteins. ST8Sia III is an α2,8-sialyltransferase that typically adds one or two sialic acid residues to glycoprotein or glycolipid substrates, can autopolysialylate its own glycans, but cannot polysialylate NCAM (46). Deletion analysis showed that amino acids 62–356 are required for PST catalytic activity. Replacement of segments of this region with corresponding STX or ST8Sia III sequences led to the suggestion that amino acids 62–127 and possibly 194–267 of PST may be required for NCAM recognition (45).Recently, Troy and co-workers (47, 48) identified a stretch of basic residues, termed the polysialyltransferase domain (PSTD), which is only observed in the two polySTs and not in other sialyltransferases. The PSTD is contiguous with SMS and extends from amino acids 246–277 in PST and 261–292 in STX. Mutation analysis demonstrated that the overall positive charge of this motif is important for activity and identified specific residues required for NCAM polysialylation (Arg²⁵², Ile²⁷⁵, Lys²⁷⁶, and Arg²⁷⁷) (47).In this study, we have scanned the critical polyST regions identified by the work of Angata et al. (45) for sequences that may be involved in protein-protein recognition and NCAM polysialylation. We identified a second polybasic motif that we named the polybasic region (PBR). The PBR is conserved in PST and STX and is located equidistant from the SML of each enzyme. It consists of 35 amino acids of which 7 are the basic amino acids Arg and Lys. We found that the replacement of two specific residues within the PBR (Arg⁸² and Arg⁹³ of PST and Arg⁹⁷ and Lys¹⁰⁸ of STX) have a greater negative effect on NCAM polysialylation than on autopolysialylation. Replacement of acidic residues surrounding PST Arg⁹³ led to a similar disparate effect on these processes. Comparison of the critical residues in both the PSTD and PBR demonstrated that the replacement of PSTD residues had an equally negative impact on both NCAM polysialylation and enzyme autopolysialylation, whereas replacement of selected PBR residues more severely impacted NCAM polysialylation, suggesting that the PBR residues may play important roles in NCAM-specific polysialylation.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1–4(Fucα1–3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1–4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1–3(Galβ1–6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome''s glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.Schistosoma blood flukes give rise to infections in over 200 million people in developing countries worldwide (1). With a Disability-Adjusted Life Years (DALY) value of more than 3 million, schistosomiasis ranks as one of the neglected tropical diseases with the highest impact on public health (2). The schistosome has a complex and intriguing lifecycle, which involves a definitive host (mammal) as well as an intermediate host (snail). Infections with Schistosoma mansoni, one of the major schistosome species infecting humans, are initiated when snail-borne cercariae penetrate intact skin. The cercariae then transform into schistosomula, which enter the vasculature of the host and mature while migrating to the portal system. Here, adult male and female worms pair, with the female worm producing hundreds of eggs each day during a life span of several years unless the infection is treated by chemotherapy. Miracidia develop inside the maturing eggs while they cross the intestinal wall over a period of several days to be excreted with the feces. Miracidia then hatch from the eggs upon contact with fresh water and infect the snail host where asexual replication takes place and eventually new cercariae are shed. Notably, many eggs get trapped in organs such as the liver, where they induce a granulomatous inflammation and organ damage, the main cause of pathology in schistosomiasis (1).Throughout their lifecycle, schistosomes express a multitude of protein- and lipid-linked glycans that play an important role in the parasite biology. The expression of many glycan elements appears to be developmentally regulated by the differential expression of glycosyltransferases during the different lifecycle stages (3). A series of papers has been published indicating that schistosome glycans play essential roles in the molecular interaction of the parasite and the host immune system, enabling survival of the parasite and allowing chronic infection to establish. For example, glycosylated soluble egg antigens (SEA) interact with the C-type lectins mannose receptor (MR), macrophage galactose-type lectin (MGL) and dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and some of these interactions lead to immunomodulatory effects of specific components of SEA via dendritic cells (DCs)¹ (4, 5). Furthermore, fucosylated egg glycolipids trigger innate immune responses of peripheral blood mononuclear cells and egg glycans are required for periovular granuloma formation in a mouse model. In addition, cercarial secretions induce alternatively activated macrophages in a carbohydrate dependent manner (6–9). Importantly, also adaptive immune responses to schistosome glycans are mounted by the human host. A large part of the antibody responses to schistosomes is directed against antigenic glycan motifs, raising the question whether they could form a basis for antischistosome vaccine strategies (10).Rapid developments in mass spectrometry-based glycan-analysis technology in the last two decades have led to several studies focused on elucidating the glycan structures of somatic and secretory schistosome preparations (11–22). Among the typical glycan elements detected in S. mansoni were unusual and antigenic Fucα1–2Fucα1–3- (DF-) motifs attached to GalNAcβ1–4GlcNAc (LacDiNAc or LDN) (12, 14, 17–19, 21), Xylβ1–2- and Fucα1–3-modified N-glycan core structures (13, 15, 17, 20), and a unique O-glycan core (Galβ1–3(Galβ1–6)GalNAc) (14, 17) (see supplemental Table S5 for a definition of glycan motifs of S. mansoni glycoconjugates). Also more widely occurring glycan elements shared with the mammalian or snail host were detected, e.g. Galβ1–4GlcNAc (LacNAc or LN), Galβ1–4(Fucα1–3)GlcNAc (Lewis X or LeX), LDN, and GalNAcβ1–4(Fucα1–3)GlcNAc (LDN-F) (23, 24). These data were generated over a long period of time, often focusing on a single schistosome life stage and a specific class of glycans only, and using various analytical techniques and strategies that make inter-study comparisons often difficult. In addition, glycosylation of the schistosomula that develop shortly after infection and are considered to be relatively vulnerable to immune attack, has remained largely unexplored (20, 25, 26), although these could be interesting therapeutic targets (27–29). Clearly, an integrated and complete overview of schistosome glycosylation was so far not available.In this study, we therefore set out to determine the overall schistosome protein- and lipid-linked glycome by analyzing a total of 16 lifecycle stages ranging from cercariae to miracidia. We analyzed the glycoprotein-derived N- and O-glycans as well as the lipid-derived glycans of these life stages by a MALDI-TOF MS-based approach complemented with fragmentation and enzyme degradation studies. Our findings give new insights in the glycobiology of parasite development and parasite–host interaction and contribute to the identification of new potential immune intervention targets.