Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.Acinetobacter baumannii is an emerging opportunistic pathogen of increasing significance to health care institutions worldwide (1–3). The growing number of identified multiple drug resistant (MDR)¹ strains (2–4), the ability of isolates to rapidly acquire resistance (3, 4), and the propensity of this agent to survive harsh environmental conditions (5) account for the increasing number of outbreaks in intensive care, burn, or high dependence health care units since the 1970s (2–5). The burden on the global health care system of MDR A. baumannii is further exacerbated by standard infection control measures often being insufficient to quell the spread of A. baumannii to high risk individuals and generally failing to remove A. baumannii from health care institutions (5). Because of these concerns, there is an urgent need to identify strategies to control A. baumannii as well as understand the mechanisms that enable its persistence in health care environments.Surface glycans have been identified as key virulence factors related to persistence and virulence within the clinical setting (6–8). Acinetobacter surface carbohydrates were first identified and studied in A. venetianus strain RAG-1, leading to the identification of a gene locus required for synthesis and export of the surface carbohydrates (9, 10). These carbohydrate synthesis loci are variable yet ubiquitous in A. baumannii (11, 12). Comparison of 12 known capsule structures from A. baumannii with the sequences of their carbohydrate synthesis loci has provided strong evidence that these loci are responsible for capsule synthesis with as many as 77 distinct serotypes identified by molecular serotyping (11). Because of the non-template driven nature of glycan synthesis, the identification and characterization of the glycans themselves are required to confirm the true diversity. This diversity has widespread implications for Acinetobacter biology as the resulting carbohydrate structures are not solely used for capsule biosynthesis but can be incorporated and utilized by other ubiquitous systems, such as O-linked protein glycosylation (13, 14).Although originally thought to be restricted to species such as Campylobacter jejuni (15, 16) and Neisseria meningitidis (17), bacterial protein glycosylation is now recognized as a common phenomenon within numerous pathogens and commensal bacteria (18, 19). Unlike eukaryotic glycosylation where robust and high-throughput technologies now exist to enrich (20–22) and characterize both the glycan and peptide component of glycopeptides (23–25), the diversity (glycan composition and linkage) within bacterial glycosylation systems makes few technologies broadly applicable to all bacterial glycoproteins. Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26, 27) and the use of multiple fragmentation approaches (28, 29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30–33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought. For example, Nothaft et al. were able to show N-linked glycosylation was widespread in the Campylobacter genus and that two broad groupings of the N-glycans existed (34).During the initial characterization of A. baumannii O-linked glycosylation the use of selective enrichment of glycopeptides followed by mass spectrometry analysis with multiple fragmentation technologies was found to be an effective means to identify multiple glycosylated substrates in the strain ATCC 17978 (14). Interestingly in this strain, the glycan utilized for protein modification was identical to a single subunit of the capsule (13) and the loss of either protein glycosylation or glycan synthesis lead to decreases in biofilm formation and virulence (13, 14). Because of the diversity in the capsule carbohydrate synthesis loci and the ubiquitous distribution of the PglL O-oligosaccharyltransferase required for protein glycosylation, we hypothesized that the glycan variability might be also extended to O-linked glycosylation. This diversity, although common in surface carbohydrates such as the lipopolysaccharide of numerous Gram-negative pathogens (35), has only recently been observed within bacterial proteins glycosylation system that are typically conserved within species (36) and loosely across genus (34, 37).In this study, we explored the diversity within the O-linked protein glycosylation systems of Acinetobacter species. Our analysis complements the recent in silico studies of A. baumannii showing extensive glycan diversity exists in the carbohydrate synthesis loci (11, 12). Employing global strategies for the analysis of glycosylation, we experimentally demonstrate that the variation in O-glycan structure extends beyond the genetic diversity predicted by the carbohydrate loci alone and targets proteins of similar properties and identity. Using this knowledge, we developed a targeted approach for the detection of protein glycosylation, enabling streamlined analysis of glycosylation within a range of genetic backgrounds. We determined that; O-linked glycosylation is widespread in clinically relevant Acinetobacter species; inter- and intra-strain heterogeneity exist within glycan structures; glycan diversity, although extensive results in the generation of glycans with similar properties and that the utilization of a single glycan for capsule and O-linked glycosylation is a general feature of A. baumannii but may not be a general characteristic of all Acinetobacter species such as A. baylyi.     

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.     

SweetSEQer,Simple de Novo Filtering and Annotation of Glycoconjugate Mass Spectra

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.Protein glycosylation is a common modification, affecting ∼50% of all expressed proteins (1). Glycosylation affects critical biological functions, including cell-cell recognition, circulating half-life, substrate binding, immunogenicity, and others (2). Regrettably, determining the exact role glycosylation plays in different biological contexts is slowed by a dearth of analytical methods and of appropriate software. Such software is crucial for performing and aiding experts in data analysis complex glycosylation.Glycopeptides are highly heterogeneous in regard to glycan composition, glycan structure, and linkage stereochemistry in addition to the tens of thousands of possible peptides. The analysis of protein glycosylation is often segmented into three distinct types of mass spectrometry experiments, which together help to resolve this complexity. The first analyzes enzymatically or chemically released glycans (which may or may not be chemically modified), and the second determines glycosylation sites after release of glycans from peptides (the resulting mass spectra allow detection of glycosylation sites and the glycans on those sites simultaneously). The third determines the glycosylation sites and the glycans on those sites simultaneously, by MS of intact glycopeptides. Frequently, researchers will perform all three types of analysis, with the first two types providing information about possible combinations of glycan structures and peptides that could be found in the third experiment. Using this MS1 information, the problem is reduced to matching masses observed with a combinatorial pool of all possible glycans and all possible glycosylated peptides within a sample; however, this combinatorial approach alone is insufficient (3), and tandem mass spectrometry can provide copious additional information to help resolve the glycopeptide content from complex samples.The similar problem of inferring peptide sequences from MS/MS spectra has received considerably more attention. Peptide inference is more constrained than glycan inference, because the chain of MS/MS peaks corresponds to a linear peptide sequence; given an MS/MS spectrum, the linear peptide sequence can be inferred through brute force or dynamic programming via de novo methods (4–6) as described in Ref. 7. Additionally, the possible search space of peptides can be dramatically lowered by using database searching (8–21) as described in Ref. 7, which compares the MS/MS spectrum to the predicted spectra from only those peptides resulting from a protein database or translated open reading frames (ORFs) of a genomic database.The possible search space of glycans is larger than the search space of peptides because, in contrast to linear peptide chains, glycans may form branching trees. Identifying glycans using database search methodologies is impractical, as it is impractical to define the database when the detailed activities of the set of glycosyltransferases are not defined. Generating an overly large database would artificially inflate the set of incompletely characterized spectra, and too small of a search space would lead to inaccurate results. Furthermore, as glycosylation is not a template-driven process, no clear choice for a database matching approach is available, and de novo sequencing is therefore a more appropriate approach.As a result, few desirable software options are available for the high throughput analysis of tandem mass spectrometry data from intact glycopeptides (as noted in a recent review (22)). In fact, manual annotation of spectra is still commonplace, despite being slow and despite the potential for disagreement between different experts. Some available software requires user-defined lists of glycan and/or peptide masses as input, which is suboptimal from a sample consumption and throughput perspective (23, 24). These lists must typically be generated by parallel experiments or simply hypothesized a priori, meaning omissions in either list may affect the results. Furthermore, some software does not work on batched input files, meaning each spectrum must be analyzed separately (23, 25–28). Moreover, there is an even greater lack of open source software for glycoproteomics, so modifying the existing software for the researchers individual applications is not easily achieved. The one open source tool that we know of (GlypID) is applicable only to the analysis of glycopeptide spectra acquired from a very specialized workflow, which requires MS1, CID, and higher-energy C-trap type dissociation (HCD) spectra (29). With that approach, oxonium ions from HCD spectra are necessary to predict the glycan class; potential peptide lists are queried by precursor m/z values (requiring accurate a priori knowledge of all modifications), and possible theoretical “N-linked” precursor m/z values are used to select candidate spectra (using templates, unlike de novo characterization). As a result, the tool is specialized and limited to analysis of “N-linked” glycopeptide spectra from very specific experimental setups.Free, open-source glycoproteomic software capable of batch analysis of general tandem mass spectrometry spectra of glycoconjugates is sorely needed. In this work, we present SweetSEQer, a tool for de novo analysis of tandem mass spectra of glycoconjugates (the most general class of spectra containing fragmentation involving sugars). Furthermore, because SweetSEQer is so general and simple, and because it does not require specific experimental setup, it is widely applicable to the analysis of general glycoconjugate spectra (e.g. it is already applicable to “O-linked” glycopeptide and glycoconjugate spectra). Moreover, because it is an open source and does not use external software, it not only eschews solving problems like MS1 deisotoping, it can also be easily customized and even used to augment and complement existing tools like GlypID (and, because we do not use a “copyleft” software license, our algorithm and code can even be added to non-open source and proprietary variants).SweetSEQer''s performance was tested on a validated, manually annotated set of glycoconjugate identifications from a urinary glycoproteomics study. Specificity was demonstrated by showing a low identification rate on negative control spectra from Escherichia coli. Annotated structures are shown to be consistent by a human expert by demonstrating a high overlap in identified glycan fragment ions, as well as a consistency between SweetSEQer''s predicted glycan graph and glycan chains produced by an expert. Our simple object-oriented python implementation is freely available (Apache 2.0 license) on line.     

The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of α-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.Alterations to the glycan structures on extracellular proteins are a common feature of many types of epithelial cancer such as pancreatic, colon, and breast cancers (1, 2). Cancer-associated glycan structures are thought to be functionally involved in many of the phenotypes characterizing cancer cells, including the ability to migrate, avoid apoptosis, evade immune destruction, and enter and exit the vasculature (3). Because proteins bearing cancer-associated glycans can be shed by tumor cells into the circulation, blood-based diagnostic tests using glycan detection may be possible. A potential advantage of using glycans for diagnostics is that carbohydrate modifications of particular proteins may be altered more frequently or more specifically in certain disease states than their underlying core protein concentrations. However, to evaluate and use such a strategy, the prevalence with which various structures appear and the specific proteins on which they appear must be better characterized.Previous studies of cancer-associated glycosylation using enzymatic, chromatographic, and mass spectrometry methods have been very effective for providing detailed information about the glycan structures produced by cancer cells, but because of the requirements for large amounts of material and the time involved to analyze each sample, these studies generally used either cell culture material or a small number of patient samples. Therefore, while many cancer-associated glycans have been identified, much remains unknown about these glycans, including how often they appear, how closely they are associated with particular disease states, and the distribution of protein carriers on which they appear.Affinity-based methods, using reagents such as lectins or glycan-binding antibodies, are a valuable complement to the above mentioned methods. Using antibodies or lectins that bind specific glycans, one may reproducibly measure the levels of those glycans over multiple samples. Although affinity-based glycosylation studies do not provide the structural detail provided by mass spectrometry and enzymatic methods, they can provide information about the biological variation of a particular motif.Lectins and glycan-binding antibodies have been used extensively in immunohistochemistry, for example in studies to examine the tissue distribution in pancreatic tumors of certain blood group carbohydrates (4, 5). Lectins have been valuable in immunoaffinity electrophoresis and blotting methods to identify cancer-associated glycan variants on major serum proteins such as α-fetoprotein (6), haptoglobin (7, 8), α₁-acid glycoprotein (9), and α₁-antitrypsin (10). Antibodies raised against particular glycan groups, such as the Thomsen-Friedenreich antigens (11), the Lewis blood group structures (12), and underglycosylated MUC1¹ (13) also have been used to study the roles of glycans in cancer. As a means of quantifying glycans on specific proteins, lectins have been used in the capture or detection of proteins in microtiter plates (14).We previously demonstrated an antibody-lectin sandwich array method (15) that is a valuable complement to the above methods and is ideal for profiling the prevalence of multiple glycans on multiple proteins. Glycan levels can be probed directly from biological samples, and many samples or detection conditions can be processed efficiently in a low volume, high throughput format (16). This method is complementary to lectin microarrays (17–19), which are useful for measuring glycan levels on individual, purified proteins; glycan microarrays (20, 21), which are used to measure the recognition of carbohydrate structures by various glycan-binding reagents; and glycoprotein arrays (22) for examining glycosylation on proteins isolated from biological samples.We applied this method to the study of glycan alterations on proteins in the circulation of pancreatic cancer patients. We sought to define the prevalence of various glycan alterations on particular protein carriers and to investigate whether those measurements have advantages for cancer diagnostics relative to measurements of core proteins. We designed antibody microarrays to target members of the mucin and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) families because some of those proteins are known to carry cancer-associated glycans. Mucins are extracellular, long-chain glycoproteins involved in the control and protection of epithelial surfaces, and the expression and glycosylation of several mucins are often altered and functionally involved in cancer (23, 24). The CEACAM family of proteins also is functionally involved in cancer, and they carry cancer-associated glycans (25, 26), but the glycans on CEACAMs are less well studied than those on mucins. By measuring both glycan levels and the core protein levels of several of these molecules, we were able to investigate whether alterations to glycans can appear at a higher rate than changes to core protein abundances. The ability to test the presence of glycan structures on multiple protein carriers in multiple samples was critical to investigating these questions.     

Comparative Performance of Four Methods for High-throughput Glycosylation Analysis of Immunoglobulin G in Genetic and Epidemiological Research

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.Glycans are important structural and functional components of the majority of proteins, but because of their structural complexity and the absence of a direct genetic template our current understanding of the role of glycans in biological processes lags significantly behind the knowledge about proteins or DNA (1, 2). However, a recent comprehensive report endorsed by the US National Academies concluded that “glycans are directly involved in the pathophysiology of every major disease and that additional knowledge from glycoscience will be needed to realize the goals of personalized medicine” (3).It is estimated that the glycome (defined as the complete set of all glycans) of a eukaryotic cell is composed of more than a million different glycosylated structures (1), which contain up to 10,000 structural glycan epitopes for interaction with antibodies, lectins, receptors, toxins, microbial adhesins, or enzymes (4). Our recent population-based studies indicated that the composition of the human plasma N-glycome varies significantly between individuals (5, 6). Because glycans have important structural and regulatory functions on numerous glycoproteins (7), the observed variability suggests that differences in glycosylation might contribute to a large part of the human phenotypic variability. Interestingly, when the N-glycome of isolated immunoglobulin G (IgG)¹ was analyzed, it was found to be even more variable than the total plasma N-glycome (8), indicating that the combined analysis of all plasma glycans released from many different glycoproteins blurs signals of protein-specific regulation of glycosylation.A number of studies have investigated the role of glycans in human disease, including autoimmune diseases and cancer (9, 10). However, most human glycan studies have been conducted with very small sample sizes. Given the complex causal pathways involved in pathophysiology of common complex disease, and thus the likely modest effect sizes associated with individual factors, the majority of these studies are very likely to be substantially underpowered. In the case of inflammatory bowel disease, only 20% of reported inflammatory bowel disease glycan associations were replicated in subsequent studies, suggesting that most are false positive findings and that there is publication bias favoring the publication of positive findings (11). This situation is similar to that which occurred in the field of genetic epidemiology in the past when many underpowered candidate gene studies were published and were later found to consist of mainly false positive findings (12, 13). It is essential, therefore, that robust and affordable methods for high-throughput analysis are developed so that adequately powered studies can be conducted and the publication of large numbers of small studies reporting false positive results (which could threaten the credibility of glycoscience) be avoided.Rapid advances of technologies for high-throughput genome analysis in the past decade enabled large-scale genome-wide association studies (GWAS). GWAS has become a reliable tool for identification of associations between genetic polymorphisms and various human diseases and traits (14). Thousands of GWAS have been conducted in recent years, but these have not included the study of glycan traits until recently. The main reason was the absence of reliable tools for high-throughput quantitative analysis of glycans that could match the measurements of genomic, biochemical, and other traits in their cost, precision, and reproducibility. However, several promising high-throughput technologies for analysis of N-glycans were developed (8, 15–20) recently. Successful implementation of high-throughput analytical techniques for glycan analysis resulted in publication of four initial GWAS of the human glycome (21–24).In this study, we compared ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR), multiplex capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography electrospray mass spectrometry (LC-ESI-MS) as tools for mid-to-high-throughput glycomics and glycoproteomics. We have analyzed IgG N-glycans by all four methods in 1201 individuals from European populations. The analysis of associations between glycans and ∼300,000 single-nucleotide genetic polymorphisms was performed and correlation between glycans and age was studied in all four data sets to identify the analytical method that shows the strongest potential to uncover biological mechanisms underlying protein glycosylation.     

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.     

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSⁿ analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSⁿ are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.Human milk offers nutrition, innate immune protection, and other developmental benefits to infants (1, 2). In addition to essential nutrients and bioactive antibodies, human milk uniquely possesses a rich pool of free-reducing glycans (oligosaccharides), most of which are unique to human milk (3, 4). Depending on the blood group status and the lactation stage of an individual, the concentration of human milk glycans (HMGs)¹ larger than lactose varies between 5 and 15 g/l, making them the third largest component of human milk after lactose and lipids (5). Over the past decades, more than 100 structurally distinct HMGs have been identified (6–9). All of these glycans originate from a lactose that is extended by type 1 (Galβ1–3GlcNAc) or type 2 (Galβ1–4GlcNAc) N-acetyllactosamine in either linear or branch forms and further modified with α-linked fucose and/or N-acetylneuraminic acid. It has been shown that HMGs are only minimally digested in the upper gastrointestinal tract and are transported intact into the lower parts of intestine (10, 11). Additionally, ∼1% to 2% of HMGs are excreted via an infant''s urine and seem to appear in the circulation (12, 13).Accumulated evidence has indicated that HMGs play multiple biological roles. In addition to having well-known prebiotic effects that promote the growth of beneficial microflora in the intestine (14, 15), HMGs are suggested to competitively interfere with pathogen attachment to the host cell surface by acting as soluble decoy receptors (16–18), and such anti-adhesive effects are often glycan specific (19). For example, α1–2 fucosylated HMGs, which arise mainly from individuals that are Secretor(+), were observed to prevent the adherence of Campylobacter jejuni to epithelial cells (20) and were associated with protection against diarrhea caused by Campylobacter, caliciviruses, and Escherichia coli toxin in breastfed infants (21–23). Sialylated HMGs were exclusive receptors for influenza viruses (24–26) and showed a capacity to inhibit cholera toxin B (27), Vibrio cholera (28), enterotoxigenic E. coli, and uropathogenic E. coli strains (29, 30). It was also proposed that HMGs might serve as anti-inflammatory components and thus contribute to the lower incidence of necrotizing enterocolitis in breastfed infants. This idea is supported by the observations that the acidic fraction of HMG inhibits leukocyte rolling, adhesion, and activation (31) and disialyllacto-N-tetraose prevents necrotizing enterocolitis in neonatal rats (32). Furthermore, a variety of cytoprotective activities of HMGs have been reported against Clostridium difficile toxins (33), Helicobacter pylori (34, 35), Streptococcus pneumonia (36), Entamoeba histolytica (37), and HIV-1-gp120 (38). Although the numerous in vitro and in vivo data provide important information about the function of HMGs, these studies have typically used HMG fraction mixtures or a small panel of defined HMGs, and therefore the bioactive HMGs were not or poorly identified.In order to better understand the interactions of HMGs with various microorganisms, it is necessary to examine the entire milk metaglycome and identify the specific bioactive components, which is not possible via traditional methods that mainly focus on compositional analysis of HMGs (39). To find an efficient route for establishing the function–structure relationship of HMGs, we applied a “shotgun glycomics” approach to generate a shotgun glycan microarray (SGM) from isolated human milk glycans of a Lewis-positive, non-secretor individual (25, 40). The functional recognition studies, along with metadata-assisted glycan sequencing (MAGS), revealed novel epitopes/receptors for anti-TRA-1 antibodies, influenza viruses, and minute viruses of mice. Our work represented the first natural glycan microarray of HMGs containing over 100 glycans. Notably, the antibody binding data showed a lack of α1,2-fucosylated HMGs on this SGM, confirming that the donor was a non-secretor (41, 42).Here we describe our studies in which we prepared a SGM containing over 200 isolated HMG targets from pooled human milk of mixed Lewis and Secretor phenotypes and investigated the binding of rotavirus (RV) cell attachment protein to them. Human RVs are the leading cause of severe gastroenteritis in children, responsible for an estimated 453,000 deaths each year worldwide (43). As with many other pathogens, RV infection is initiated by the interaction with specific cellular glycans. The VP8* domain of the RV outer capsid spike protein VP4 mediates this process (44), but the identity of VP8* receptors is quite controversial. It was believed that VP8* recognized either terminal sialic acid or internal sialic acid, mainly based on crystallographic and NMR studies (45–48). However, recently a human strain (HAL1166) with a P[14] VP8* was found to bind to A-type histo-blood group antigen (49), a neonatal strain with a P[11] VP8* bound to type 2 precursor glycans (50), and several other P types recognized secretor-related antigens Lewis b and H type 1 (51). These studies indicate that sialic acid might not be required by all RVs and that the glycan receptors are genotype-dependent. The infectivity of a porcine RV was inhibited by sialyl HMGs in vitro (52); however, there are limited data on human RVs. Here, we demonstrate that the VP8* of two different human neonatal RVs and an additional bovine strain bound to HMGs independent of sialic acid and that each VP8* demonstrated a unique glycan-binding specificity.     

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen''s ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host''s stomach. Although Hp''s glycans are linked to pathogenesis, Hp''s glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp''s glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp''s azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.Helicobacter pylori (Hp)¹ infection poses a significant health risk to humans worldwide. The Gram-negative, pathogenic bacterium Hp colonizes the gastric tract of more than 50% of humans (1). Approximately 15% of infected individuals develop duodenal ulcers and 1% of infected individuals develop gastric cancer (2). Current treatment to clear infection requires “triple therapy” (3), a combination of multiple antibiotics that is often associated with negative side effects (4). Because of poor patient compliance and the evolution of antibiotic resistance, existing antibiotics are no longer effective at eradicating Hp infection (4). New treatment methods are needed to eliminate Hp from the human gastric tract.Recent work has focused on gaining insights into the pathogenesis of Hp to aid the development of new treatments. The most recent findings in this area have conclusively revealed that glycosylation of proteins in Hp is required for pathogenesis. Hp use complex flagella, comprised of flagellin proteins, to navigate the host''s gastric mucosa (5, 6). The flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, found exclusively in mucosal-associated pathogens (Hp (7), Campylobacter jejuni (8) and Pseudomonas aeruginosa (9)). This modification is absolutely essential for the formation of functional flagella on Hp (7, 10). Deletion of any one of the enzymes in the pseudaminic acid biosynthetic pathway results in Hp that lack flagella, are nonmotile, and are unable to colonize the host''s stomach (7). Although pseudaminic acid is critical for Hp virulence, it is absent from humans (11, 12). Therefore, insights into Hp''s pathogenesis have revealed that Hp''s glycan pseudaminic acid is a bona fide target of therapeutic intervention. This is one of a number of examples linking protein glycosylation to virulence in medically significant bacterial pathogens (13, 14).Despite these findings, Hp''s glycome remains poorly understood overall. Only the two flagellin glycoproteins have been firmly characterized in Hp (7) to date. Nine other candidate glycoproteins have been identified in Hp, but their glycosylation status has not been biochemically confirmed (15). The relative paucity of information regarding Hp''s glycoproteins is due in part to the previously held belief that protein glycosylation could not occur in bacteria (13, 16, 17). However, even after Szymanski (18, 19), Koomey (20), Guerry (21), Logan (7), Comstock and others (13, 16, 17) disproved this belief by firmly establishing the synthesis of glycoproteins in bacteria, the study of bacterial glycoproteins has presented unique challenges for analytical study (14, 22). For example, the unusual structures of bacterial glycans, which often contain amino- and deoxy-carbohydrates exclusively found in bacteria (12, 23–25), hampers their identification using existing tools. Though methods such as the use of glycan-binding reagents (20, 24, 26, 27) and periodic acid/hydrazide glycan labeling (15) have successfully detected glycoproteins in a range of bacteria, they present limitations. Glycan binding-based methods are often limited because of the unavailability of lectins or antibodies with binding specificity for glycosylated proteins in the bacteria of interest (14, 22). Periodic acid/hydrazide-based labeling is plagued by a lack of specificity for glycosylated proteins (15). Thus, an efficient and robust approach to discover Hp''s glycoproteins is needed.In previous work, we established that the chemical technique known as metabolic oligosaccharide engineering (MOE), which was developed by Bertozzi (28, 29), Reutter (30), and others for the study of mammalian glycoproteins, is a powerful approach to label and detect Hp''s glycoproteins (31). Briefly, Hp metabolically processes the unnatural, azide-containing sugar peracetylated N-azidoacetylglucosamine (Ac₄GlcNAz) (32), an analog of the common metabolic precursor N-acetylglucosamine (GlcNAc), into cellular glycoproteins (Fig. 1). Elaboration of azide-labeled glycoproteins via Staudinger ligation (33) with a phosphine probe conjugated to a FLAG peptide (Phos-FLAG) (34) followed by visualization with an anti-FLAG antibody (Fig. 1) revealed a glycoprotein fingerprint containing a large number of as-yet unidentified Hp glycoproteins that merit further investigation (31).Open in a separate windowFig. 1.Metabolic oligosaccharide engineering facilitates labeling and detection of Hp''s glycoproteins. Supplementation of Hp with Ac₄GlcNAz leads to metabolic labeling of Hp''s N-linked and O-linked glycoproteins with azides. Azide-modified glycoproteins covalently labeled with Phos-FLAG can be detected via Western blot analysis with anti-FLAG antibody to yield Hp''s glycoprotein fingerprint, which contains a large number of as-yet unidentified glycoproteins.Here we describe a glycoproteomic identification strategy for the selective detection, isolation, and discovery of Hp''s glycoproteins. In particular, we demonstrate that glycan metabolic labeling coupled with mass spectrometry analysis is an efficient and robust chemical approach to identify novel glycoproteins in Hp. This work characterizes glycosylated virulence factors in Hp, thus opening the door to new vaccination and antibiotic therapies to eradicate Hp infection. Broadly, this work validates metabolic oligosaccharide engineering as a complementary method to discover bacterial glycoproteins.     

The Human Scavenger Receptor CD36: GLYCOSYLATION STATUS AND ITS ROLE IN TRAFFICKING AND FUNCTION*

Human CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidized low density lipoprotein and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation, and 10 putative glycosylation sites situated in the large extracellular loop of the protein have been identified; however, their utilization and role in the folding and function of the protein have not been characterized. Using mass spectrometry on purified and peptide N-glycosidase F-deglycosylated CD36 and also by comparing the electrophoretic mobility of different glycosylation site mutants, we have determined that 9 of the 10 sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells established that glycosylation is necessary for trafficking to the plasma membrane. Minimally glycosylated mutants that supported trafficking were identified and indicated the importance of carboxyl-terminal sites Asn-247, Asn-321, and Asn-417. However, unlike SRBI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally glycosylated mutants appear to be predominantly core-glycosylated, indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified low density lipoprotein.Human CD36, originally identified in platelets as glycoprotein IV (1), is a class B scavenger receptor localized to the plasma membrane. It is not expressed ubiquitously but is present in a variety of different cells and tissue types including epithelial cells (2), macrophages (3), endothelial cells of the microvasculature (4), and smooth muscle (5). Its function is complex, and its involvement in different disease scenarios, such as cancer (6), atherosclerosis (3, 7, 8), malaria (9), and insulin resistance (10), most likely reflects the interaction of the receptor with a particular ligand in a specific cell type. For example, CD36 expressed in monocytic macrophages functions as a scavenger receptor for the uptake of oxidized LDL² (3, 11). Under certain physiological conditions, this results in the lipid loading of macrophages at the site of tissue damage in the arterial wall, leading to foam cell formation and plaque development, a key early stage in the pathogenesis of atherosclerosis (8, 12). In fat and muscle cells, CD36 plays an essential role in lipid homeostasis by uptake of long chain fatty acids (13). In this case CD36 deficiency has been linked to disorders in lipid metabolism, giving rise to increased incidences of insulin resistance and cardiomyopathies (11, 14, 15).Although much is known about the function of CD36, less is known about its structure. CD36 has no bacterial homologues but is a member of a protein family that also includes the mammalian proteins LIMPII (16), CLA-1 (17), SRBI (18), and the Drosophila proteins Croquemort (19) and emp (20). The sequence of 471 amino acids has two short hydrophobic regions at the carboxyl and amino termini separated by a large hydrophilic region (21); however, the topology of the protein is unclear with both ditopic (22) and type I (23) topological models proposed. Both are consistent in predicting that the large hydrophilic region is extracellular, which is clearly supported by epitope mapping studies (24). The protein is heavily modified post-translationally. The six extracellular cysteines, which are highly conserved within the orthologous CD36 subfamily, have been shown to be linked by disulfide bonds in bovine Cd36 (25), and the remaining four cysteines, two at each terminus, are palmitoylated (26), lending credence to the ditopic topological model.CD36 is also modified by N-linked glycosylation, which accounts for the observation that the protein migrates with an apparent molecular mass of 78–94 kDa on SDS-PAGE (4, 27) despite a theoretical mass for the polypeptide of 53 kDa. N-Linked glycosylation is a common modification of extracellular and secreted proteins, and defects in the glycosylation pathways lead to a wide range of serious diseases known collectively as congenital disorders of glycosylation (28). Glycosylation can be important for correct folding of proteins (29, 30) either by directly inducing and/or stabilizing the tertiary fold of the polypeptide (31) or via an affinity for lectin chaperones such as calnexin or calreticulin (32). Glycosylation has also been shown to be important for the trafficking of certain glycoproteins through affinity for lectin transport machinery (33). The glycosylation status of bovine Cd36 has already been determined with all eight putative sites shown to be glycosylated (34). Human and bovine CD36 are 83% identical (93% when similar residues are included) and share 7 glycosylation sites (human has 10 putative glycosylation sites). In the related mouse SRBI, which is 33% identical (54% similar) to human CD36, there are 11 putative N-linked glycosylation sites, only 3 of which are shared with the human protein. Site-directed mutagenesis of each of the 11 sites independently in SRBI in an otherwise wild type protein indicates that all are glycosylated, with two (Asn-108 and Asn-173) important for either trafficking or folding. Mutagenesis of either of these two residues resulted in very little cell surface expression of the protein (35); however, neither site is conserved in human CD36.To gain further understanding of the role of glycosylation of CD36, we used mutagenesis and biophysical analysis (mass spectrometry and gel electrophoresis) to identify unequivocally which glycosylation sites are occupied in human CD36. Antibody and ligand binding studies with these mutant proteins also provided insights into the role of glycosylation and site occupancy in the trafficking and function of the protein.     

Laserspray Ionization, a New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.Tissue imaging by mass spectrometry (MS) is proving useful in areas such as detecting tumor margins, determining sites of high drug uptake, and mapping signaling molecules in brain tissue (1–8). Imaging using secondary ion mass spectrometry is well established but is only marginally useful with intact molecular mass measurements from biological tissue (9–11). Matrix-assisted laser desorption/ionization (MALDI)-MS operating under vacuum conditions has been used for tissue imaging with success, especially for abundant components such as membrane lipids, drug metabolites, and proteins (12–14). Spatial resolution of ∼20 μm has been achieved (15), and the MALDI-MS method has been applied in an attempt to shed light on Parkinson disease (16, 17), muscular dystrophy (18), obesity, and cancer (12, 19).Unfortunately, there are disadvantages in using vacuum-based MS for tissue imaging in relation to analysis of unadulterated tissue. Also, the mass spectrometers used in these studies frequently have much lower mass resolution and mass accuracy than are available with atmospheric pressure ionization (API)¹ instruments and are not as widely available. Because the vacuum ionization methods produce singly charged ions, mass-selected fragmentation methods provide only limited information, especially for proteins. In addition, no advanced fragmentation such as electron transfer dissociation (ETD) (20–22) is available for confident protein confirmation or identification. Atmospheric pressure (AP) MALDI can be coupled to high performance mass spectrometers but suffers from sensitivity issues for tissue imaging where high spatial resolution is desired (23). AP MALDI also primarily produces singly charged ions (24, 25). Thus, mass and cross-section analysis of intact proteins has yet to be accomplished using AP MALDI because of intrinsic mass range limitations of API instruments, which frequently have a mass-to-charge (m/z) limit of <4000. Thus, new improved methods of mass-specific tissue imaging, especially at AP, are needed.The potential of laserspray ionization (LSI) (Scheme 1) (26–33) for protein tissue analysis is reported here. LSI has advantages relative to other MS-based methods, including speed of analysis, laser ablation of small volumes, more relevant AP conditions, extended mass range and improved fragmentation through multiple charging, and the ability to obtain cross-section data for proteins on appropriate instrumentation. The applicability of LSI for high mass compounds on high performance API mass spectrometers (Orbitrap Exactive and SYNAPT G2) has been demonstrated producing ESI-like multiply protonated ions (26–28). The first experiments showing sequence analysis by ETD using the LSI method were successfully carried out on a Thermo Fisher Scientific (San Jose, CA) LTQ-ETD mass spectrometer (26). Nearly complete sequence coverage was obtained for ubiquitin, an important regulatory protein. Applying ETD fragmentation to LSI-MS analyses potentially provides a new method for studying biological processes, including the mapping of phosphorylation, glycosylation, and ubiquitination sites from intact proteins and directly from tissue.Open in a separate windowScheme 1.Overview of LSI-MS operated in transmission geometry.Furthermore, unlike ESI and related ESI-based methods such as desorption-ESI (34), the LSI method has been shown to allow analysis of lipids in tissue from ablated areas <80 μm (30). In comparison with literature reports for AP MALDI at the same stage of development (35), LSI is more than an order of magnitude more sensitive and is capable of analyzing proteins on high resolution mass spectrometers as was demonstrated by obtaining full-acquisition mass spectra at 100,000 mass resolution (FWHH, m/z 200) after application of only 20 fmol of bovine pancreas insulin in the matrix 2,5-dihydroxyacetophenone (2,5-DHAP) onto a glass microscope slide (33). The analysis speed of LSI was demonstrated by obtaining mass spectra of five samples in 8 s (32). Here, we show the utility of LSI for intact peptide and protein analyses directly from mouse brain tissue. The ability to obtain a protein mass spectrum directly from mouse brain tissue in a single laser shot at 100,000 mass resolution and with ETD fragmentation is demonstrated.     

Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification.Asparagine (N)-linked glycosylation is an essential post-translational modification of secretory and membrane proteins in eukaryota and also occurs in archaea and some bacteria (1). Oligosaccharyltransferase (OTase)¹ is an integral membrane protein that catalyzes N-glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum (ER) (2). Eukaryotic OTase is physically associated with the translocon (3) and transfers oligosaccharide from a dolichol-pyrophosphate carrier onto asparagine side-chains of substrate polypeptides (2). The efficiency of glycosylation of asparagine residues is dramatically increased if they are present in glycosylation “sequons” (N-x-T/S; x ≠ P), the peptide recognition motifs of the catalytic site of OTase (4). After the transfer of glycan to protein, the presence of N-glycosylation assists efficient glycoprotein folding in the ER intrinsically and by locally recruiting the disulfide isomerase ERp57 through the lectins calnexin and calreticulin (5). Correctly folded glycoproteins are free to traffic through the Golgi, where further modification and extension of glycan structures can occur (6). The precise glycan structures present on mature glycoproteins are often vital for their biological functions, including those involved in immune response, embryonic development, and cancer (6–8).OTase in Baker''s yeast, Saccharomyces cerevisiae, is a multiprotein complex consisting of eight subunits (2). The catalytic site is located in Stt3p, and other protein subunits whose functions have been investigated are required for the integrity of the complex and for regulation of substrate specificity. It has been proposed that the requirement of Ost1p (human ribophorin I) for efficient glycosylation of a subset of integral membrane proteins (9) is due to direct physical association (10) to retain potential substrates in close proximity to Stt3p (11). The details and mechanisms of this association are not known. Ost3p and Ost6p are homologous proteins, and the incorporation of either into OTase defines two isoforms of the enzyme with distinct protein substrate specificities (Fig. 1) (12–14). Efficient glycosylation of some asparagine residues requires Ost3p-OTase, whereas others require Ost6p-OTase (15). A model of Ost3p/Ost6p function in N-glycosylation site selection has been proposed (16) in which the ER-lumenal peptide-binding grooves transiently tether nascent polypeptide non-covalently or through mixed disulfides, inhibiting local protein folding and increasing the efficiency of glycosylation of nearby asparagine residues. Aspects of this model, including mixed-disulfide formation (17) and non-covalent peptide binding (18), have been tested in vitro. Although it has been established that Ost3p-OTase and Ost6p-OTase have different polypeptide substrate preferences in vivo (12–15, 19), the physiological relevance and details of any interactions between Ost3p/Ost6p and substrate polypeptide are unclear.Open in a separate windowFig. 1.Overview of experimental manipulation of yeast OTase isoforms. A, OTase in wild-type yeast has eight subunits, with two isoforms defined by incorporation of either of the homologous Ost3p or Ost6p subunits. Yeast with only (B) Ost3p-OTase or (C) Ost6p-OTase was constructed via genomic deletion of OST3 and OST6 with overexpression of either Ost3p or Ost6p. D, yeast with a single variant OTase isoform was constructed through overexpression of variant Ost3p or Ost6p, for example, Ost3_Q103K,Q106K.Here, we used in vivo yeast genetics, glycoproteomics, and in vitro biochemistry to identify the precise sites of interaction between Ost3p/Ost6p and substrate polypeptides that affect the efficiency of N-glycosylation at diverse asparagines. Based on these data, we present a model in which transient binding of nascent polypeptide by Ost3p/Ost6p isolates newly translocating polypeptide from pre-translocated polypeptide, resulting in the formation of a short flexible loop of glycosylation-competent polypeptide substrate in close proximity to the active site of OTase for efficient modification.     

The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome: IDENTIFICATION, GLYCOSITE OCCUPANCY, AND MEMBRANE ORIENTATION*

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function. One of the best examples of endogenous repair mechanisms involves skeletal muscle, which has innate regenerative capacity (for reviews, see Refs. 1–4). Skeletal muscle repair begins with satellite cells, a heterogeneous population of mitotically quiescent cells located in the basal lamina that surrounds adult skeletal myofibers (5, 6), that, when activated, rapidly proliferate (7). The progeny of activated satellite cells, known as myogenic precursor cells or myoblasts, undergo several rounds of division prior to withdrawal from the cell cycle. This is followed by fusion to form terminally differentiated multinucleated myotubes and skeletal myofibers (7, 8). These cells effectively repair or replace damaged cells or contribute to an increase in skeletal muscle mass.The molecular mechanisms that regulate differentiation of satellite cells and myoblasts toward myofibers are not fully understood, although it is known that the cell surface proteome plays an important biological role in skeletal muscle differentiation. Examples include how cell surface proteins modulate myoblast elongation, orientation, and fusion (for a review, see Ref. 8). The organization and fusion of myoblasts is mediated, in part, by cadherins (for reviews, see Refs. 9 and 10), which enhance skeletal muscle differentiation and are implicated in myoblast fusion (11). Neogenin, another cell surface protein, is also a likely regulator of myotube formation via the netrin ligand signal transduction pathway (12, 13), and the family of sphingosine 1-phosphate receptors (Edg receptors) are known key signal transduction molecules involved in regulating myogenic differentiation (14–17). Given the important role of these proteins, identifying and characterizing the cell surface proteins present on myoblasts in a more comprehensive approach could provide insights into the molecular mechanisms involved in skeletal muscle development and repair. The identification of naturally occurring cell surface proteins (i.e. markers) could also foster the enrichment and/or characterization of cell intermediates during differentiation that could be useful therapeutically.Although it is possible to use techniques such as flow cytometry, antibody arrays, and microscopy to probe for known proteins on the cell surface in discrete populations, these methods rely on a priori knowledge of the proteins present on the cell surface and the availability/specificity of an antibody. Proteomics approaches coupled with mass spectrometry offer an alternative approach that is antibody-independent and allows for the de novo discovery of proteins on the surface. One approach, which was used in the current study, exploits the fact that a majority of the cell surface proteins are glycosylated (18). The method uses hydrazide chemistry (19) to immobilize and enrich for glycoproteins/glycopeptides, and previous studies using this chemistry have successfully identified soluble glycoproteins (20–24) as well as cell surface glycoproteins (25–28). A recently optimized hydrazide chemistry strategy by Wollscheid et al. (29) termed cell surface capturing (CSC)¹ technology, reports the ability to identify cell surface (plasma membrane) proteins specifically with little (<15%) contamination from non-cell surface proteins. The specificity stems from the fact that the oligosaccharide structure is labeled using membrane-impermeable reagents while the cells are intact rather than after cell lysis. Consequently, only extracellular oligosaccharides are labeled and subsequently captured. Utilizing information regarding the glycosylation site then allows for a rapid elimination of nonspecifically captured proteins (i.e. non-cell surface proteins) during the data analysis process, a feature that makes this approach unique to methods where no label or tag is used. Additionally, the CSC technology provides information about glycosylation site occupancy (i.e. whether a potential N-linked glycosylation site is actually glycosylated), which is important for determining the protein orientation within the membrane and, therefore, antigen selection and antibody design.To uncover information about the cell surface of myoblasts and to identify potential markers of myoblast differentiation, we used the CSC technology on the mouse myoblast C2C12 cell line model system (30, 31). This adherent cell line derived from satellite cells has routinely been used as a model for skeletal muscle development (e.g. Refs. 1, 32, and 33), skeletal muscle differentiation (e.g. Refs. 34–36), and studying muscular dystrophy (e.g. Refs. 37–39). Additionally, these cells have been used in cell-based therapies (e.g. Refs. 40–42). Using the CSC technology, 128 cell surface N-linked glycoproteins were identified, including several that were found to change in overall abundance as the myoblasts differentiate toward myotubes. The current data also confirmed the occupancy of 235 N-linked glycosites of which 226 were previously unconfirmed. The new information provided by the current study is expected to facilitate the development of useful tools for studying the differentiation of myoblasts toward myotubes.